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Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis

Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for... Published online 16 March 2008 Nucleic Acids Research, 2008, Vol. 36, No. 8 2677–2689 doi:10.1093/nar/gkn123 Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis 1 1 2,3 2,3,4,5 Christoph Jo¨ chl , Mathieu Rederstorff , Jana Hertel , Peter F. Stadler , 2 6 6 1, Ivo L. Hofacker , Markus Schrettl , Hubertus Haas and Alexander Hu¨ ttenhofer * Innsbruck Biocenter, Division of Genomics and RNomics – Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Institute for Theoretical Chemistry, University of Vienna, Wa¨ hringerstr. 17, A-1090 Wien, Austria, Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, University of Leipzig, Hartelstraße 16-18, D-04107 Leipzig, Germany, Santa Fe Institute, Santa Fe, NM 87501, USA, Fraunhofer Institut fuer Zelltherapie und Immunologie,Deutscher Platz 5e, 04103 Leipzig, Germany and Innsbruck Biocenter, Division of Molecular Biology – Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria Received December 7, 2007; Revised February 4, 2008; Accepted March 4, 2008 ABSTRACT down-regulate protein synthesis in a filamentous fungus. Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organ- isms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus INTRODUCTION fumigatus. To that aim, we experimentally screened Cells from all organisms, studied to date, contain two for ncRNAs, expressed under various growth con- different kinds of RNA species, the protein-encoding ditions or during specific developmental stages, messenger RNAs (mRNAs) as well as non-protein-coding by generating a specialized cDNA library from RNAs (ncRNAs). In contrast to mRNAs, ncRNAs are size-selected small RNA species. Our screen not translated into proteins, but have important cellular revealed 30 novel ncRNA candidates from known functions, either on their own or in complex with proteins ncRNA classes such as small nuclear RNAs (1–6). Functions of ncRNAs range from RNA processing, (snRNAs) and C/D box-type small nucleolar RNAs modification, transcriptional regulation, mRNA stability (C/D box snoRNAs). Additionally, several candidates and translation up to protein secretion (2). Reported sizes of many known ncRNAs are generally well below sizes of for H/ACA box snoRNAs could be predicted by a mRNAs and range from 21–22-nt long microRNAs (7,8) bioinformatical screen. We also identified 15 candi- to about 500 nt [e.g. telomerase RNA (9)]. In addition, dates for ncRNAs, which could not be assigned to also very large ncRNAs, including the 17-kb long human any known ncRNA class. Some of these ncRNA Xist RNA (10,11) or the 108-kb long mouse Air RNA (12) species are developmentally regulated implying a have been observed. possible novel function in A. fumigatus develop- Recently, whole genome screens in eukaryal organisms ment. Surprisingly, in addition to full-length tRNAs, have revealed a large number of ncRNAs which have been we also identified 5’-or3’-halves of tRNAs, only, shown to regulate gene expression by novel mechanisms which are likely generated by tRNA cleavage within such as RNA interference, gene co-suppression, gene the anti-codon loop. We show that conidiation silencing, imprinting and DNA methylation (8,13–15). induces tRNA cleavage resulting in tRNA depletion Evidence for the involvement of ncRNAs exerting critical within conidia. Since conidia represent the rest- functions during vegetative growth, development or cell ing state of A. fumigatus we propose that conidial differentiation as well as in diseases, such as carcinogen- tRNA depletion might be a novel mechanism to esis, is becoming increasingly clear (16,17). *To whom correspondence should be addressed. Tel: +43 512 9003 70250; Fax: +43 512 9003 73100; Email: alexander.huettenhofer@i-med.ac.at Correspondence may also be addressed to Hubertus Haas. Tel: +43 512 9003 70205; Email: hubertus.haas@i-med.ac.at 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2678 Nucleic Acids Research, 2008, Vol. 36, No. 8 Several single-cellular eukaryal organisms have been according to Pontecorvo et al. (31) containing 1% (wt/vol) studied in the past, revealing a plethora of novel ncRNAs glucose as carbon source and 20 mM glutamine as a (18–20). A bioinformatical analysis of the fungal genomes nitrogen source. For liquid growth A. fumigatus was from seven different yeast species provided a significant cultured at 378C up to the indicated time point either in number of evolutionarily conserved, structured ncRNAs, AMM or in Aspergillus complete media (ACM) compris- suggesting their roles in post-transcriptional regulation ing 2% (wt/vol) glucose, 0.2% tryptone (wt/vol), 0.1% (21). In contrast, identification and functions of ncRNAs yeast extract (wt/vol) and 0.1% casamino acids (wt/vol). in filamentous fungi, such as Aspergillus species, have not Media contained 10mM FeSO and respectively for iron- been studied. depleted conditions, iron was omitted. For nitrogen Most filamentous fungi are saprophytes playing impor- starvation, 18-h AMM cultures were harvested and shifted tant roles in carbon and nitrogen recycling. Moreover, for another 6 h into AMM lacking glutamine. several members of this fungal group are well known for production of biotechnological important secondary Growth conditions for conidiation of A. fumigatus metabolites, as producers of toxins, or as facultative ATCC46645 pathogens for plants and animals. Infections with filamen- For synchronized asexual developmental A. fumigatus was tous fungi have emerged as an increasing risk for immuno- grown in liquid ACM for 18 h (32). Then, mycelia were suppressed patients. Aspergillus fumigatus accounts for harvested by filtering and transferred to solid ACM, were most of these infections, termed invasive aspergillosis, and conidiation is induced (32). Samples for RNA isolation can be regarded as the most common airborne fungal were collected after 6, 12, 24, 48 and 72 h of growth on pathogen. Specific diagnostics as well as therapeutic solid ACM. possibilities are limited (22–24). Hence, the mortality rate of invasive aspergillosis ranges between 30 and 90%, Generation of an A. fumigatus cDNA library depending on the immune status of the host (22,23). Its global ubiquity as well as the infectious cycle of this Aspergillus fumigatus was cultured under various condi- pathogen is perpetuated by prolific production of asexual tions to ensure expression of also growth-regulated spores (termed conidia) from specialized aeral hyphae ncRNAs. Total RNA was extracted from harvested (termed conidiophores). Conidial germination, e.g. in the mycelia of A. fumigatus by the TRI-zol method (Gibco human lung, following spore inhalation represents the BRL) (33). Subsequently, equal amounts of total RNAs initiating event of pulmonary disease. Three important were pooled and size-fractionated by denaturing 8% steps can be distinguished during spore germination: PAGE (7 M urea, 1 TBE buffer). RNAs in the size activation of the resting spore to appropriate environ- range between 15 and 500 nt were excised from the gel, mental conditions, isotropic growth that involves water passively eluted and ethanol-precipitated. RNAs were uptake and wall growth (termed swelling) and polarized poly(C)-tailed employing poly(A) polymerase from yeast growth that results in the formation of a germ tube from (USB). C-tailed RNAs were ligated to a 19-nt long 5 which the new mycelium originates (25). Conidia are linker by T4 RNA ligase, as described previously (29). dormant, metabolically inactive cells, which can be stored RNAs from the library were subsequently converted into for extended periods. The combined presence of air, water cDNAs by RT–PCR as described, employing complemen- and a carbon source induces germination with the first tary primers to 5 linkers and the poly(C) tail (29), and measurable activities being trehalose breakdown and cloned into pGEM-T vector (Promega). translation (26). Aspergillus fumigatus cells contain a haploid nuclear Dot-blot hybridization genome of 28.9 Mb in size, distributed into eight chromo- Aspergillus fumigatus library-derived cDNA clones were somes (27) and a circular mitochondrial genome exhibit- PCR-amplified using the primers M13 and M13 reverse. ing a size of 32 kb. Apart from ribosomal RNAs (rRNAs) Two micro litres of diluted (1:20) and denatured (918C, and transfer RNAs (tRNAs), no other ncRNAs have yet 2 min) PCR products were spotted onto a nylon mem- been annotated and characterized within the A. fumigatus brane (Hybond N , Amersham), cross linked employing genome (27). However, knowledge on the number and the STRATAGENE UV crosslinker (120 mJ/cm ) and functions of ncRNAs is vital for understanding cell pre-hybridized for 2 h in 1 M sodium phosphate buffer functions in A. fumigatus and could potentially open up (pH 6.2) with 7% SDS. Oligonucleotides, complementary new avenues for the development of novel anti-fungal to known and most abundant ncRNAs were [g- P]ATP drugs. Thus, for the experimental identification of novel end-labelled by T4 polynucleotide kinase. All six oligo- ncRNA species in A. fumigatus we generated a specialized nucleotide probes were added to the hybridization tube cDNA library comprising small ncRNA species sized from and hybridization was carried out at 528C in hybridization 20 to –500 nt (28,29). buffer (178 mM Na HPO , NaH PO , pH 6.2, 7% SDS) 2 4 2 4 for 12 h. Blots were washed twice: at room temperature in 2 SSC buffer, 0.1% SDS for 10 min and subsequently at MATERIAL AND METHODS hybridization temperature in 0.1 SSC, 0.1% SDS for Strain and growth conditions 10 min. Afterwards blots were rinsed in desalted water. Aspergillus fumigatus wild-type ATCC46645 (30) was Following stringency washes membranes were exposed to maintained on solid Aspergillus minimal media (AMM) Kodak MS-1 film from 30 min to 2 days. Nucleic Acids Research, 2008, Vol. 36, No. 8 2679 Sequence analysis of cDNA library (http://genome.jgi-psf.org/Aspni1/Aspni1.home.html) sequence conservation of snRNAs and novel ncRNA cDNA clones were sequenced using the M13 reverse candidates was analysed in the genomes of A. niger, primer and the BigDye terminator cycle sequencing A. oryzae, A. fumigatus and A. nidulans (Supplementary reaction kit (PE Applied Biosystems). Sequencing reac- Data, Figures 1 and 4). Since U3 snoRNA could not be tions were run on an ABI Prism 3100 (Perkin Elmer) found by means of a BLASTN search, a computationally capillary sequencer. Subsequently, sequences were ana- more expensive strategy was employed. The semi-local lysed with the LASERGENE sequence analysis program variant of Gotoh’s dynamic programming algorithm (40) package (DNASTAR). In this analysis, cDNA sequences was implemented in a memory-efficient scanning version were compared to each other using the Lasergene Seqman in the C programming language (parameters: match +3, II program package to identify identical sequences mismatch 1, gap opening 8 and gap extension 2). (DNASTAR). Following a BLASTN search against the GenBank database (NCBI, http://www.ncbi.nlm.nih.gov/ BLAST) was performed using standard parameters (i.e. RESULTS AND DISCUSSION match/mismatch score: 1,-2 and linear gap costs) which cDNA library construction and analysis were adjusted automatically for short input sequences. All RNA sequences, which were not annotated in the Aspergillus fumigatus was grown under seven different database and showed a perfect match within the culture conditions (see Materials and methods section). A. fumigatus genome (34), were treated as potential Liquid complete medium (ACM) is the optimal growth candidates for novel ncRNAs. condition resulting in maximal vegetative proliferation. In comparison, minimal medium demands expression of an Northern blot analysis increased number of anabolic pathways. Starvation for iron (AMM-Fe) or nitrogen (AMM-N) was found to Total RNA was either size-separated on 1.2% agarose– induce virulence traits (41,42). In contrast to liquid 2.2 M formaldehyde gels and blotted onto Hybond N cultures (vegetative growth), plate cultures induce con- membranes (Amersham) as described earlier (35), or size- idiation, i.e. the formation of infectious propagules (43). fractionated on denaturing polyacrylamide gels (PAGE). These different growth conditions are reported to induce For PAGE, 3–40mg of total RNA isolated from different different mRNA transcriptomes and thus, in addition, are growth conditions (see above) was denatured for 1 min at likely to also induce the expression of differentially 958C, separated on a 8% denaturing polyacrylamide gel expressed ncRNA species. Total RNA from each culture (7 M urea, 1 TBE buffer) and transferred onto a nylon condition was isolated separately and converted into membrane (Hybond N , Amersham) using the Bio-Rad cDNA as previously described (29). semi-dry blotting apparatus (Trans-blot SD; Bio-Rad). Subsequently, we screened about 7200 cDNA clones for After immobilizing of RNAs using the STRATAGENE identification of novel ncRNA species by dot-blot UV crosslinker (120 mJ/cm ), nylon membranes were pre- hybridization employing labelled oligonucleotides directed incubated for 2 h in 1 M sodium phosphate buffer (pH 6.2) against the most abundant known ncRNAs, as described with 7% SDS. Oligonucleotides from 18 to 35 nt in size, previously (44). This approach yielded 3120 cDNA complementary to potentially novel RNA species, were sequences, which were subjected to further bioinformatical end-labelled with [g- P]ATP and T4 polynucleotide analysis (see Materials and methods section). Thereby, kinase. Depending on the T of the respective oligonu- 58.2% of cDNA sequences corresponded to nuclear cleotides, hybridization was carried out from 42 to 588Cin or mitochondrial rRNA fragments. tRNAs or tRNA hybridization buffer (178 mM Na HPO , NaH PO ,pH 2 4 2 4 cleavage fragments (see below) represented about 23.0% 6.2, 7% SDS) for 12 h. Blots were washed twice, once at of cDNA clones (Figure 1A). About 4.9% of cDNA room temperature in 2 SSC buffer, 0.1% SDS for 10 min sequences corresponded to three different snRNAs and subsequently at the respective hybridization tempera- (U1, U5 and U6 snRNA), which had escaped annotation ture in 0.1 SSC, 0.1% SDS for 1–10 min. Membranes in the current release of the A. fumigatus genome (27). were exposed to Kodak MS-1 film from 15 min to 5 days. Candidates for small nucleolar RNA (snoRNA) made up 11.3% of all cDNA clones. Due to the lack of conserved Bioinformatical methods sequence or structure motifs the remaining 1.2% of cDNA To obtain secondary structure predictions and conserva- clones could not be assigned to any class of known tion of A. fumigatus ncRNAs, sequences were mapped to ncRNAs and thus might represent entirely novel ncRNAs the genomes of related species via BLAST (36) search in A. fumigatus (Figure 1A). –3 (E-value: 10 ). The homologous sequences were then Subsequently, we confirmed expression and sizes of aligned using a dynamic programming alignment algo- ncRNAs by northern blot analysis. To that aim, total rithm as implemented in CLUSTAL W (37) and out of the RNA isolated from different growth conditions or multiple sequence alignment the secondary structure developmental stages of A. fumigatus, was employed in was predicted using the folding routines from the Vienna northern blot analysis to investigate expression levels of RNA package (38,39). Genomes of Aspergillus oryzae, ncRNAs (Figures 2 and 3). In our final analysis, we only Aspergillus nidulans and Aspergillus niger were listed those novel ncRNAs for which unambiguous downloaded from the NCBI database. Employing a northern blot signals were obtained, with the notable BLASTN search in the JGI genome browser of A. niger exception of some snoRNA species (Table 1, see below), 2680 Nucleic Acids Research, 2008, Vol. 36, No. 8 Figure 1. (A) Distribution of 3120 cDNA clones from the A. fumigatus expression library encoding ncRNA candidates. The abundance of different ncRNA species is shown as percentage of total clones. (B) Numbers of ncRNA candidates derived from the A. fumigatus cDNA library are indicated in brackets. Figure 2. Northern blot analysis of A. fumigatus snRNAs and selected snoRNA candidates. Designation of clones is indicated on the left. Sizes of ncRNAs, as estimated by comparison with an internal RNA marker, are indicated on the right. Total RNA was isolated from A. fumigatus mycelia grown under seven different conditions and at different time points: AMM, minimal medium; ACM, complete medium; AMM-Fe, iron starvation; AMM-N, nitrogen starvation. Liquid cultures resemble vegetative growth and plate cultures induce conidiogenesis. In-gel ethidium bromide-stained 5.8 S rRNA and 5 S rRNA serve as loading controls. (A) U1-1/U1-2, U5 and U6 snRNAs, respectively. (B) C/D box snoRNAs with predicted targets. (C) C/D box snoRNAs without predicted targets. which were computationally confirmed by the presence of cerevisae homologues shows highly conserved regions in canonical sequence and structure motifs. the loops of the hairpins. Compared to the consensus secondary structure as reported by the Rfam database (45) the Aspergillus snRNA structures show the same stem– snRNA candidates loop distribution and arrangement (Supplementary Data Four different cDNA clones representing putative snRNA Figure 1A–D). candidates were annotated as U1-1, U1-2, U5 and U6 Except for nuclear encoded 26S, 18S, 5.8S rRNAs and snRNAs, respectively, by comparison to the RNA family tRNAs, U1-1 snRNA appeared as the most abundant database of alignments and Covariance Models (45). clone in the library (Table 1). Three identical truncated Sequences of U1-1 and U1-2 snRNAs differ by 1 nt, only cDNA clones of U6 snRNA, mapping to two different loci (A or T at position 128, respectively), and exhibit different within the A. fumigatus genome, were also identified in our genomic locations, implying that they represent two screen (Table 1). distinct isoforms of U1 snRNA (Table 1). A comparison Expression of A. fumigatus U1, U5 and U6 snRNAs of the predicted secondary structures of A. fumigatus could be confirmed by northern blot analysis (Figure 2A). snRNAs U1-1, U1-2, U5 and U6 snRNAs and their Sizes, as estimated by comparison to an internal RNA homologues with the corresponding Saccharomyces marker, were determined as 130 nt, 100 nt or 105 nt, Nucleic Acids Research, 2008, Vol. 36, No. 8 2681 Figure 3. Northern blot analysis of ncRNA candidates from unknown ncRNA classes. Designation of clones is indicated on the left. Sizes of ncRNAs, as estimated by comparison with an internal RNA marker, are indicated on the right. Growth conditions and loading controls (5.8 S rRNA and 5 S rRNA) as described in Figure 2. As an additional loading control, U1 snRNA was included in northern blot analysis. respectively (Figure 2A). The absence of U2 and U4 verified by northern blot analysis (Supplementary Data, snRNAs in our screen might be explained by RNA Figure 2A). It is noteworthy, however, that sizes of U2 and modifications or structural constraints, impeding reverse U4 snRNAs, as estimated by northern blot analysis, differ transcription into cDNAs. from their bioinformatical predicted sizes due to the fact 0 0 Therefore, we employed, as a bioinformatical approach, that precise 5 - and 3 termini cannot be obtained by a BLASTN search of all known U2 and U4 snRNAs as computational analysis. annotated in the Rfam database, including all yeast snRNA genes. Indeed, we were able to predict U2 and snoRNA candidates U4 homologues within the A. fumigatus genome Two classes of small nucleolar ncRNAs (snoRNAs) have (Supplementary Data Figure 1B and D). However, been detected in eukaryal as well as in archaeal species A. fumigatus U2 and U4 snRNAs appeared to be less (49–52): C/D box snoRNAs, which guide 2 -O-methyla- conserved on the sequence level than expected and could tion of ribosomal, spliceosomal and tRNAs (the latter in only be partially aligned. Manually extending the align- Archaea, only), and H/ACA snoRNAs which guide ment yielded a set of nearly perfect U2 and U4 snRNA pseudouridylation in these RNA species (53). In our sequences in A. fumigatus and related species (e.g. screen, we identified 27 candidates for C/D box snoRNAs A. niger). In addition, we were able to compute based on conserved sequence or structural motifs by the interaction structure of the U4–U6 complex (Supple- employing the SnoReport program (54). mentary Data Figure 1D) using the RNAalifold All C/D box snoRNAs from our screen contained bona program (47). fide sequence motifs of canonical snoRNAs, namely C, D , A comparison of the predicted consensus secondary C and D boxes, respectively (1,55). We noticed, however, structures to annotated secondary structures in the Rfam that all C/D box snoRNAs from A. fumigatus lacked the database and in (48) shows that these computational canonical terminal stem structure, in contrast to mamma- candidates match perfectly, although they show high lian C/D snoRNAs. This is in agreement with previous variance in their primary sequence (Supplementary Data, Figure 1A–C). This is consistently observed for the observations on archaeal C/D snoRNAs (56) or C/D evolution of ncRNAs, since these genes are mainly con- snoRNAs from the slime mold Dyctiostelium discoideum, served on the secondary structure level. Table 3 indicates which are also devoid of a terminal stem (57). levels of conservation within all identified snRNA candi- Surprisingly, the abundant U3 C/D snoRNA was dates in A. fumigatus. In addition to their bioinformatical missing among these candidates. A standard BLASTN identification, expression of U2 and U4 snRNAs was search also failed to identify U3 snoRNA candidates 2682 Nucleic Acids Research, 2008, Vol. 36, No. 8 Table 1. Candidates for snRNAs or snoRNAs Name Copies cDNA (nt) Northern Location Putative target Accession blot (nt) number snRNAs U1-1 157 132 130 Intergenic; Afu1g06980/Afu1g07000 AM921915 U1-2 67 132 130 Intergenic; Afu4g12490/Afu4g12500 AM921916 U5 19 99 100 Intergenic; Afu6g12670/Afu6g12680 AM921917 U6 3 50 105 Intergenic; Afu4g12500/Afu4g12520 AM921918 Intergenic; Afu2g10150/Afu2g10160 C/D box snoRNAs with predicted target Afu-34 18 84 n.d. Intergenic; Afu2g15970/Afu2g15980 Am1131 and Gm2506 in 26S AM921919 Afu-191 11 92 90 Intergenic; Afu1g10270/Afu1g10280 Gm75 in 5.8S and Am32 AM921920 in U2 snRNA Afu-190 8 107 110 Intergenic; Afu4g11320/Afu4g11330 Gm557 in 18S AM921921 Afu-198 8 130 n.d. Intergenic; Afu1g02700/Afu1g02680 Cm2856 and Um 2859 in 26S AM921922 Afu-294 7 85 80 Intergenic; Afu1g09750/Afu1g09760 Cm1851 in 26S; Am43 in 5.8S AM921923 Afu-264 4 103 n.d. Intergenic; Afu7g05290/Afu7g05300 Gm2770 and Gm2773 in 26S; AM921924 Afu-277 4 100 n.d. Intergenic; Afu1g09740/Afu1g09760 Um2706 in 26S; Am97 in AM921925 18S; Cm47 in 5.8S Afu-263 3 96 85 Intron1 - Exon2 -3 UTR (sense); Am815 and Gm906 in 26S AM921926 Afu-188 2 84 n.d. Intergenic; Afu4g11310/Afu4g11320 Am25 and Um26 in 18S AM921927 Afu-200 2 91 n.d. Intron 3 (sense); Afu1g12390 Am415 and Gm1422 in 18S AM921928 Afu-304 2 106 95 Intron 2 (sense); Afu1g09800 Cm2925 in 26S AM921929 Afu-380 2 87 n.d. Intergenic; Afu4g06780/Afu4g06770 Um2701 in 26S AM921930 Afu-513 2 129 n.d. Intergenic; Afu4g11310/Afu4g11320 Gm1338 and Gm3719 in 26S AM921931 Afu-328 1 75 75 Intron 2 (sense); Afu6g04570 Gm2792 in 26S AM921932 Afu-335 1 97 100 Intron 2 (sense); Afu1g04840 Cm2316 in 26S AM921933 Afu-438 1 83 n.d. Intergenic; Afu2g15980/Afu2g15970 Am2877 in 26S AM921934 Afu-455 1 92 n.d. Intergenic; Afu1g09760/Afu1g09740 Gm1122 in 18S AM921935 C/D box snoRNAs without predicted target (orphan snoRNAs) Afu-40 123 90 90 Intergenic; Afu4g11310/Afu4g11320 AM921936 Afu-514 74 105 n.d. Intergenic; Afu6g03830/Afu6g03840 AM921937 Afu-515 40 89 n.d. Intergenic; Afu4g11310/Afu4g11320 AM921938 Afu-199 26 79 80 Intron 1 (sense); Afu7g02320 AM921939 Afu-298 3 93 90 Intergenic; Afu1g05080/Afu1g05100 AM921940 Afu-300 3 88 90 Intergenic; Afu4g11310/Afu4g11320 AM921941 Afu-215 2 24 85 Intergenic; Afu5g12870/Afu5g12880 AM921942 Afu-511 2 164 160 Intergenic; Afu1g03400/Afu1g03410 AM921943 Afu-210 1 24 75 Intron 2 (sense); Afu2g03610 AM921944 Afu-265 1 23 n.d. Intergenic; Afu3g02340/Afu3g02370 AM921945 Copies: number of independent cDNA clones obtained from each ncRNA candidate; cDNA (nt): length of cDNA assessed by sequencing; northern blot (nt): length of a ncRNA candidate estimated by northern blot analysis (n.d., expression was not determined); location: accession numbers of genes flanking the ncRNA (intergenic) or accession number of the gene, which the ncRNA is derived from (sense or antisense to exon or intron); putative target: refers to the predicted modified nucleotides within rRNAs; accession number: accession number of the ncRNA sequence in DDBJ/ EMBL/GeneBank databases. within the A. fumigatus genome. Thus, we applied a Afu-263, extends beyond a predicted intron–exon border computationally more sophisticated method, employing within the 3 -untranslated region (UTR; Figure 1B) of a the semi-local variant of the Gotoh dynamic programming hypothetical protein, encoded by the Afu3g14080 gene. algorithm (parameters: match +3, mismatch –1, gap However, this might be due to a wrong annotation of opening –8 and gap extension –2; see Materials and Afu3g14080 as a protein-coding gene, consistent with its methods section). By this method, we were able to also unusually small transcript length of 96 nt. Comparison of identify a U3 snoRNA candidate and verify its expression Afu-263 with the Rfam database reveals homology to by northern blot analysis (Supplementary Data, Figure 2B S. cerevisiae snR60. In addition, Afu-263 is also conserved and C). in the genome of the related fungus A.oryzae. Most C/D snoRNAs identified in our screen, with some The remaining 20 candidates map to intergenic regions. exceptions (Table 1) are within the size range of a canonical Twelve snoRNA candidates from this group are present as C/D snoRNAs (i.e. from 80 to 100 nt; Table 1). Identifica- singletons, whereas the remaining eight candidates are tion of some larger C/D box snoRNAs in our library is in distributed in two clusters, comprised of three and five agreement with previous findings in Oryza sativa (58) or snoRNA genes, respectively. A similar clustered gene Trypanosoma brucei (59) (Table 1). From the 27 C/D organization has been previously observed in S. cerevisiae snoRNA candidates, six are intron-derived (Table 1, (60–63). The first snoRNA cluster is located on chromo- Figure 1B) as observed for most mammalian and plant some 1 between protein-coding genes Afu1g09740 and snoRNAs (53). One C/D box snoRNA candidate, Afu1g09760 and comprises the C/D box snoRNAs Nucleic Acids Research, 2008, Vol. 36, No. 8 2683 Table 2. Candidates for entirely novel ncRNAs Name Copies cDNA (nt) Northern blot (nt) Location Accession number Afu-182 17 219 300 Intergenic; Afu4g07680/Afu4g07690 AM921946 Afu-202 5 266 270 Intergenic; Afu1g10420/Afu1g10430 AM921947 Afu-67 2 21 600/190 Intergenic; Afu4g01630/Afu4g02610 AM921948 Afu-254 2 163 90/420 Intergenic; Afu7g04110/Afu7g04120 AM921949 Afu-203 1 111 110 Intron 1 (sense); Afu3g14240 AM921950 Afu-222 1 22 535/250 Intergenic; Afu4g12410/Afu4g12420 AM921951 Afu-262 1 25 225 Intergenic; Afu1g10370/Afu1g10380 AM921952 Afu-309 1 318 320 Intergenic; Afu4g10430/Afu4g10420 AM921953 Afu-318 1 24 70 Intergenic; Afu7g01590/Afu7g01600 AM921954 Afu-322 1 46 75/35 Intergenic; Afu2g13250/Afu2g13240 AM921955 Afu-336 1 28 130 Intergenic; Afu1g09740/Afu1g09760 AM921956 Afu-364 1 18 100 Intergenic; Afu1g13820/Afu1g13830; AM921957 Intergenic; Afu3g03090/Afu3g03130; Exon4-Intron4; Afu3g02730; Intergenic; Afu6g11780/Afu6g11790 Afu-448 1 29 315 Intergenic; Afu1g11550/Afu1g11540 AM921958 Afu-465 1 17 270 Intron 2 (sense); Afu4g08930 AM921959 Afu-484 1 41 600 Exon2 (antisense); Afu3g07170 AM921960 Copies: number of independent cDNA clones, obtained from each ncRNA candidate; cDNA (nt): length of cDNA assessed by sequencing; northern blot (nt): length of a ncRNA candidate estimated by northern blot analysis; location: accession numbers of genes flanking the ncRNA (intergenic) or accession number of the gene, which the ncRNA is derived from (sense or antisense to exon or intron); accession number: accession number of the ncRNA sequence in the DDBJ/EMBL/GeneBank databases. Afu-455, Afu-277 and Afu-294. The second cluster maps receptor 2C mRNA, (65). Whether some of these orphan to chromosome 4 between the protein-coding genes snoRNAs from A. fumigatus might fulfil similar functions Afu4g11310 and Afu4g11320 and contains five C/D in mRNA targeting needs to be determined. Expression analysis of snoRNAs by northern blot box snoRNAs, Afu-40, Afu-188, Afu-300, Afu-513 and analysis revealed that most snoRNAs are equally Afu-515. Database searches with genomes from the expressed under most growth conditions with some related moulds A. nidulans and A. oryzae revealed their exceptions, (Figure 2B and C). Down-regulation during conservation in both filamentous fungi. SnoRNA clusters iron starvation, as observed for Afu-328, might implicate have been detected in a multitude of different eukaryal iron-related functions; iron starvation, for example, down- organisms, including S. cerevisiae, which may suggest an regulates transcription of genes encoding iron-containing ancient origin of this gene arrangement (62,63). proteins or iron-consuming pathways in A. nidulans (66). By employing the Snoscan Server 1.0 program (64) we Likewise, down-regulation of expression during conidio- identified putative targets for 17 out of 27 C/D box genesis, as observed for Afu-335, Afu-199 or Afu-511, snoRNAs (Table 1 and Figure 1B). As a probabilistic suggests their developmental regulation. search model for filamentous fungi is currently not In contrast to C/D box snoRNAs, we were so far unable available, the search model for target evaluation was to experimentally identify any representatives for adjusted to S. cerevisiae. As potential target sequences we H/ACA box snoRNAs. Therefore, we applied the considered the previously identified canonical targets, i.e. snoReport program (54) to chromosome 1 of the 26 S, 18 S, 5.8 S rRNAs and all snRNAs [identified by our A. fumigatus genome and were indeed able to bioinfor- screen (i.e. U1, U5 or U6 snRNA, respectively)]. Most of matically identify candidates for H/ACA snoRNAs. The the C/D box snoRNAs were predicted to guide methyla- majority of those candidates show a canonical H/ACA tion of 26 S rRNA, and, to a lesser extent, also 18 S and secondary structure and both of the sequence motifs H 5.8 S rRNAs. Several snoRNA candidates are predicted and ACA (for examples see Supplementary Data, to guide two different rRNA modifications, while Figure 3A–E). C/D box snoRNA Afu-277 is predicted to guide methyla- tion of three RNAs, i.e. 26 S rRNA, 18 S rRNAs and 5.8 S Candidates for entirely novel ncRNAs rRNA, respectively (Table 1). No C/D box snoRNAs targeting U1, U5 or U6 snRNAs were found in our screen. Due to the lack of conserved sequence motifs 1.2% of For the remaining 10 C/D box snoRNA candidates, no cDNA clones, amounting to 15 different cDNA sequences, rRNA or snRNA targets could be identified (Table 1 and could not be assigned to any class of known ncRNAs and Figure 1B). Hence, these were termed ‘orphan snoRNAs’ thus might encode entirely novel ncRNAs in A. fumigatus. in agreement with earlier findings of similar snoRNAs in Northern blot analysis verified expression of all 15 other species including mouse (1,44). Some orphan ncRNA candidates (Figure 3) and the genomic location snoRNAs were represented by numerous cDNA clones could be determined for all 15 candidates (Table 2). (Table 1), indicating their high abundance in A. fumigatus. Expression of ncRNA candidates was verified by Interestingly, MBII-52 an abundant orphan snoRNA northern blot analysis, pointing towards a significant from mouse brain, was proposed to target the serotonin abundance within A. fumigatus. Interestingly, many of 2684 Nucleic Acids Research, 2008, Vol. 36, No. 8 Table 3. Conservation level of snRNA candidates in Aspergillus species snRNAs Location in A.fumigatus Conserved in related species (% sequence identity) U1-1 chr1:1991385-1991564 A.nidulans (78), A.oryzae (94), A.niger (90) U1-2 chr4:3279813-3279991 A.nidulans (88), A.oryzae (91), A.niger (93) U2-1 chr3:3242037-3242312 A.nidulans (76), A.oryzae (85), A.niger (75) U2-2 chr1:3223904-3224180 A.nidulans (88), A.oryzae (84), A.niger (74) U4 chr1:1274827-1275331 A.nidulans (49), A.oryzae (53), A.niger (53) U5 chr6:3202420-3202638 A.nidulans (44), A.oryzae (60), A.niger (64) U6 chr1:1651441-1651760 A.nidulans (42), A.oryzae (39), A.niger (43) chr2:2598365-2598684 A.nidulans (50), A.oryzae (54), A.niger (52) chr4:3281001-3281320 A.nidulans (42), A.oryzae (49), A.niger (67) This table shows the snRNA candidates, their exact location in the A.fumigatus genome and the sequence identity in percent to the homologous candidates in related species. Although the sequence identity for U4 and U5 is relatively low, all sequences are able to fold into an appropriate secondary structure. For the consensus structures and conservation curves see Supplementary Data Figure 1 A–D. Table 4. Conservation, annotation and secondary structure prediction of novel ncRNA candidates Name Location in A. fumigatus Conservation Structure conservation Annotation Afu-182 chr4:1997138-1996922 A.niger, A.oryzae, A.nidulans + Afu-202 chr1:2714678-2714414 A.niger, A.oryzae, A.nidulans + Afu-67 chr4:444973-444954 –  550 nt upstream of rRNA operon chr4:706179-706160 – Afu-254 chr7:927348-927204 – Afu-203 chr3:3785105-3785214 – Afu-222 chr4:3252985-3252964 – Afu-262 chr1:2675372-2675395 – Afu-309 chr4:2732230-2732547 A.niger, A.oryzae + Afu-318 chr7:415029-415006 – Afu-322 chr2:3404938-3404893 – Afu-336 chr1:2523503-2523530 A.niger, A.oryzae, A.nidulans + Afu-364 chr1:3693906-3693889 A.niger + chr3:710476-710493 A.niger + chr3:836808-836825 A.niger + chr6:2935513 2935530 A.niger + Afu-448 chr1:3048137-3048109 A.niger, A.oryzae Afu-465 chr4:2316203-2316188 – Afu-484 chr3:1802200-1802240 – Results of computational approach to annotate the 15 novel ncRNA candidates. Afu-67 exists in two copies in A.fumigatus and both of these copies are located 550 nt upstream of the rRNA operon that also exists in two copies. None of the others could be annotated to a known gene. In contrast Afu-182, Afu-202, Afu-309 and Afu-336 are highly conserved among the Aspergillus species both in sequence and secondary structure over their complete length of around 220–340 nt. For the secondary structure graphs and conservation curves see Supplementary Data Figure 4 A–D. these candidates, appear differentially expressed under A. oryzae and A. niger (Table 4) over their entire length growth conditions tested, which might indicate their from 220 to 350 nt. Hence, consensus secondary structures involvement in regulatory processes. In particular, expres- could be predicted employing the JGI genome browser for sion of Afu-336, Afu-364 and Afu-448 appears to be A. niger (http://genome.jgi-psf.org; Supplementary Data down-regulated following growth on plates for 48 h, Figure 4A–D). One conserved candidate is located in the indicating developmental regulation. In several cases, vicinity of a known ncRNA gene: Afu-67 is present in two northern blot analysis revealed signals of larger sizes copies within the A. fumigatus genome and is located compared to sizes as determined by cDNA cloning. This 550-nt upstream of the rRNA operon. None of the might be explained by cDNA sequences representing remaining novel ncRNA candidates could be assigned to partial sequence fragments of full-length ncRNAs. In any known ncRNA gene or function. addition, for some cDNA clones several bands could be A large number of known ncRNAs function as anti- observed, the larger ones potentially reflecting precursor sense RNAs targeting mRNAs (67). Thus, it would be forms of mature ncRNA candidates. desirable to identify potential targets for novel ncRNAs in No obvious conserved sequence or structure motifs A. fumigatus. However, in the absence of any hints on the could be identified among these candidates. However, six location of anti-sense boxes, this is currently a difficult candidates turned out to be highly conserved in related task. As an exception, Afu-484 ncRNA is transcribed in Aspergillus species. In particular, Afu-182, Afu-202, anti-sense orientation to Exon 2 of the PIGC mRNA and Afu-309 and Afu-336 are conserved in A. nidulans, thus might regulate the translation or stability of the Nucleic Acids Research, 2008, Vol. 36, No. 8 2685 iron-dependence of metabolism within this cellular compartment. Interestingly, from cytoplasmic tRNAs, 16 were also represented as partial sequences in the cDNA library by several identical clones, corresponding either to the 5 -or 3 -halves of tRNAs (Figure 5A). The majority of tRNA 5 -halves contained the anti-codon sequence at their 3 -ends, whereas sequences of cDNAs encoding the tRNA 3 -halves started right after the anti-codon (Figure 5A). This strongly suggests an endonucleolytic cleavage of tRNAs within their anti-codon loop, at a position 3 adjacent to the anti-codon. For cytoplasmic Gln His tRNA and tRNA we confirmed these stable cleavage intermediates by northern blot analysis employing oligo- 0 0 nucleotides directed against the 5 - and the 3 -halves of the tRNAs (Figure 5A, and data not shown). Thereby, the estimated sizes of the northern blot signals from the 0 0 5 - and the 3 -halves of tRNAs are in agreement with the lengths of the corresponding cloned cDNAs. In addition, Gln Gly His for cytoplasmic tRNA , tRNA and tRNA we verified cleavage sites 3 adjacent to the anti-codon by Figure 4. Northern blot analysis of selected nuclear and mitochondrial encoded tRNAs. Total RNA was isolated from A. fumigatus mycelia primer extension (data not shown). grown under iron-depleted (AMM-Fe) or iron-repleted conditions. We next investigated whether cleavage of tRNAs was Loading controls (5.8 S rRNA, 5 S rRNA and U1 snRNA) as described developmentally regulated in A. fumigatus. To synchro- in Figure 3. nize conidiation (43), mycelia from 18-h liquid cultures were transferred to plates and RNA was isolated at different time points (Figure 5A). Indeed, northern blot mRNA. Interestingly, under conditions where A. fumiga- Gln analysis of tRNA revealed cleavage products of the tus undergoes conidiation (ACM plate cultures at 48 hr) expected sizes (see above) from 6 to 24-h after the transfer expression of Afu-484 decreases while expression of the from liquid to plate culturing. In comparison to full-length corresponding mRNA increases, consistent with a regula- tRNAs, tRNA-halves are expected to undergo rapid tion of stability of the PIGC mRNA by Afu-484 degradation by exonucleases. Thus, the high abundance (Supplementary Data, Figure 4E). of these tRNA cleavage products, as determined by northern blot analysis, is unexpected and therefore tRNA cleavage: a novel mechanism to regulate protein might even reflect a lower estimate of tRNA cleavage at synthesis? these time points, only. At the 48- and 72-h time points Gln (where conidia production is maximal) tRNA levels tRNAs corresponded to 23% of all cDNA clones from the were significantly decreased and in conidia largely expression library (Figure 1A). All nuclear encoded Gln 0 0 decreased. The absence of 5-or3 -halves of tRNA at Met Trp tRNAs except cytoplasmic tRNA , tRNA and these time points probably reflects an increase in Asp tRNA were identified. This suggests a reasonable exonuclease activity, thus rapidly degrading the unstable good coverage of the cDNA library for ncRNA species, tRNA intermediates during conidiogenesis (Figure 5A). all the more so, since tRNAs, because of their highly As a control for conidiation, the expression of the stable tertiary structure and their base modifications, are conidiation-specific transcription factor brlA was mea- generally refractory to reverse transcription and cDNA sured (32). Consistently, brlA transcripts were detected in cloning (44). plate cultures with a maximum at 48 to 72 h, but not in From mitochondrial tRNAs, only two different cDNA liquid cultures and in conidia (Figure 5A). Ser Lys clones were obtained encoding tRNA and tRNA . We then analysed whether tRNA cleavage during Both tRNA species are encoded by the mitochondrial conidiogenesis is only restricted to tRNAs or also involves genome of A. fumigatus (34). Interestingly, in contrast to other abundant ncRNA species. To that aim, we nuclear encoded tRNAs, expression of the two mitochon- investigated expression levels of various house keeping drial tRNA species was down-regulated by 2.1 and ncRNAs during A. fumigatus development (Figure 5B). 3.7-fold, respectively, in media lacking iron (Figure 4). Northern blot analysis and in-gel ethidium bromide Within mitochondria, several enzymes containing iron- staining of total RNA (24) revealed that all large and sulphur clusters are present; also, the final step of haeme small ribosomal RNAs (i.e. 26 S, 18 S, 5.8 S and 5 S biosynthesis, the incorporation of iron into protoporphy- rRNAs, respectively), as well as spliceosomal U1 snRNA rinogen IX, takes place (68). Hence, these cell organelles showed comparable abundance at all developmental are the primary consumers of cellular iron. It is tempting stages. In contrast, levels of all selected tRNAs, either to speculate that down-regulation of mitochondrial tRNA from the nuclear or mitochondrial genome, were strongly levels represents a mechanism for decreasing mitochon- reduced in conidia, compared to hyphae, as shown by drial activity during iron starvation due to the strong northern blot analysis (Figure 5B). 2686 Nucleic Acids Research, 2008, Vol. 36, No. 8 Gln 0 0 Figure 5. (A) Upper: alignment of cDNA sequences from the cDNA library, representing 5 and 3 -halves of cytoplasmic tRNA . The anti-codon is Gln 0 0 boxed in red. Northern blot analysis of cytoplasmic tRNA (bottom) employing oligonucleotide probes directed against 5 - and 3 -halves of Gln Gln 0 0 tRNA . Northern blot signals correspond to full-length tRNA (75 nt) or 5 or 3 cleavage products (36 nt or 39 nt), respectively. Lower: total RNAs were isolated from A. fumigatus conidia and from mycelia undergoing conidiogenesis (solid ACM, 6 h, 12 h, 18 h, 24 h, 48 h and 72 h), germination (liquid ACM 6 h) and vegetative growth (liquid ACM 12 h). Conidiogenesis is indicated by expression of the brlA gene. The proposed site of endonucleolytic cleavage within the anti-codon loop is indicated by a red triangle; (B) Comparison of expression levels of most abundant ncRNA species by in-gel ethidium bromide staining or by northern blot analysis from germination to hyphal growth; expression levels of the entire tRNA fraction is indicated by a red arrow; total RNA was isolated from A. fumigatus conidia, germinating conidia (3 h and 6 h) and hyphae (9–21 h), respectively. Levels of all tRNAs steadily increase during germina- to nutritional stress, like amino acid deprivation, by the tion (Figure 5A: right lanes 6 and 12 h time points; so-called stringent response, which primarily results in Figure 5B: 3-, 6- and 9-h time points). Under these inhibition of RNA synthesis (70). The effector of the 0 0 conditions, cleavage products are not detectable, consis- stringent control is guanosine 3 ,5 -bisdiphosphate tent with suppression of conidiogenesis in liquid culture. (ppGpp), synthesized by the ribosome-associated RelA Thereby, the 3-, 6- and 9-h time points resemble conidial protein. Upon amino acid deprivation, uncharged tRNAs swelling, conidial germ tube formation and hyphal bind to the ribosome, which triggers increased ppGpp growth, respectively (Figure 5B). Maximal tRNA levels synthesis by RelA, resulting in changes in gene expression are observed during hyphal growth from 9 to 15 h. The including inhibition of promoters for ribosomal and most significant lower abundance of tRNAs in conidia, tRNA operons and stimulation of promoters for many compared to other ncRNAs, can readily be detected by amino acid biosynthesis operons (71). As an alternative in-gel ethidium bromide staining of total RNA (indicated mechanism, in the single-cellular organism Tetrahymena, by a red arrow, Figure 5B). it has recently been reported that amino acid deprivation We currently envision two alternative models for a triggers endonucleolytic cleavage within several positions decrease of total tRNA levels during conidiogenesis: (i) a in the anti-codon loop of functional tRNAs (72). This decrease in the de novo synthesis of tRNAs or (i) an suggests that anti-codon loop cleavage reduces the increase in tRNA cleavage. At this point, we favour the accumulation of uncharged tRNAs as a part of a specific second model, which is consistent with the observed response induced by starvation (72). tRNA halves derived from cleavage within the anti-codon Inhibition of protein synthesis by a similar mechanism, loop of tRNAs (see above). What would be the function of i.e. tRNA cleavage, is also found for Escherichia coli cells tRNA degradation in fungi during conidiogenesis? Since infected by the phage T4. Here, it was demonstrated that filamentous fungi usually start their asexual life cycle as an E. coli-encoded anti-codon-specific endonuclease, Lys metabolically inactive asexual spores, the conidia (69), this termed ACNase, cleaves tRNA within its anti-codon requires stalling of protein synthesis (25). loop as a ‘suicide-response’ to T4 phage infection. This Several different mechanisms for stalling of protein lesion could deplete the infected cell of functional Lys synthesis have been identified. For example, bacteria react tRNA , inhibit translation of late T4 proteins and, Nucleic Acids Research, 2008, Vol. 36, No. 8 2687 consequently, contain the infection (73). Interestingly, the conidia is achieved by cleavage and subsequent degrada- T4 phage employs an RNA repair mechanism to offset the tion of tRNAs in A. fumigatus. To verify this model, damage, namely by religation of both tRNA halves future experiments have to address the identification of the tRNA cleavage activity in A. fumigatus. It is tempting to through polynucleotide kinase and RNA ligase (73). It should be noted that the tRNA cleavage mechanism, speculate whether similar mechanisms to regulate protein proposed for A. fumigatus, differs from the one described synthesis are also employed in resting states of other, Lys for E. coli: here the ACNase cleaves tRNA , only, at a multi-cellular, organisms, such as plants. position 5 to the wobble base of the anti-codon, while the proposed endonucleolytic cleavage in A. fumigatus occurs SUPPLEMENTARY DATA at all tRNAs investigated but 3 adjacent to the anti- codon. Hence, endonucleases involved in this process Supplementary Data are available at NAR Online. might differ significantly from each other between the two species, consistent with the lack of ACN homologues found in A. fumigatus employing a BlastP database search ACKNOWLEDGEMENTS (data not shown). This work was supported by the Fonds zur Fo¨ rderung der By employing this mechanism, resuming protein syn- wissenschaftlichen Forschung (FWF grant P171370) and thesis in A. fumigatus, would initially require de novo an Austrian genome research (Gen-Au grant D 110420- transcription of tRNAs, only, followed by rapid protein 011-013) to A.H. and a FWF grant (P18606) to H.H. We synthesis which would ensure a fast response to growth thank Norbert Polacek for helpful discussions and reading stimuli. Accordingly, the initiation of the fungal germina- of the manuscript. Funding to pay the Open Access tion process is reported to be associated with the onset of publication charges for this article was provided by Gen- protein synthesis (25). Au grant D 110420-011-013. Conflict of interest statement. None declared. 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Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis

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Abstract

Published online 16 March 2008 Nucleic Acids Research, 2008, Vol. 36, No. 8 2677–2689 doi:10.1093/nar/gkn123 Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis 1 1 2,3 2,3,4,5 Christoph Jo¨ chl , Mathieu Rederstorff , Jana Hertel , Peter F. Stadler , 2 6 6 1, Ivo L. Hofacker , Markus Schrettl , Hubertus Haas and Alexander Hu¨ ttenhofer * Innsbruck Biocenter, Division of Genomics and RNomics – Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Institute for Theoretical Chemistry, University of Vienna, Wa¨ hringerstr. 17, A-1090 Wien, Austria, Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, University of Leipzig, Hartelstraße 16-18, D-04107 Leipzig, Germany, Santa Fe Institute, Santa Fe, NM 87501, USA, Fraunhofer Institut fuer Zelltherapie und Immunologie,Deutscher Platz 5e, 04103 Leipzig, Germany and Innsbruck Biocenter, Division of Molecular Biology – Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria Received December 7, 2007; Revised February 4, 2008; Accepted March 4, 2008 ABSTRACT down-regulate protein synthesis in a filamentous fungus. Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organ- isms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus INTRODUCTION fumigatus. To that aim, we experimentally screened Cells from all organisms, studied to date, contain two for ncRNAs, expressed under various growth con- different kinds of RNA species, the protein-encoding ditions or during specific developmental stages, messenger RNAs (mRNAs) as well as non-protein-coding by generating a specialized cDNA library from RNAs (ncRNAs). In contrast to mRNAs, ncRNAs are size-selected small RNA species. Our screen not translated into proteins, but have important cellular revealed 30 novel ncRNA candidates from known functions, either on their own or in complex with proteins ncRNA classes such as small nuclear RNAs (1–6). Functions of ncRNAs range from RNA processing, (snRNAs) and C/D box-type small nucleolar RNAs modification, transcriptional regulation, mRNA stability (C/D box snoRNAs). Additionally, several candidates and translation up to protein secretion (2). Reported sizes of many known ncRNAs are generally well below sizes of for H/ACA box snoRNAs could be predicted by a mRNAs and range from 21–22-nt long microRNAs (7,8) bioinformatical screen. We also identified 15 candi- to about 500 nt [e.g. telomerase RNA (9)]. In addition, dates for ncRNAs, which could not be assigned to also very large ncRNAs, including the 17-kb long human any known ncRNA class. Some of these ncRNA Xist RNA (10,11) or the 108-kb long mouse Air RNA (12) species are developmentally regulated implying a have been observed. possible novel function in A. fumigatus develop- Recently, whole genome screens in eukaryal organisms ment. Surprisingly, in addition to full-length tRNAs, have revealed a large number of ncRNAs which have been we also identified 5’-or3’-halves of tRNAs, only, shown to regulate gene expression by novel mechanisms which are likely generated by tRNA cleavage within such as RNA interference, gene co-suppression, gene the anti-codon loop. We show that conidiation silencing, imprinting and DNA methylation (8,13–15). induces tRNA cleavage resulting in tRNA depletion Evidence for the involvement of ncRNAs exerting critical within conidia. Since conidia represent the rest- functions during vegetative growth, development or cell ing state of A. fumigatus we propose that conidial differentiation as well as in diseases, such as carcinogen- tRNA depletion might be a novel mechanism to esis, is becoming increasingly clear (16,17). *To whom correspondence should be addressed. Tel: +43 512 9003 70250; Fax: +43 512 9003 73100; Email: alexander.huettenhofer@i-med.ac.at Correspondence may also be addressed to Hubertus Haas. Tel: +43 512 9003 70205; Email: hubertus.haas@i-med.ac.at 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2678 Nucleic Acids Research, 2008, Vol. 36, No. 8 Several single-cellular eukaryal organisms have been according to Pontecorvo et al. (31) containing 1% (wt/vol) studied in the past, revealing a plethora of novel ncRNAs glucose as carbon source and 20 mM glutamine as a (18–20). A bioinformatical analysis of the fungal genomes nitrogen source. For liquid growth A. fumigatus was from seven different yeast species provided a significant cultured at 378C up to the indicated time point either in number of evolutionarily conserved, structured ncRNAs, AMM or in Aspergillus complete media (ACM) compris- suggesting their roles in post-transcriptional regulation ing 2% (wt/vol) glucose, 0.2% tryptone (wt/vol), 0.1% (21). In contrast, identification and functions of ncRNAs yeast extract (wt/vol) and 0.1% casamino acids (wt/vol). in filamentous fungi, such as Aspergillus species, have not Media contained 10mM FeSO and respectively for iron- been studied. depleted conditions, iron was omitted. For nitrogen Most filamentous fungi are saprophytes playing impor- starvation, 18-h AMM cultures were harvested and shifted tant roles in carbon and nitrogen recycling. Moreover, for another 6 h into AMM lacking glutamine. several members of this fungal group are well known for production of biotechnological important secondary Growth conditions for conidiation of A. fumigatus metabolites, as producers of toxins, or as facultative ATCC46645 pathogens for plants and animals. Infections with filamen- For synchronized asexual developmental A. fumigatus was tous fungi have emerged as an increasing risk for immuno- grown in liquid ACM for 18 h (32). Then, mycelia were suppressed patients. Aspergillus fumigatus accounts for harvested by filtering and transferred to solid ACM, were most of these infections, termed invasive aspergillosis, and conidiation is induced (32). Samples for RNA isolation can be regarded as the most common airborne fungal were collected after 6, 12, 24, 48 and 72 h of growth on pathogen. Specific diagnostics as well as therapeutic solid ACM. possibilities are limited (22–24). Hence, the mortality rate of invasive aspergillosis ranges between 30 and 90%, Generation of an A. fumigatus cDNA library depending on the immune status of the host (22,23). Its global ubiquity as well as the infectious cycle of this Aspergillus fumigatus was cultured under various condi- pathogen is perpetuated by prolific production of asexual tions to ensure expression of also growth-regulated spores (termed conidia) from specialized aeral hyphae ncRNAs. Total RNA was extracted from harvested (termed conidiophores). Conidial germination, e.g. in the mycelia of A. fumigatus by the TRI-zol method (Gibco human lung, following spore inhalation represents the BRL) (33). Subsequently, equal amounts of total RNAs initiating event of pulmonary disease. Three important were pooled and size-fractionated by denaturing 8% steps can be distinguished during spore germination: PAGE (7 M urea, 1 TBE buffer). RNAs in the size activation of the resting spore to appropriate environ- range between 15 and 500 nt were excised from the gel, mental conditions, isotropic growth that involves water passively eluted and ethanol-precipitated. RNAs were uptake and wall growth (termed swelling) and polarized poly(C)-tailed employing poly(A) polymerase from yeast growth that results in the formation of a germ tube from (USB). C-tailed RNAs were ligated to a 19-nt long 5 which the new mycelium originates (25). Conidia are linker by T4 RNA ligase, as described previously (29). dormant, metabolically inactive cells, which can be stored RNAs from the library were subsequently converted into for extended periods. The combined presence of air, water cDNAs by RT–PCR as described, employing complemen- and a carbon source induces germination with the first tary primers to 5 linkers and the poly(C) tail (29), and measurable activities being trehalose breakdown and cloned into pGEM-T vector (Promega). translation (26). Aspergillus fumigatus cells contain a haploid nuclear Dot-blot hybridization genome of 28.9 Mb in size, distributed into eight chromo- Aspergillus fumigatus library-derived cDNA clones were somes (27) and a circular mitochondrial genome exhibit- PCR-amplified using the primers M13 and M13 reverse. ing a size of 32 kb. Apart from ribosomal RNAs (rRNAs) Two micro litres of diluted (1:20) and denatured (918C, and transfer RNAs (tRNAs), no other ncRNAs have yet 2 min) PCR products were spotted onto a nylon mem- been annotated and characterized within the A. fumigatus brane (Hybond N , Amersham), cross linked employing genome (27). However, knowledge on the number and the STRATAGENE UV crosslinker (120 mJ/cm ) and functions of ncRNAs is vital for understanding cell pre-hybridized for 2 h in 1 M sodium phosphate buffer functions in A. fumigatus and could potentially open up (pH 6.2) with 7% SDS. Oligonucleotides, complementary new avenues for the development of novel anti-fungal to known and most abundant ncRNAs were [g- P]ATP drugs. Thus, for the experimental identification of novel end-labelled by T4 polynucleotide kinase. All six oligo- ncRNA species in A. fumigatus we generated a specialized nucleotide probes were added to the hybridization tube cDNA library comprising small ncRNA species sized from and hybridization was carried out at 528C in hybridization 20 to –500 nt (28,29). buffer (178 mM Na HPO , NaH PO , pH 6.2, 7% SDS) 2 4 2 4 for 12 h. Blots were washed twice: at room temperature in 2 SSC buffer, 0.1% SDS for 10 min and subsequently at MATERIAL AND METHODS hybridization temperature in 0.1 SSC, 0.1% SDS for Strain and growth conditions 10 min. Afterwards blots were rinsed in desalted water. Aspergillus fumigatus wild-type ATCC46645 (30) was Following stringency washes membranes were exposed to maintained on solid Aspergillus minimal media (AMM) Kodak MS-1 film from 30 min to 2 days. Nucleic Acids Research, 2008, Vol. 36, No. 8 2679 Sequence analysis of cDNA library (http://genome.jgi-psf.org/Aspni1/Aspni1.home.html) sequence conservation of snRNAs and novel ncRNA cDNA clones were sequenced using the M13 reverse candidates was analysed in the genomes of A. niger, primer and the BigDye terminator cycle sequencing A. oryzae, A. fumigatus and A. nidulans (Supplementary reaction kit (PE Applied Biosystems). Sequencing reac- Data, Figures 1 and 4). Since U3 snoRNA could not be tions were run on an ABI Prism 3100 (Perkin Elmer) found by means of a BLASTN search, a computationally capillary sequencer. Subsequently, sequences were ana- more expensive strategy was employed. The semi-local lysed with the LASERGENE sequence analysis program variant of Gotoh’s dynamic programming algorithm (40) package (DNASTAR). In this analysis, cDNA sequences was implemented in a memory-efficient scanning version were compared to each other using the Lasergene Seqman in the C programming language (parameters: match +3, II program package to identify identical sequences mismatch 1, gap opening 8 and gap extension 2). (DNASTAR). Following a BLASTN search against the GenBank database (NCBI, http://www.ncbi.nlm.nih.gov/ BLAST) was performed using standard parameters (i.e. RESULTS AND DISCUSSION match/mismatch score: 1,-2 and linear gap costs) which cDNA library construction and analysis were adjusted automatically for short input sequences. All RNA sequences, which were not annotated in the Aspergillus fumigatus was grown under seven different database and showed a perfect match within the culture conditions (see Materials and methods section). A. fumigatus genome (34), were treated as potential Liquid complete medium (ACM) is the optimal growth candidates for novel ncRNAs. condition resulting in maximal vegetative proliferation. In comparison, minimal medium demands expression of an Northern blot analysis increased number of anabolic pathways. Starvation for iron (AMM-Fe) or nitrogen (AMM-N) was found to Total RNA was either size-separated on 1.2% agarose– induce virulence traits (41,42). In contrast to liquid 2.2 M formaldehyde gels and blotted onto Hybond N cultures (vegetative growth), plate cultures induce con- membranes (Amersham) as described earlier (35), or size- idiation, i.e. the formation of infectious propagules (43). fractionated on denaturing polyacrylamide gels (PAGE). These different growth conditions are reported to induce For PAGE, 3–40mg of total RNA isolated from different different mRNA transcriptomes and thus, in addition, are growth conditions (see above) was denatured for 1 min at likely to also induce the expression of differentially 958C, separated on a 8% denaturing polyacrylamide gel expressed ncRNA species. Total RNA from each culture (7 M urea, 1 TBE buffer) and transferred onto a nylon condition was isolated separately and converted into membrane (Hybond N , Amersham) using the Bio-Rad cDNA as previously described (29). semi-dry blotting apparatus (Trans-blot SD; Bio-Rad). Subsequently, we screened about 7200 cDNA clones for After immobilizing of RNAs using the STRATAGENE identification of novel ncRNA species by dot-blot UV crosslinker (120 mJ/cm ), nylon membranes were pre- hybridization employing labelled oligonucleotides directed incubated for 2 h in 1 M sodium phosphate buffer (pH 6.2) against the most abundant known ncRNAs, as described with 7% SDS. Oligonucleotides from 18 to 35 nt in size, previously (44). This approach yielded 3120 cDNA complementary to potentially novel RNA species, were sequences, which were subjected to further bioinformatical end-labelled with [g- P]ATP and T4 polynucleotide analysis (see Materials and methods section). Thereby, kinase. Depending on the T of the respective oligonu- 58.2% of cDNA sequences corresponded to nuclear cleotides, hybridization was carried out from 42 to 588Cin or mitochondrial rRNA fragments. tRNAs or tRNA hybridization buffer (178 mM Na HPO , NaH PO ,pH 2 4 2 4 cleavage fragments (see below) represented about 23.0% 6.2, 7% SDS) for 12 h. Blots were washed twice, once at of cDNA clones (Figure 1A). About 4.9% of cDNA room temperature in 2 SSC buffer, 0.1% SDS for 10 min sequences corresponded to three different snRNAs and subsequently at the respective hybridization tempera- (U1, U5 and U6 snRNA), which had escaped annotation ture in 0.1 SSC, 0.1% SDS for 1–10 min. Membranes in the current release of the A. fumigatus genome (27). were exposed to Kodak MS-1 film from 15 min to 5 days. Candidates for small nucleolar RNA (snoRNA) made up 11.3% of all cDNA clones. Due to the lack of conserved Bioinformatical methods sequence or structure motifs the remaining 1.2% of cDNA To obtain secondary structure predictions and conserva- clones could not be assigned to any class of known tion of A. fumigatus ncRNAs, sequences were mapped to ncRNAs and thus might represent entirely novel ncRNAs the genomes of related species via BLAST (36) search in A. fumigatus (Figure 1A). –3 (E-value: 10 ). The homologous sequences were then Subsequently, we confirmed expression and sizes of aligned using a dynamic programming alignment algo- ncRNAs by northern blot analysis. To that aim, total rithm as implemented in CLUSTAL W (37) and out of the RNA isolated from different growth conditions or multiple sequence alignment the secondary structure developmental stages of A. fumigatus, was employed in was predicted using the folding routines from the Vienna northern blot analysis to investigate expression levels of RNA package (38,39). Genomes of Aspergillus oryzae, ncRNAs (Figures 2 and 3). In our final analysis, we only Aspergillus nidulans and Aspergillus niger were listed those novel ncRNAs for which unambiguous downloaded from the NCBI database. Employing a northern blot signals were obtained, with the notable BLASTN search in the JGI genome browser of A. niger exception of some snoRNA species (Table 1, see below), 2680 Nucleic Acids Research, 2008, Vol. 36, No. 8 Figure 1. (A) Distribution of 3120 cDNA clones from the A. fumigatus expression library encoding ncRNA candidates. The abundance of different ncRNA species is shown as percentage of total clones. (B) Numbers of ncRNA candidates derived from the A. fumigatus cDNA library are indicated in brackets. Figure 2. Northern blot analysis of A. fumigatus snRNAs and selected snoRNA candidates. Designation of clones is indicated on the left. Sizes of ncRNAs, as estimated by comparison with an internal RNA marker, are indicated on the right. Total RNA was isolated from A. fumigatus mycelia grown under seven different conditions and at different time points: AMM, minimal medium; ACM, complete medium; AMM-Fe, iron starvation; AMM-N, nitrogen starvation. Liquid cultures resemble vegetative growth and plate cultures induce conidiogenesis. In-gel ethidium bromide-stained 5.8 S rRNA and 5 S rRNA serve as loading controls. (A) U1-1/U1-2, U5 and U6 snRNAs, respectively. (B) C/D box snoRNAs with predicted targets. (C) C/D box snoRNAs without predicted targets. which were computationally confirmed by the presence of cerevisae homologues shows highly conserved regions in canonical sequence and structure motifs. the loops of the hairpins. Compared to the consensus secondary structure as reported by the Rfam database (45) the Aspergillus snRNA structures show the same stem– snRNA candidates loop distribution and arrangement (Supplementary Data Four different cDNA clones representing putative snRNA Figure 1A–D). candidates were annotated as U1-1, U1-2, U5 and U6 Except for nuclear encoded 26S, 18S, 5.8S rRNAs and snRNAs, respectively, by comparison to the RNA family tRNAs, U1-1 snRNA appeared as the most abundant database of alignments and Covariance Models (45). clone in the library (Table 1). Three identical truncated Sequences of U1-1 and U1-2 snRNAs differ by 1 nt, only cDNA clones of U6 snRNA, mapping to two different loci (A or T at position 128, respectively), and exhibit different within the A. fumigatus genome, were also identified in our genomic locations, implying that they represent two screen (Table 1). distinct isoforms of U1 snRNA (Table 1). A comparison Expression of A. fumigatus U1, U5 and U6 snRNAs of the predicted secondary structures of A. fumigatus could be confirmed by northern blot analysis (Figure 2A). snRNAs U1-1, U1-2, U5 and U6 snRNAs and their Sizes, as estimated by comparison to an internal RNA homologues with the corresponding Saccharomyces marker, were determined as 130 nt, 100 nt or 105 nt, Nucleic Acids Research, 2008, Vol. 36, No. 8 2681 Figure 3. Northern blot analysis of ncRNA candidates from unknown ncRNA classes. Designation of clones is indicated on the left. Sizes of ncRNAs, as estimated by comparison with an internal RNA marker, are indicated on the right. Growth conditions and loading controls (5.8 S rRNA and 5 S rRNA) as described in Figure 2. As an additional loading control, U1 snRNA was included in northern blot analysis. respectively (Figure 2A). The absence of U2 and U4 verified by northern blot analysis (Supplementary Data, snRNAs in our screen might be explained by RNA Figure 2A). It is noteworthy, however, that sizes of U2 and modifications or structural constraints, impeding reverse U4 snRNAs, as estimated by northern blot analysis, differ transcription into cDNAs. from their bioinformatical predicted sizes due to the fact 0 0 Therefore, we employed, as a bioinformatical approach, that precise 5 - and 3 termini cannot be obtained by a BLASTN search of all known U2 and U4 snRNAs as computational analysis. annotated in the Rfam database, including all yeast snRNA genes. Indeed, we were able to predict U2 and snoRNA candidates U4 homologues within the A. fumigatus genome Two classes of small nucleolar ncRNAs (snoRNAs) have (Supplementary Data Figure 1B and D). However, been detected in eukaryal as well as in archaeal species A. fumigatus U2 and U4 snRNAs appeared to be less (49–52): C/D box snoRNAs, which guide 2 -O-methyla- conserved on the sequence level than expected and could tion of ribosomal, spliceosomal and tRNAs (the latter in only be partially aligned. Manually extending the align- Archaea, only), and H/ACA snoRNAs which guide ment yielded a set of nearly perfect U2 and U4 snRNA pseudouridylation in these RNA species (53). In our sequences in A. fumigatus and related species (e.g. screen, we identified 27 candidates for C/D box snoRNAs A. niger). In addition, we were able to compute based on conserved sequence or structural motifs by the interaction structure of the U4–U6 complex (Supple- employing the SnoReport program (54). mentary Data Figure 1D) using the RNAalifold All C/D box snoRNAs from our screen contained bona program (47). fide sequence motifs of canonical snoRNAs, namely C, D , A comparison of the predicted consensus secondary C and D boxes, respectively (1,55). We noticed, however, structures to annotated secondary structures in the Rfam that all C/D box snoRNAs from A. fumigatus lacked the database and in (48) shows that these computational canonical terminal stem structure, in contrast to mamma- candidates match perfectly, although they show high lian C/D snoRNAs. This is in agreement with previous variance in their primary sequence (Supplementary Data, Figure 1A–C). This is consistently observed for the observations on archaeal C/D snoRNAs (56) or C/D evolution of ncRNAs, since these genes are mainly con- snoRNAs from the slime mold Dyctiostelium discoideum, served on the secondary structure level. Table 3 indicates which are also devoid of a terminal stem (57). levels of conservation within all identified snRNA candi- Surprisingly, the abundant U3 C/D snoRNA was dates in A. fumigatus. In addition to their bioinformatical missing among these candidates. A standard BLASTN identification, expression of U2 and U4 snRNAs was search also failed to identify U3 snoRNA candidates 2682 Nucleic Acids Research, 2008, Vol. 36, No. 8 Table 1. Candidates for snRNAs or snoRNAs Name Copies cDNA (nt) Northern Location Putative target Accession blot (nt) number snRNAs U1-1 157 132 130 Intergenic; Afu1g06980/Afu1g07000 AM921915 U1-2 67 132 130 Intergenic; Afu4g12490/Afu4g12500 AM921916 U5 19 99 100 Intergenic; Afu6g12670/Afu6g12680 AM921917 U6 3 50 105 Intergenic; Afu4g12500/Afu4g12520 AM921918 Intergenic; Afu2g10150/Afu2g10160 C/D box snoRNAs with predicted target Afu-34 18 84 n.d. Intergenic; Afu2g15970/Afu2g15980 Am1131 and Gm2506 in 26S AM921919 Afu-191 11 92 90 Intergenic; Afu1g10270/Afu1g10280 Gm75 in 5.8S and Am32 AM921920 in U2 snRNA Afu-190 8 107 110 Intergenic; Afu4g11320/Afu4g11330 Gm557 in 18S AM921921 Afu-198 8 130 n.d. Intergenic; Afu1g02700/Afu1g02680 Cm2856 and Um 2859 in 26S AM921922 Afu-294 7 85 80 Intergenic; Afu1g09750/Afu1g09760 Cm1851 in 26S; Am43 in 5.8S AM921923 Afu-264 4 103 n.d. Intergenic; Afu7g05290/Afu7g05300 Gm2770 and Gm2773 in 26S; AM921924 Afu-277 4 100 n.d. Intergenic; Afu1g09740/Afu1g09760 Um2706 in 26S; Am97 in AM921925 18S; Cm47 in 5.8S Afu-263 3 96 85 Intron1 - Exon2 -3 UTR (sense); Am815 and Gm906 in 26S AM921926 Afu-188 2 84 n.d. Intergenic; Afu4g11310/Afu4g11320 Am25 and Um26 in 18S AM921927 Afu-200 2 91 n.d. Intron 3 (sense); Afu1g12390 Am415 and Gm1422 in 18S AM921928 Afu-304 2 106 95 Intron 2 (sense); Afu1g09800 Cm2925 in 26S AM921929 Afu-380 2 87 n.d. Intergenic; Afu4g06780/Afu4g06770 Um2701 in 26S AM921930 Afu-513 2 129 n.d. Intergenic; Afu4g11310/Afu4g11320 Gm1338 and Gm3719 in 26S AM921931 Afu-328 1 75 75 Intron 2 (sense); Afu6g04570 Gm2792 in 26S AM921932 Afu-335 1 97 100 Intron 2 (sense); Afu1g04840 Cm2316 in 26S AM921933 Afu-438 1 83 n.d. Intergenic; Afu2g15980/Afu2g15970 Am2877 in 26S AM921934 Afu-455 1 92 n.d. Intergenic; Afu1g09760/Afu1g09740 Gm1122 in 18S AM921935 C/D box snoRNAs without predicted target (orphan snoRNAs) Afu-40 123 90 90 Intergenic; Afu4g11310/Afu4g11320 AM921936 Afu-514 74 105 n.d. Intergenic; Afu6g03830/Afu6g03840 AM921937 Afu-515 40 89 n.d. Intergenic; Afu4g11310/Afu4g11320 AM921938 Afu-199 26 79 80 Intron 1 (sense); Afu7g02320 AM921939 Afu-298 3 93 90 Intergenic; Afu1g05080/Afu1g05100 AM921940 Afu-300 3 88 90 Intergenic; Afu4g11310/Afu4g11320 AM921941 Afu-215 2 24 85 Intergenic; Afu5g12870/Afu5g12880 AM921942 Afu-511 2 164 160 Intergenic; Afu1g03400/Afu1g03410 AM921943 Afu-210 1 24 75 Intron 2 (sense); Afu2g03610 AM921944 Afu-265 1 23 n.d. Intergenic; Afu3g02340/Afu3g02370 AM921945 Copies: number of independent cDNA clones obtained from each ncRNA candidate; cDNA (nt): length of cDNA assessed by sequencing; northern blot (nt): length of a ncRNA candidate estimated by northern blot analysis (n.d., expression was not determined); location: accession numbers of genes flanking the ncRNA (intergenic) or accession number of the gene, which the ncRNA is derived from (sense or antisense to exon or intron); putative target: refers to the predicted modified nucleotides within rRNAs; accession number: accession number of the ncRNA sequence in DDBJ/ EMBL/GeneBank databases. within the A. fumigatus genome. Thus, we applied a Afu-263, extends beyond a predicted intron–exon border computationally more sophisticated method, employing within the 3 -untranslated region (UTR; Figure 1B) of a the semi-local variant of the Gotoh dynamic programming hypothetical protein, encoded by the Afu3g14080 gene. algorithm (parameters: match +3, mismatch –1, gap However, this might be due to a wrong annotation of opening –8 and gap extension –2; see Materials and Afu3g14080 as a protein-coding gene, consistent with its methods section). By this method, we were able to also unusually small transcript length of 96 nt. Comparison of identify a U3 snoRNA candidate and verify its expression Afu-263 with the Rfam database reveals homology to by northern blot analysis (Supplementary Data, Figure 2B S. cerevisiae snR60. In addition, Afu-263 is also conserved and C). in the genome of the related fungus A.oryzae. Most C/D snoRNAs identified in our screen, with some The remaining 20 candidates map to intergenic regions. exceptions (Table 1) are within the size range of a canonical Twelve snoRNA candidates from this group are present as C/D snoRNAs (i.e. from 80 to 100 nt; Table 1). Identifica- singletons, whereas the remaining eight candidates are tion of some larger C/D box snoRNAs in our library is in distributed in two clusters, comprised of three and five agreement with previous findings in Oryza sativa (58) or snoRNA genes, respectively. A similar clustered gene Trypanosoma brucei (59) (Table 1). From the 27 C/D organization has been previously observed in S. cerevisiae snoRNA candidates, six are intron-derived (Table 1, (60–63). The first snoRNA cluster is located on chromo- Figure 1B) as observed for most mammalian and plant some 1 between protein-coding genes Afu1g09740 and snoRNAs (53). One C/D box snoRNA candidate, Afu1g09760 and comprises the C/D box snoRNAs Nucleic Acids Research, 2008, Vol. 36, No. 8 2683 Table 2. Candidates for entirely novel ncRNAs Name Copies cDNA (nt) Northern blot (nt) Location Accession number Afu-182 17 219 300 Intergenic; Afu4g07680/Afu4g07690 AM921946 Afu-202 5 266 270 Intergenic; Afu1g10420/Afu1g10430 AM921947 Afu-67 2 21 600/190 Intergenic; Afu4g01630/Afu4g02610 AM921948 Afu-254 2 163 90/420 Intergenic; Afu7g04110/Afu7g04120 AM921949 Afu-203 1 111 110 Intron 1 (sense); Afu3g14240 AM921950 Afu-222 1 22 535/250 Intergenic; Afu4g12410/Afu4g12420 AM921951 Afu-262 1 25 225 Intergenic; Afu1g10370/Afu1g10380 AM921952 Afu-309 1 318 320 Intergenic; Afu4g10430/Afu4g10420 AM921953 Afu-318 1 24 70 Intergenic; Afu7g01590/Afu7g01600 AM921954 Afu-322 1 46 75/35 Intergenic; Afu2g13250/Afu2g13240 AM921955 Afu-336 1 28 130 Intergenic; Afu1g09740/Afu1g09760 AM921956 Afu-364 1 18 100 Intergenic; Afu1g13820/Afu1g13830; AM921957 Intergenic; Afu3g03090/Afu3g03130; Exon4-Intron4; Afu3g02730; Intergenic; Afu6g11780/Afu6g11790 Afu-448 1 29 315 Intergenic; Afu1g11550/Afu1g11540 AM921958 Afu-465 1 17 270 Intron 2 (sense); Afu4g08930 AM921959 Afu-484 1 41 600 Exon2 (antisense); Afu3g07170 AM921960 Copies: number of independent cDNA clones, obtained from each ncRNA candidate; cDNA (nt): length of cDNA assessed by sequencing; northern blot (nt): length of a ncRNA candidate estimated by northern blot analysis; location: accession numbers of genes flanking the ncRNA (intergenic) or accession number of the gene, which the ncRNA is derived from (sense or antisense to exon or intron); accession number: accession number of the ncRNA sequence in the DDBJ/EMBL/GeneBank databases. Afu-455, Afu-277 and Afu-294. The second cluster maps receptor 2C mRNA, (65). Whether some of these orphan to chromosome 4 between the protein-coding genes snoRNAs from A. fumigatus might fulfil similar functions Afu4g11310 and Afu4g11320 and contains five C/D in mRNA targeting needs to be determined. Expression analysis of snoRNAs by northern blot box snoRNAs, Afu-40, Afu-188, Afu-300, Afu-513 and analysis revealed that most snoRNAs are equally Afu-515. Database searches with genomes from the expressed under most growth conditions with some related moulds A. nidulans and A. oryzae revealed their exceptions, (Figure 2B and C). Down-regulation during conservation in both filamentous fungi. SnoRNA clusters iron starvation, as observed for Afu-328, might implicate have been detected in a multitude of different eukaryal iron-related functions; iron starvation, for example, down- organisms, including S. cerevisiae, which may suggest an regulates transcription of genes encoding iron-containing ancient origin of this gene arrangement (62,63). proteins or iron-consuming pathways in A. nidulans (66). By employing the Snoscan Server 1.0 program (64) we Likewise, down-regulation of expression during conidio- identified putative targets for 17 out of 27 C/D box genesis, as observed for Afu-335, Afu-199 or Afu-511, snoRNAs (Table 1 and Figure 1B). As a probabilistic suggests their developmental regulation. search model for filamentous fungi is currently not In contrast to C/D box snoRNAs, we were so far unable available, the search model for target evaluation was to experimentally identify any representatives for adjusted to S. cerevisiae. As potential target sequences we H/ACA box snoRNAs. Therefore, we applied the considered the previously identified canonical targets, i.e. snoReport program (54) to chromosome 1 of the 26 S, 18 S, 5.8 S rRNAs and all snRNAs [identified by our A. fumigatus genome and were indeed able to bioinfor- screen (i.e. U1, U5 or U6 snRNA, respectively)]. Most of matically identify candidates for H/ACA snoRNAs. The the C/D box snoRNAs were predicted to guide methyla- majority of those candidates show a canonical H/ACA tion of 26 S rRNA, and, to a lesser extent, also 18 S and secondary structure and both of the sequence motifs H 5.8 S rRNAs. Several snoRNA candidates are predicted and ACA (for examples see Supplementary Data, to guide two different rRNA modifications, while Figure 3A–E). C/D box snoRNA Afu-277 is predicted to guide methyla- tion of three RNAs, i.e. 26 S rRNA, 18 S rRNAs and 5.8 S Candidates for entirely novel ncRNAs rRNA, respectively (Table 1). No C/D box snoRNAs targeting U1, U5 or U6 snRNAs were found in our screen. Due to the lack of conserved sequence motifs 1.2% of For the remaining 10 C/D box snoRNA candidates, no cDNA clones, amounting to 15 different cDNA sequences, rRNA or snRNA targets could be identified (Table 1 and could not be assigned to any class of known ncRNAs and Figure 1B). Hence, these were termed ‘orphan snoRNAs’ thus might encode entirely novel ncRNAs in A. fumigatus. in agreement with earlier findings of similar snoRNAs in Northern blot analysis verified expression of all 15 other species including mouse (1,44). Some orphan ncRNA candidates (Figure 3) and the genomic location snoRNAs were represented by numerous cDNA clones could be determined for all 15 candidates (Table 2). (Table 1), indicating their high abundance in A. fumigatus. Expression of ncRNA candidates was verified by Interestingly, MBII-52 an abundant orphan snoRNA northern blot analysis, pointing towards a significant from mouse brain, was proposed to target the serotonin abundance within A. fumigatus. Interestingly, many of 2684 Nucleic Acids Research, 2008, Vol. 36, No. 8 Table 3. Conservation level of snRNA candidates in Aspergillus species snRNAs Location in A.fumigatus Conserved in related species (% sequence identity) U1-1 chr1:1991385-1991564 A.nidulans (78), A.oryzae (94), A.niger (90) U1-2 chr4:3279813-3279991 A.nidulans (88), A.oryzae (91), A.niger (93) U2-1 chr3:3242037-3242312 A.nidulans (76), A.oryzae (85), A.niger (75) U2-2 chr1:3223904-3224180 A.nidulans (88), A.oryzae (84), A.niger (74) U4 chr1:1274827-1275331 A.nidulans (49), A.oryzae (53), A.niger (53) U5 chr6:3202420-3202638 A.nidulans (44), A.oryzae (60), A.niger (64) U6 chr1:1651441-1651760 A.nidulans (42), A.oryzae (39), A.niger (43) chr2:2598365-2598684 A.nidulans (50), A.oryzae (54), A.niger (52) chr4:3281001-3281320 A.nidulans (42), A.oryzae (49), A.niger (67) This table shows the snRNA candidates, their exact location in the A.fumigatus genome and the sequence identity in percent to the homologous candidates in related species. Although the sequence identity for U4 and U5 is relatively low, all sequences are able to fold into an appropriate secondary structure. For the consensus structures and conservation curves see Supplementary Data Figure 1 A–D. Table 4. Conservation, annotation and secondary structure prediction of novel ncRNA candidates Name Location in A. fumigatus Conservation Structure conservation Annotation Afu-182 chr4:1997138-1996922 A.niger, A.oryzae, A.nidulans + Afu-202 chr1:2714678-2714414 A.niger, A.oryzae, A.nidulans + Afu-67 chr4:444973-444954 –  550 nt upstream of rRNA operon chr4:706179-706160 – Afu-254 chr7:927348-927204 – Afu-203 chr3:3785105-3785214 – Afu-222 chr4:3252985-3252964 – Afu-262 chr1:2675372-2675395 – Afu-309 chr4:2732230-2732547 A.niger, A.oryzae + Afu-318 chr7:415029-415006 – Afu-322 chr2:3404938-3404893 – Afu-336 chr1:2523503-2523530 A.niger, A.oryzae, A.nidulans + Afu-364 chr1:3693906-3693889 A.niger + chr3:710476-710493 A.niger + chr3:836808-836825 A.niger + chr6:2935513 2935530 A.niger + Afu-448 chr1:3048137-3048109 A.niger, A.oryzae Afu-465 chr4:2316203-2316188 – Afu-484 chr3:1802200-1802240 – Results of computational approach to annotate the 15 novel ncRNA candidates. Afu-67 exists in two copies in A.fumigatus and both of these copies are located 550 nt upstream of the rRNA operon that also exists in two copies. None of the others could be annotated to a known gene. In contrast Afu-182, Afu-202, Afu-309 and Afu-336 are highly conserved among the Aspergillus species both in sequence and secondary structure over their complete length of around 220–340 nt. For the secondary structure graphs and conservation curves see Supplementary Data Figure 4 A–D. these candidates, appear differentially expressed under A. oryzae and A. niger (Table 4) over their entire length growth conditions tested, which might indicate their from 220 to 350 nt. Hence, consensus secondary structures involvement in regulatory processes. In particular, expres- could be predicted employing the JGI genome browser for sion of Afu-336, Afu-364 and Afu-448 appears to be A. niger (http://genome.jgi-psf.org; Supplementary Data down-regulated following growth on plates for 48 h, Figure 4A–D). One conserved candidate is located in the indicating developmental regulation. In several cases, vicinity of a known ncRNA gene: Afu-67 is present in two northern blot analysis revealed signals of larger sizes copies within the A. fumigatus genome and is located compared to sizes as determined by cDNA cloning. This 550-nt upstream of the rRNA operon. None of the might be explained by cDNA sequences representing remaining novel ncRNA candidates could be assigned to partial sequence fragments of full-length ncRNAs. In any known ncRNA gene or function. addition, for some cDNA clones several bands could be A large number of known ncRNAs function as anti- observed, the larger ones potentially reflecting precursor sense RNAs targeting mRNAs (67). Thus, it would be forms of mature ncRNA candidates. desirable to identify potential targets for novel ncRNAs in No obvious conserved sequence or structure motifs A. fumigatus. However, in the absence of any hints on the could be identified among these candidates. However, six location of anti-sense boxes, this is currently a difficult candidates turned out to be highly conserved in related task. As an exception, Afu-484 ncRNA is transcribed in Aspergillus species. In particular, Afu-182, Afu-202, anti-sense orientation to Exon 2 of the PIGC mRNA and Afu-309 and Afu-336 are conserved in A. nidulans, thus might regulate the translation or stability of the Nucleic Acids Research, 2008, Vol. 36, No. 8 2685 iron-dependence of metabolism within this cellular compartment. Interestingly, from cytoplasmic tRNAs, 16 were also represented as partial sequences in the cDNA library by several identical clones, corresponding either to the 5 -or 3 -halves of tRNAs (Figure 5A). The majority of tRNA 5 -halves contained the anti-codon sequence at their 3 -ends, whereas sequences of cDNAs encoding the tRNA 3 -halves started right after the anti-codon (Figure 5A). This strongly suggests an endonucleolytic cleavage of tRNAs within their anti-codon loop, at a position 3 adjacent to the anti-codon. For cytoplasmic Gln His tRNA and tRNA we confirmed these stable cleavage intermediates by northern blot analysis employing oligo- 0 0 nucleotides directed against the 5 - and the 3 -halves of the tRNAs (Figure 5A, and data not shown). Thereby, the estimated sizes of the northern blot signals from the 0 0 5 - and the 3 -halves of tRNAs are in agreement with the lengths of the corresponding cloned cDNAs. In addition, Gln Gly His for cytoplasmic tRNA , tRNA and tRNA we verified cleavage sites 3 adjacent to the anti-codon by Figure 4. Northern blot analysis of selected nuclear and mitochondrial encoded tRNAs. Total RNA was isolated from A. fumigatus mycelia primer extension (data not shown). grown under iron-depleted (AMM-Fe) or iron-repleted conditions. We next investigated whether cleavage of tRNAs was Loading controls (5.8 S rRNA, 5 S rRNA and U1 snRNA) as described developmentally regulated in A. fumigatus. To synchro- in Figure 3. nize conidiation (43), mycelia from 18-h liquid cultures were transferred to plates and RNA was isolated at different time points (Figure 5A). Indeed, northern blot mRNA. Interestingly, under conditions where A. fumiga- Gln analysis of tRNA revealed cleavage products of the tus undergoes conidiation (ACM plate cultures at 48 hr) expected sizes (see above) from 6 to 24-h after the transfer expression of Afu-484 decreases while expression of the from liquid to plate culturing. In comparison to full-length corresponding mRNA increases, consistent with a regula- tRNAs, tRNA-halves are expected to undergo rapid tion of stability of the PIGC mRNA by Afu-484 degradation by exonucleases. Thus, the high abundance (Supplementary Data, Figure 4E). of these tRNA cleavage products, as determined by northern blot analysis, is unexpected and therefore tRNA cleavage: a novel mechanism to regulate protein might even reflect a lower estimate of tRNA cleavage at synthesis? these time points, only. At the 48- and 72-h time points Gln (where conidia production is maximal) tRNA levels tRNAs corresponded to 23% of all cDNA clones from the were significantly decreased and in conidia largely expression library (Figure 1A). All nuclear encoded Gln 0 0 decreased. The absence of 5-or3 -halves of tRNA at Met Trp tRNAs except cytoplasmic tRNA , tRNA and these time points probably reflects an increase in Asp tRNA were identified. This suggests a reasonable exonuclease activity, thus rapidly degrading the unstable good coverage of the cDNA library for ncRNA species, tRNA intermediates during conidiogenesis (Figure 5A). all the more so, since tRNAs, because of their highly As a control for conidiation, the expression of the stable tertiary structure and their base modifications, are conidiation-specific transcription factor brlA was mea- generally refractory to reverse transcription and cDNA sured (32). Consistently, brlA transcripts were detected in cloning (44). plate cultures with a maximum at 48 to 72 h, but not in From mitochondrial tRNAs, only two different cDNA liquid cultures and in conidia (Figure 5A). Ser Lys clones were obtained encoding tRNA and tRNA . We then analysed whether tRNA cleavage during Both tRNA species are encoded by the mitochondrial conidiogenesis is only restricted to tRNAs or also involves genome of A. fumigatus (34). Interestingly, in contrast to other abundant ncRNA species. To that aim, we nuclear encoded tRNAs, expression of the two mitochon- investigated expression levels of various house keeping drial tRNA species was down-regulated by 2.1 and ncRNAs during A. fumigatus development (Figure 5B). 3.7-fold, respectively, in media lacking iron (Figure 4). Northern blot analysis and in-gel ethidium bromide Within mitochondria, several enzymes containing iron- staining of total RNA (24) revealed that all large and sulphur clusters are present; also, the final step of haeme small ribosomal RNAs (i.e. 26 S, 18 S, 5.8 S and 5 S biosynthesis, the incorporation of iron into protoporphy- rRNAs, respectively), as well as spliceosomal U1 snRNA rinogen IX, takes place (68). Hence, these cell organelles showed comparable abundance at all developmental are the primary consumers of cellular iron. It is tempting stages. In contrast, levels of all selected tRNAs, either to speculate that down-regulation of mitochondrial tRNA from the nuclear or mitochondrial genome, were strongly levels represents a mechanism for decreasing mitochon- reduced in conidia, compared to hyphae, as shown by drial activity during iron starvation due to the strong northern blot analysis (Figure 5B). 2686 Nucleic Acids Research, 2008, Vol. 36, No. 8 Gln 0 0 Figure 5. (A) Upper: alignment of cDNA sequences from the cDNA library, representing 5 and 3 -halves of cytoplasmic tRNA . The anti-codon is Gln 0 0 boxed in red. Northern blot analysis of cytoplasmic tRNA (bottom) employing oligonucleotide probes directed against 5 - and 3 -halves of Gln Gln 0 0 tRNA . Northern blot signals correspond to full-length tRNA (75 nt) or 5 or 3 cleavage products (36 nt or 39 nt), respectively. Lower: total RNAs were isolated from A. fumigatus conidia and from mycelia undergoing conidiogenesis (solid ACM, 6 h, 12 h, 18 h, 24 h, 48 h and 72 h), germination (liquid ACM 6 h) and vegetative growth (liquid ACM 12 h). Conidiogenesis is indicated by expression of the brlA gene. The proposed site of endonucleolytic cleavage within the anti-codon loop is indicated by a red triangle; (B) Comparison of expression levels of most abundant ncRNA species by in-gel ethidium bromide staining or by northern blot analysis from germination to hyphal growth; expression levels of the entire tRNA fraction is indicated by a red arrow; total RNA was isolated from A. fumigatus conidia, germinating conidia (3 h and 6 h) and hyphae (9–21 h), respectively. Levels of all tRNAs steadily increase during germina- to nutritional stress, like amino acid deprivation, by the tion (Figure 5A: right lanes 6 and 12 h time points; so-called stringent response, which primarily results in Figure 5B: 3-, 6- and 9-h time points). Under these inhibition of RNA synthesis (70). The effector of the 0 0 conditions, cleavage products are not detectable, consis- stringent control is guanosine 3 ,5 -bisdiphosphate tent with suppression of conidiogenesis in liquid culture. (ppGpp), synthesized by the ribosome-associated RelA Thereby, the 3-, 6- and 9-h time points resemble conidial protein. Upon amino acid deprivation, uncharged tRNAs swelling, conidial germ tube formation and hyphal bind to the ribosome, which triggers increased ppGpp growth, respectively (Figure 5B). Maximal tRNA levels synthesis by RelA, resulting in changes in gene expression are observed during hyphal growth from 9 to 15 h. The including inhibition of promoters for ribosomal and most significant lower abundance of tRNAs in conidia, tRNA operons and stimulation of promoters for many compared to other ncRNAs, can readily be detected by amino acid biosynthesis operons (71). As an alternative in-gel ethidium bromide staining of total RNA (indicated mechanism, in the single-cellular organism Tetrahymena, by a red arrow, Figure 5B). it has recently been reported that amino acid deprivation We currently envision two alternative models for a triggers endonucleolytic cleavage within several positions decrease of total tRNA levels during conidiogenesis: (i) a in the anti-codon loop of functional tRNAs (72). This decrease in the de novo synthesis of tRNAs or (i) an suggests that anti-codon loop cleavage reduces the increase in tRNA cleavage. At this point, we favour the accumulation of uncharged tRNAs as a part of a specific second model, which is consistent with the observed response induced by starvation (72). tRNA halves derived from cleavage within the anti-codon Inhibition of protein synthesis by a similar mechanism, loop of tRNAs (see above). What would be the function of i.e. tRNA cleavage, is also found for Escherichia coli cells tRNA degradation in fungi during conidiogenesis? Since infected by the phage T4. Here, it was demonstrated that filamentous fungi usually start their asexual life cycle as an E. coli-encoded anti-codon-specific endonuclease, Lys metabolically inactive asexual spores, the conidia (69), this termed ACNase, cleaves tRNA within its anti-codon requires stalling of protein synthesis (25). loop as a ‘suicide-response’ to T4 phage infection. This Several different mechanisms for stalling of protein lesion could deplete the infected cell of functional Lys synthesis have been identified. For example, bacteria react tRNA , inhibit translation of late T4 proteins and, Nucleic Acids Research, 2008, Vol. 36, No. 8 2687 consequently, contain the infection (73). Interestingly, the conidia is achieved by cleavage and subsequent degrada- T4 phage employs an RNA repair mechanism to offset the tion of tRNAs in A. fumigatus. To verify this model, damage, namely by religation of both tRNA halves future experiments have to address the identification of the tRNA cleavage activity in A. fumigatus. It is tempting to through polynucleotide kinase and RNA ligase (73). It should be noted that the tRNA cleavage mechanism, speculate whether similar mechanisms to regulate protein proposed for A. fumigatus, differs from the one described synthesis are also employed in resting states of other, Lys for E. coli: here the ACNase cleaves tRNA , only, at a multi-cellular, organisms, such as plants. position 5 to the wobble base of the anti-codon, while the proposed endonucleolytic cleavage in A. fumigatus occurs SUPPLEMENTARY DATA at all tRNAs investigated but 3 adjacent to the anti- codon. Hence, endonucleases involved in this process Supplementary Data are available at NAR Online. might differ significantly from each other between the two species, consistent with the lack of ACN homologues found in A. fumigatus employing a BlastP database search ACKNOWLEDGEMENTS (data not shown). This work was supported by the Fonds zur Fo¨ rderung der By employing this mechanism, resuming protein syn- wissenschaftlichen Forschung (FWF grant P171370) and thesis in A. fumigatus, would initially require de novo an Austrian genome research (Gen-Au grant D 110420- transcription of tRNAs, only, followed by rapid protein 011-013) to A.H. and a FWF grant (P18606) to H.H. We synthesis which would ensure a fast response to growth thank Norbert Polacek for helpful discussions and reading stimuli. Accordingly, the initiation of the fungal germina- of the manuscript. Funding to pay the Open Access tion process is reported to be associated with the onset of publication charges for this article was provided by Gen- protein synthesis (25). Au grant D 110420-011-013. Conflict of interest statement. None declared. 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Published: May 16, 2008

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