TY - JOUR
AU - Sivasubbu, Sridhar
AB - Abstract The utility of model organisms to understand the function of a novel transcript/genes has allowed us to delineate their molecular mechanisms in maintaining cellular homeostasis. Organisms such as zebrafish have contributed a lot in the field of developmental and disease biology. Attributable to advancement and deep transcriptomics, many new transcript isoforms and non-coding RNAs such as long noncoding RNA (lncRNA) and circular RNAs (circRNAs) have been identified and cataloged in multiple databases and many more are yet to be identified. Various methods and tools have been utilized to identify lncRNAs/circRNAs in zebrafish using deep sequencing of transcriptomes as templates. Functional analysis of a few candidates such as tie1-AS, ECAL1 and CDR1as in zebrafish provides a prospective outline to approach other known or novel lncRNA/circRNA. New genetic alteration tools like TALENS and CRISPRs have helped in probing for the molecular function of lncRNA/circRNA in zebrafish. Further latest improvements in experimental and computational techniques offer the identification of lncRNA/circRNA counterparts in humans and zebrafish thereby allowing easy modeling and analysis of function at cellular level. zebrafish, lncRNA, circRNA, function, conservation Introduction ‘Nature has been generous to science and has provided us with many model systems’—the famous quote by Sydney Brenner highlights the importance of model organisms in providing important biological insights. For decades, many model organisms have been employed to understand the developmental processes and disease mechanisms of humans due to their shared evolutionary ancestry and homology. For the past 50 years, a tiny aquatic animal called zebrafish has increasingly been used in the developmental biology field due to its numerous unique features. Among other hallmark features, higher fecundity rate, the optical transparency of embryos and the resemblance with human physiological systems have allowed real-time monitoring of developmental and pathophysiological phenomenon’s. The zebrafish genome also shares high similarity with the human genome, sharing about 71% of gene orthologs [1]. Chemical mutagenesis screens in zebrafish have allowed functional discoveries and characterization of multiple genes and novel pathways involved in embryonic development [2–4]. Additionally, various high-throughput forward and reverse genetic screens including the use of genome editing tools have enabled the creation of a large number of human disease models for gaining mechanistic understanding [5–14]. In the era of next-generation sequencing, the high-throughput data-led discoveries of novel transcripts and their variants have been made possible with the improvements in throughput of nucleic acid sequencing, adoption of sequencing to understand key genomic processes as well as computational approaches to analyze them. A deep understanding of these newly identified transcripts is expected to provide us with novel insights into biology. A large number of transcriptomic profiles using deep-sequencing have identified multiple new protein-coding transcript isoforms as well as non-coding RNAs in zebrafish [15]. While protein coding transcripts remain the mainstay for understanding core biological processes, noncoding RNAs have gained popularity in recent years due to their implication in the functional regulation of various developmental and disease pathways. Noncoding-RNAs (ncRNAs) are notable for their diversity in occurrences and functions. NcRNAs can also be further sub-categorized based on their sizes and structures, such as long ncRNA (lncRNA), micro-RNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar-RNAs (snoRNAs), small nuclear-RNA (snRNA), circular-RNA (circRNA) and transfer-RNA (tRNA). In this review, we will be focusing on understanding the importance of the most dominating class of ncRNA i.e. lncRNA and circRNA in the development of zebrafish using various in silico and molecular approaches. The current definition of lncRNA and circRNA Long non-coding RNAs (lncRNAs) constitute a diverse class of RNA isoforms that are >200 nucleotides in length with no obvious protein coding potential, having very low cellular expression levels, however displaying high cell/tissue specificity [16–18]. These transcripts are thought to be functionally driven to regulate the host genome either by direct or indirect biomolecular interactions with their RNA, DNA, or protein counterpart [19]. Large-scale functional annotation studies, such as ENCODE [20] and FANTOM [21, 22], have cataloged a large set of lncRNAs. A number of candidate lncRNAs have also been characterized as a regulator for various genomic interactions involving chromatin remodeling, looping, and scaffolding of chromatin complexes [23]. Increasingly many of lncRNAs or their genomic loci are being implicated with diseases (such as cancer, cardiovascular, inflammatory, etc.) and developmental processes [24]. The discovery of lncRNAs have been made possible through high-throughput transcriptome studies that have enabled them to be cataloged. These candidate lncRNA transcripts upon characterization were suggested to be involved in the development and tissue biogenesis of zebrafish [15, 25–27]. Recent evidence suggests that there are around 21 128 lncRNA transcripts in zebrafish [28]. However, only a handful (<0.1%) of these transcripts have been functionally characterized to understand their physiological relevance. With the improvement of deep RNA-sequencing techniques and the advancement of new bioinformatics tools, the prospect of identifying many more novel and functional lncRNAs has been high. circRNAs on the other hand are transcript isoforms that are formed due to non-canonical splicing wherein covalent bonding of back spliced 5′ donor end and 3′ acceptor junctions of a transcript result in circularization of the RNA [29]. These circular transcript isoforms are thought to be stable due to their closed structure, which constrains their exonucleases-mediated degradation. Although, being a byproduct of noncanonical-splicing, the circRNAs comprise 20% of the total transcriptome and the majority of them fall into the non-protein-coding component [30]. However, the capability of circRNA to encode for proteins is questionable, as few candidates that possess the internal-ribosome entry site (IRES) and may produce small peptides or proteins [31]. In recent years, deep sequencing of transcriptomes has identified large sets of circRNAs in different developmental stages and tissues biogenesis of zebrafish [32–34]. The functional characterization of these circRNAs in zebrafish can provide many insights into their conserved role in development and disease. LncRNA and circRNA databases for zebrafish Databases are a rich source of information for genomic and functional annotation of various transcripts and their regulatory interactions. Presently there are eight lncRNA- and three circRNA-specific databases for zebrafish (Table 1). Put together, these databases have reported more than thousands of lncRNAs identified in zebrafish tissues as well as different developmental stages. NONCODE is one of the early databases which has cataloged about 4852 lncRNAs transcript isoforms belonging to 3503 lncRNA genes in zebrafish [35]. Similarly, deepBase v2.0 includes lncRNAs annotations from eight developmental stages of zebrafish (2–4 cells, 1000 cells, dome, shield, bud, 28 h post-fertilization (hpf), 48 hpf and 120 hpf) contributing to 851 lncRNAs [36]. Another database developed by our group zflncRNApedia catalogues 2267 lncRNAs from three major zebrafish lncRNA studies including Ulitsky et al., Pauli et al., and Kausik et al. as well as some manually curated sources [25–27, 37]. A recent database called ZFLNC, extensively compiled lncRNA data from NCBI, Ensembl, NONCODE, zflncRNApedia, literature, and also performed analysis of RNAseq data independently and identifies 13 604 lncRNA genes contributing to 21 128 lncRNA transcripts [28]. Genetic and structural variation in the lncRNA loci has been shown to alter their structure and function [38]. Databases such as LncVar classified these associated genetic variations of 6303 lncRNAs identified from zebrafish [39]. This dataset could be a useful resource to understand the effect of genetic variants on lncRNA and disease progression. Another database called RegenDbase comprises a specific lncRNA dataset from heart and fin regeneration which could be explored to understand the regulatory aspect of lncRNAs in regeneration [40]. Table 1 Details of existing lncRNA and circRNA online databases for zebrafish S.No . Database . lncRNA/CircRNA . URL . Total no. of transcripts . Ref. . 1. NCBI lncRNA https://www.ncbi.nlm.nih.gov/ 4869 [44] 2. Ensembl   http://asia.ensembl.org/Danio_rerio/Info/Index 4133 [45] 3. NONCODE   http://www.noncode.org/ 4852 [35] 4. zflncRNApedia   http://genome.igib.res.in/zflncRNApedia/ 2267 [37] 5. LncVar   http://159.226.118.31/LncVar/ 6303 [39] 6. ZFLNC   http://zflnc.org/ 21,128 [28] 7. RegenDbase   https://regendbase.org 2820* [40] 8. deepBase2.0   http://biocenter.sysu.edu.cn/deepBase/ 851 [36] 9. zfcircdb circRNA http://clingen.igib.res.in/zfcircdb/ 4205 [43] 10. CIRCpedia v2   https://www.picb.ac.cn/rnomics/circpedia/ 914 [42] 11. circRNome   http://clingen.igib.res.in/circRNome/ 12,360 [41] S.No . Database . lncRNA/CircRNA . URL . Total no. of transcripts . Ref. . 1. NCBI lncRNA https://www.ncbi.nlm.nih.gov/ 4869 [44] 2. Ensembl   http://asia.ensembl.org/Danio_rerio/Info/Index 4133 [45] 3. NONCODE   http://www.noncode.org/ 4852 [35] 4. zflncRNApedia   http://genome.igib.res.in/zflncRNApedia/ 2267 [37] 5. LncVar   http://159.226.118.31/LncVar/ 6303 [39] 6. ZFLNC   http://zflnc.org/ 21,128 [28] 7. RegenDbase   https://regendbase.org 2820* [40] 8. deepBase2.0   http://biocenter.sysu.edu.cn/deepBase/ 851 [36] 9. zfcircdb circRNA http://clingen.igib.res.in/zfcircdb/ 4205 [43] 10. CIRCpedia v2   https://www.picb.ac.cn/rnomics/circpedia/ 914 [42] 11. circRNome   http://clingen.igib.res.in/circRNome/ 12,360 [41] Open in new tab Table 1 Details of existing lncRNA and circRNA online databases for zebrafish S.No . Database . lncRNA/CircRNA . URL . Total no. of transcripts . Ref. . 1. NCBI lncRNA https://www.ncbi.nlm.nih.gov/ 4869 [44] 2. Ensembl   http://asia.ensembl.org/Danio_rerio/Info/Index 4133 [45] 3. NONCODE   http://www.noncode.org/ 4852 [35] 4. zflncRNApedia   http://genome.igib.res.in/zflncRNApedia/ 2267 [37] 5. LncVar   http://159.226.118.31/LncVar/ 6303 [39] 6. ZFLNC   http://zflnc.org/ 21,128 [28] 7. RegenDbase   https://regendbase.org 2820* [40] 8. deepBase2.0   http://biocenter.sysu.edu.cn/deepBase/ 851 [36] 9. zfcircdb circRNA http://clingen.igib.res.in/zfcircdb/ 4205 [43] 10. CIRCpedia v2   https://www.picb.ac.cn/rnomics/circpedia/ 914 [42] 11. circRNome   http://clingen.igib.res.in/circRNome/ 12,360 [41] S.No . Database . lncRNA/CircRNA . URL . Total no. of transcripts . Ref. . 1. NCBI lncRNA https://www.ncbi.nlm.nih.gov/ 4869 [44] 2. Ensembl   http://asia.ensembl.org/Danio_rerio/Info/Index 4133 [45] 3. NONCODE   http://www.noncode.org/ 4852 [35] 4. zflncRNApedia   http://genome.igib.res.in/zflncRNApedia/ 2267 [37] 5. LncVar   http://159.226.118.31/LncVar/ 6303 [39] 6. ZFLNC   http://zflnc.org/ 21,128 [28] 7. RegenDbase   https://regendbase.org 2820* [40] 8. deepBase2.0   http://biocenter.sysu.edu.cn/deepBase/ 851 [36] 9. zfcircdb circRNA http://clingen.igib.res.in/zfcircdb/ 4205 [43] 10. CIRCpedia v2   https://www.picb.ac.cn/rnomics/circpedia/ 914 [42] 11. circRNome   http://clingen.igib.res.in/circRNome/ 12,360 [41] Open in new tab Three major high-throughput studies have reported circular RNAs from zebrafish tissues and developmental stages [32–34]. A database developed by our group circRNome [41] provides a compiled resource of 12 360 circRNA from these three major studies. It also provides details of 45 candidate circRNA, which have been previously validated. The CIRCpedia v2 database also annotates 911 circular RNAs from zebrafish using the CIRCexplorer2 annotation tool [42]. We also compiled a database called ZFCircDB that catalogs 4205 circRNAs identified from a study by Sharma et al. [34, 43]. Genome-wide sequencing approach to identify lncRNA and circRNA in zebrafish Zebrafish have been previously probed for lncRNA/circRNA across different tissue or cell-types, developmental stages, and regenerative timepoints [25–27, 32–34, 46]. Many more possibilities to explore for functional lncRNA/circRNA in the different processes such as organogenesis, disease pathophysiology, mitochondrial-nuclear transport or metabolic processes [27, 47–51] still remain open. Presently, two types of RNA-sequencing protocols are widely used to identify genome-wide lncRNAs in zebrafish. These two protocols are referred as ‘strand-specific RNA sequencing’ and ‘non-stranded RNA sequencing’. The ‘strand-specific RNA sequencing yields information about the loci and retained strand, whereas “non-strand RNA sequencing” ’ only provides the details of the loci without any strand information. One significant shortcoming of the ‘non-stranded RNA-sequencing protocol’ is that it is difficult to differentiate the expression of overlapping genes/antisense-lncRNAs accurately [52]. The major classification of lncRNA is based on their genetic proximity to protein-coding genes and the directionality of transcription. Hence, the recent advancement of ‘strand-specific RNA-sequencing’ provides strand specificity to the lncRNA loci. Additionally, the strand specificity provides additional features to known annotations and improves accuracy of expression profiling. These features provide an advantage in classifying these lncRNAs into categories of antisense, intronic, intergenic, bidirectional and overlapping RNAs [53]. Similarly, it is difficult to capture and distinguish circular RNAs as these have low expression. Also, circRNAs do not have free 5′ or 3′ ends due to which techniques such as rapid amplification of complementary DNA ends (RACE) or poly-A enrichment are not effective. circRNA isoforms are resistant to exonuclease enzymes (RNAse-R), therefore, prior treatment of total RNA with RNAse-R enzyme can enrich circRNAs for sequencing and identification [54]. Past discoveries have estimated that 40% of the lncRNAs and all circRNAs are non-polyadenylated [55, 56]. Hence, it is advised to perform total RNA-sequencing after removing ribosomal-RNAs (ribominus-RNA sequencing) than the conventional polyadenylated-RNA sequencing approach to identify genome-wide lncRNA/circRNA from zebrafish samples. Computational tools for analysis of lncRNA and circRNA lncRNA A number of studies have contributed to the existing lncRNA repertoire, but no gold standard pipeline to identify lncRNAs from zebrafish is still established. In brief, lncRNA transcripts are identified based on two main properties,( i) length of the transcript should be >200 nucleotides and (ii) absence of coding potential. Majorly RNA-sequencing analysis tools like STAR and HISAT2 perform sequencing reads based alignment over the zebrafish reference genome to quantitatively estimate the abundance of the transcripts [57, 58]. Whereas analysis tools like Kallisto and Salmon use pseudo-alignment methods based on expectation–maximization algorithms to assign RNA-sequencing reads to the set of compatible transcripts and then estimate their abundance [59, 60]. Upon comparison, it was observed that pseudo-alignment methods are much faster, utilize less computational-time and resources than the alignment-based method. The expression of known lncRNAs is estimated by annotating the alignment files using annotation data from UCSC or Ensembl or NCBI or in combination [44, 45, 61, 62]. lncRNA expression analysis poses multiple challenges such as low abundance, rough annotation, less information about their functions compared to protein-coding genes. In common practice, low expressed genes with reads per kilobase per million mapped reads (RPKM) < 0.3 are also filtered out to increase the reliability. Multiple bioinformatics tools are available for calculating the differential expression of lncRNAs, which includes the Cufflinks package, DESeq2, edgrR, DEsingle, ShrinkBayes and lncDIFF [63–67]. To identify novel lncRNA isoforms in zebrafish, the de novo approach is utilized and few bioinformatic filters are applied to it. These in silico filters help in annotating the transcript as lncRNA. Major filters utilized in annotating lncRNAs are the length of the transcript, which should be >200 bp that is followed by estimating the coding potential using tools like coding potential calculator (CPC2), coding potential assessment tool (CPAT) and PhyloCSF [68–70]. Also, the prediction of any open reading frame (ORF) present on the transcript is analyzed against the Pfam database using tools like HAMMERv3.3 [71, 72]. All the transcripts with length > 200 bp with no viable coding potential are then considered to be putative lncRNAs, which are then annotated using publicly available lncRNA databases. Transcript with no previous annotation in the database is then termed as a putative novel lncRNA. Figure 1 summarizes briefly the discovery and validations of lncRNAs from zebrafish samples. A detailed step by step protocol for lncRNA identification and annotation can also be found in our previously published book chapter by our group [27, 73] (Figure 1). Figure 1 Open in new tabDownload slide Schematic of lncRNA and circRNA sequencing and computational approach for their discovery. Total RNA from the zebrafish sample is processed and sequenced using the NGS platform and the output data is processed for alignment after quality check. LncRNA and circRNA have different annotation approaches. LncRNA are annotated by directly mapping the reads and then applying lncRNA specific filters for length and coding potentiality and then validated using RACE and other molecular techniques. Other hand circRNAs are annotated by aligning the unmapped reads to the genome and identifying the back-splice junctions and further validated it by PCR using divergent primers. Figure 1 Open in new tabDownload slide Schematic of lncRNA and circRNA sequencing and computational approach for their discovery. Total RNA from the zebrafish sample is processed and sequenced using the NGS platform and the output data is processed for alignment after quality check. LncRNA and circRNA have different annotation approaches. LncRNA are annotated by directly mapping the reads and then applying lncRNA specific filters for length and coding potentiality and then validated using RACE and other molecular techniques. Other hand circRNAs are annotated by aligning the unmapped reads to the genome and identifying the back-splice junctions and further validated it by PCR using divergent primers. circRNA From 2013 to now, more than 25 different circular RNA annotation tools have been published that differ based on their algorithms to identify back-splice junctions, alignment tools, annotation of circular RNAs precision, sensitivity and computational usage [74]. To identify circRNAs, all the reads aligned to the reference are discarded and unmapped reads are processed for putative circRNA annotation using multiple tools [74]. One approach is a pseudo-reference-based strategy that utilizes the existing gene model to identify discordant reads, but the only drawback is the chance of missing a few candidates. This approach is mainly used by annotating tools like CIRCexplorer2, KNIFE, NCLscan and UROBOROUS [75–78]. Another approach employs a de-novo-based strategy where it identifies novel splice sites with overlapping back-splice junctions. This approach has a disadvantage of identifying false positive junctions. Annotation tools like Findcirc, segemehl, DCC, PcircRNA_finder, CIRI and CIRCexplorer deploy this approach in annotating circRNA [79–84]. Previous studies that identified circRNAs in zebrafish have exploited annotation tools like FindCirc, CIRI and segemehl. Upon validation of selective circRNA candidates using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and divergent primers shows that these tools can predict circRNA efficiently in zebrafish [32–34]. Following this, a comparison study was performed by Hansen et al. [85, 86] using five different annotation tools which include CIRI, MapSplice, CIRCexplorer, circRNA_finder and FindCirc. They observed a high false-positive circRNA candidate with the algorithms independently in a range of 59–79% (except CIRCexplorer and MapSplice). Upon pairing the tools at random, they observed that the false-positive rate was decreased (8–12%), and when they combined all the algorithms the false discovery rate further decreased (~6%). The study concluded that no single tool is perfect [85, 86]. While performing annotation of circRNA using multiple annotation tools, one should be cautious of not using the output alignment files from one pipeline to another. Each annotation pipeline is designed based on its specific alignment tool and not using it as per recommendations can create difficulties in the annotation [87]. The general pipeline for circRNAs identification has been shown in Figure 1. The detailed step-by-step description of circRNA identification and annotation using zebrafish samples can also be found in a book chapter published previously by our group [88]. The functions of lncRNAs and circRNAs in zebrafish The discovery of lncRNAs and circRNAs has provided an additional regulatory layer for maintaining cellular physiology. Tissue-specific expression studies of lncRNA and circRNA in zebrafish from our lab also indicates that these transcripts could have an important function in maintaining homeostasis across different biological system [27, 34] (Table 2). Large databases like ZFLNC cataloged around 21 128 lncRNA transcripts [28] and circRNome [41] cataloged information of about 12 360 circRNAs in zebrafish (Figure 2). Although these databases provide rich resources to researchers, only selected candidate transcripts are functionally validated for their role in the development and disease pathophysiology. A prime functional understanding of lncRNA is associated with local gene regulation of its neighboring genes [18]. Mostly antisense-lncRNAs in proximity to a protein-coding gene tends to regulate its expression in a cis state. In zebrafish, an antisense-lncRNA at the 3′ untranslated region (3′UTR) of tie-1 locus was identified and was termed as tie-1AS. Molecular characterization of the locus revealed that the tie-1AS forms an RNA–RNA hybrid interaction with the tie-1 messenger RNA (mRNA) [89]. Interestingly, overexpression of tie-1AS in zebrafish leads to intersegmental vessels (ISV) defects due to loss of vascular integrity. Further, RNA-pulldown using biotinylated RNA probes pointed out an RNA‑protein interaction of Elva1 protein with tie-1AS. This interaction of Elva1 and tie-1AS tends to regulate the tie-1 RNA spatiotemporally in vascular patterning of the brain, making it an essential regulation during the development of zebrafish [90]. Similarly, antisense-lncRNA durga was observed to modulate its neighboring protein-coding transcript kalrna to affect the development of dendrites in zebrafish neuronal primary cell culture [91]. The other class of lncRNA which does not regulate or affect the expression of their neighboring genes is classified as trans-acting lncRNA. They regulate the function of distant genes or are associated with the global regulation of transcripts or protein. An lncRNA in humans known as CAT7 was identified upon pulldown of BMI1 for capturing PCR2 complex to identify RNA-Protein interactions [92]. Its zebrafish counterpart zcal7l, upon knockdown, showed a microcephaly phenotype similar to protein-coding gene bmi1a/b deficiency. CAT7 lncRNA exemplified the role of lncRNA as a regulator of polycomb-group proteins and their downstream targets. Other conserved lncRNAs such as TERMINATOR, PUNISHER and ALIEN are shown to exhibit their role in the development of zebrafish [93]. Impairment of development during gastrulation and low survival was observed upon morpholino mediated knockdown of the TERMINATOR transcript. Similarly, several anatomical defects including vascular patterning and heart defects were observed in zebrafish with the knockdown of the ALIEN lncRNA transcript. The knockdown of PUNISHER manifested vasculature defects in branching and vessel formation. Recently identified lncRNA-ECAL1 in zebrafish was characterized to be a sponge for Cldn5b 3′UTR target miRNA-23a [94]. Impairment of ECAL1 leads to impairment of cerebrovascular networking, which affects the blood–brain barrier. Tissue-specific functional studies of candidate lncRNAs in zebrafish indicate that different lncRNAs are required to perform specific functions to maintain homeostasis of biological systems. For example, in the cardiovascular system, specific lncRNAs like tie1-AS, TERMINATOR, PUNISHER, ALIEN or ECAL1 have been associated with distinct functions within the system such as maintaining vascular permeability or heart and vascular patterning. Moreover, a large number of tissue-specific lncRNA have been identified and need functional validation to be associated with their biological system. Table 2 Descriptions of lncRNA and circRNA identified and their studies in zebrafish S.No . lncRNA . Mode of action(cis/trans) . Type of conservation with humans . Genetic manipulation (mutant/morphant/overexpression) . Biological system effected . Phenotype/specific tissue . Molecular model . ref. . 1. Malat1 trans Syntenic Morphant/poly-A insertion Cardiovascular system Lethal, ear defects, smaller body size and eyes, curved body, swollen pericardium, and pigmentation reduction – [96, 97] 2. Tie1-AS cis Syntenic Overexpression/CRISPRi/Morphant   Intersegmental vessels patterning defect, temporal and spatial brain vascular patterning RNA–RNA, RNAProtein (Elva), stability [89, 90] 3. TERMINATOR trans Syntenic Morphant   >70% lethality, developmental arrest, and severe cardiovascular defects – [93] 4. ALIEN trans Syntenic Morphant   Defective vascular patterning, branching defect in da and ISV, defective cardiac chamber formation – [93] 5. PUNISHER trans Syntenic Morphant   Defects in vascular branching and vessel formation, – [93] 6. ECAL1 trans – Morphant / mutant   Impaired vascular integrity miRNA23a sponge [94] 7. TUNA (megamind) trans Sequence Morphant / mutant Nervous system Impaired locomotor and CNS function lncRNA-Protein (PTBP1, hnRNP-K, and NCL) complex recruited to promoters [25, 98] 8. Durga cis Syntenic Overexpression   Impaired dendrites formation – [91] 9. cyrano trans Sequence Morphant / mutant   Defective Embryogenesis and brain morphogenesis miRNA 7 regulator and HuR protein sponge [25, 99, 100] 10. CAT7l cis Sequence and syntenic Morphant   Microcephaly and death at 5dpf lncRNA-protein (bmi1a/b) complex and Regulates bmi1a/b targets (PRC1 complex protein) [92] 11. lncrps25 trans Syntenic and sequence Morphant   Motor neuron defect Regulates olig2 expression [103] 12. CDR1as trans Unknown Circular plasmid   Defective midbrain development miRNA7 sponge [79] 13. sox2-ot – Syntenic –   Dynamic expression in the central nervous system – [25] 14. PU.1 AS cis Syntenic shRNA Immune System Immune gene regulation – [101] 15. THOR trans sequence Mutant Reproductive system Fertility defect, resistance to melanoma development lncRNA-protein (IGF2BP1) interaction [102] 16. nc-sox4a – – Mutant – – – [96] 17. lnc-pou2af1 – – Mutant – – – [96] S.No . lncRNA . Mode of action(cis/trans) . Type of conservation with humans . Genetic manipulation (mutant/morphant/overexpression) . Biological system effected . Phenotype/specific tissue . Molecular model . ref. . 1. Malat1 trans Syntenic Morphant/poly-A insertion Cardiovascular system Lethal, ear defects, smaller body size and eyes, curved body, swollen pericardium, and pigmentation reduction – [96, 97] 2. Tie1-AS cis Syntenic Overexpression/CRISPRi/Morphant   Intersegmental vessels patterning defect, temporal and spatial brain vascular patterning RNA–RNA, RNAProtein (Elva), stability [89, 90] 3. TERMINATOR trans Syntenic Morphant   >70% lethality, developmental arrest, and severe cardiovascular defects – [93] 4. ALIEN trans Syntenic Morphant   Defective vascular patterning, branching defect in da and ISV, defective cardiac chamber formation – [93] 5. PUNISHER trans Syntenic Morphant   Defects in vascular branching and vessel formation, – [93] 6. ECAL1 trans – Morphant / mutant   Impaired vascular integrity miRNA23a sponge [94] 7. TUNA (megamind) trans Sequence Morphant / mutant Nervous system Impaired locomotor and CNS function lncRNA-Protein (PTBP1, hnRNP-K, and NCL) complex recruited to promoters [25, 98] 8. Durga cis Syntenic Overexpression   Impaired dendrites formation – [91] 9. cyrano trans Sequence Morphant / mutant   Defective Embryogenesis and brain morphogenesis miRNA 7 regulator and HuR protein sponge [25, 99, 100] 10. CAT7l cis Sequence and syntenic Morphant   Microcephaly and death at 5dpf lncRNA-protein (bmi1a/b) complex and Regulates bmi1a/b targets (PRC1 complex protein) [92] 11. lncrps25 trans Syntenic and sequence Morphant   Motor neuron defect Regulates olig2 expression [103] 12. CDR1as trans Unknown Circular plasmid   Defective midbrain development miRNA7 sponge [79] 13. sox2-ot – Syntenic –   Dynamic expression in the central nervous system – [25] 14. PU.1 AS cis Syntenic shRNA Immune System Immune gene regulation – [101] 15. THOR trans sequence Mutant Reproductive system Fertility defect, resistance to melanoma development lncRNA-protein (IGF2BP1) interaction [102] 16. nc-sox4a – – Mutant – – – [96] 17. lnc-pou2af1 – – Mutant – – – [96] Open in new tab Table 2 Descriptions of lncRNA and circRNA identified and their studies in zebrafish S.No . lncRNA . Mode of action(cis/trans) . Type of conservation with humans . Genetic manipulation (mutant/morphant/overexpression) . Biological system effected . Phenotype/specific tissue . Molecular model . ref. . 1. Malat1 trans Syntenic Morphant/poly-A insertion Cardiovascular system Lethal, ear defects, smaller body size and eyes, curved body, swollen pericardium, and pigmentation reduction – [96, 97] 2. Tie1-AS cis Syntenic Overexpression/CRISPRi/Morphant   Intersegmental vessels patterning defect, temporal and spatial brain vascular patterning RNA–RNA, RNAProtein (Elva), stability [89, 90] 3. TERMINATOR trans Syntenic Morphant   >70% lethality, developmental arrest, and severe cardiovascular defects – [93] 4. ALIEN trans Syntenic Morphant   Defective vascular patterning, branching defect in da and ISV, defective cardiac chamber formation – [93] 5. PUNISHER trans Syntenic Morphant   Defects in vascular branching and vessel formation, – [93] 6. ECAL1 trans – Morphant / mutant   Impaired vascular integrity miRNA23a sponge [94] 7. TUNA (megamind) trans Sequence Morphant / mutant Nervous system Impaired locomotor and CNS function lncRNA-Protein (PTBP1, hnRNP-K, and NCL) complex recruited to promoters [25, 98] 8. Durga cis Syntenic Overexpression   Impaired dendrites formation – [91] 9. cyrano trans Sequence Morphant / mutant   Defective Embryogenesis and brain morphogenesis miRNA 7 regulator and HuR protein sponge [25, 99, 100] 10. CAT7l cis Sequence and syntenic Morphant   Microcephaly and death at 5dpf lncRNA-protein (bmi1a/b) complex and Regulates bmi1a/b targets (PRC1 complex protein) [92] 11. lncrps25 trans Syntenic and sequence Morphant   Motor neuron defect Regulates olig2 expression [103] 12. CDR1as trans Unknown Circular plasmid   Defective midbrain development miRNA7 sponge [79] 13. sox2-ot – Syntenic –   Dynamic expression in the central nervous system – [25] 14. PU.1 AS cis Syntenic shRNA Immune System Immune gene regulation – [101] 15. THOR trans sequence Mutant Reproductive system Fertility defect, resistance to melanoma development lncRNA-protein (IGF2BP1) interaction [102] 16. nc-sox4a – – Mutant – – – [96] 17. lnc-pou2af1 – – Mutant – – – [96] S.No . lncRNA . Mode of action(cis/trans) . Type of conservation with humans . Genetic manipulation (mutant/morphant/overexpression) . Biological system effected . Phenotype/specific tissue . Molecular model . ref. . 1. Malat1 trans Syntenic Morphant/poly-A insertion Cardiovascular system Lethal, ear defects, smaller body size and eyes, curved body, swollen pericardium, and pigmentation reduction – [96, 97] 2. Tie1-AS cis Syntenic Overexpression/CRISPRi/Morphant   Intersegmental vessels patterning defect, temporal and spatial brain vascular patterning RNA–RNA, RNAProtein (Elva), stability [89, 90] 3. TERMINATOR trans Syntenic Morphant   >70% lethality, developmental arrest, and severe cardiovascular defects – [93] 4. ALIEN trans Syntenic Morphant   Defective vascular patterning, branching defect in da and ISV, defective cardiac chamber formation – [93] 5. PUNISHER trans Syntenic Morphant   Defects in vascular branching and vessel formation, – [93] 6. ECAL1 trans – Morphant / mutant   Impaired vascular integrity miRNA23a sponge [94] 7. TUNA (megamind) trans Sequence Morphant / mutant Nervous system Impaired locomotor and CNS function lncRNA-Protein (PTBP1, hnRNP-K, and NCL) complex recruited to promoters [25, 98] 8. Durga cis Syntenic Overexpression   Impaired dendrites formation – [91] 9. cyrano trans Sequence Morphant / mutant   Defective Embryogenesis and brain morphogenesis miRNA 7 regulator and HuR protein sponge [25, 99, 100] 10. CAT7l cis Sequence and syntenic Morphant   Microcephaly and death at 5dpf lncRNA-protein (bmi1a/b) complex and Regulates bmi1a/b targets (PRC1 complex protein) [92] 11. lncrps25 trans Syntenic and sequence Morphant   Motor neuron defect Regulates olig2 expression [103] 12. CDR1as trans Unknown Circular plasmid   Defective midbrain development miRNA7 sponge [79] 13. sox2-ot – Syntenic –   Dynamic expression in the central nervous system – [25] 14. PU.1 AS cis Syntenic shRNA Immune System Immune gene regulation – [101] 15. THOR trans sequence Mutant Reproductive system Fertility defect, resistance to melanoma development lncRNA-protein (IGF2BP1) interaction [102] 16. nc-sox4a – – Mutant – – – [96] 17. lnc-pou2af1 – – Mutant – – – [96] Open in new tab Figure 2 Open in new tabDownload slide Genomic distribution of protein-coding genes, lncRNAs and circular RNAs across zebrafish genome. The peaks in the circos plot represent the counts of the transcripts per bin (1 Mb). Similar abundance pattern is observed for protein-coding genes and lncRNAs. CircRNA are observed to be abundant in selective locations in the genome. Figure 2 Open in new tabDownload slide Genomic distribution of protein-coding genes, lncRNAs and circular RNAs across zebrafish genome. The peaks in the circos plot represent the counts of the transcripts per bin (1 Mb). Similar abundance pattern is observed for protein-coding genes and lncRNAs. CircRNA are observed to be abundant in selective locations in the genome. Circular RNAs are still at the initial stage of discovery with not many functional studies. Selected candidate studies have shown that circRNAs are implicated in different developmental processes and disease conditions. At present, only a handful of circRNA has been functionally characterized for their molecular function. An in-depth study by Memczak et al. showed that a conserved circRNA called cerebellar degeneration-related protein 1 antisense (CDR1as) acts as a sponge for a highly conserved miRNA-7 [79]. CDR1as comprise multiple binding sites for miRNA-7 and inhibit it from binding to its targets. In zebrafish, overexpression of CDR1as leads to the reduction of the midbrain size, which correlated to the miRNA-7 knockout phenotype [79]. However, a lot of circRNAs have been identified in different developmental stages and tissues of zebrafish, none of them have been characterized for their molecular function yet. On the other hand, various circRNAs have been characterized in cell lines and other model systems to perform myriads of molecular functions such as miRNA-sponge, protein-sponge, transcription or translation regulator. Some of the circRNA were also observed to have internal-ribosome entry sites (IRES) for potential small-peptide generation [95]. Genetic engineering tools and molecular techniques to target lncRNAs/circRNAs in zebrafish Functional characterization of the annotated lncRNA and circRNA aids in understanding the regulatory molecular mechanism of these transcripts. Techniques such as genetic alterations (overexpression or downregulation) of the transcript help in understanding their physiological relevance. Many tools have been previously deployed in manipulating the zebrafish genome so far [96]. Genetic alteration of lncRNAs/circRNAs has always been a complicated task as the majority of lncRNAs or circRNAs originate from an overlapping locus with the protein-coding genes. In the past, the antisense-oligo based approach was considered ideal for targeting the transcripts directly, and for more than a decade, these modified antisense-oligos called morpholinos were extensively used for modulating the zebrafish genes [104, 105]. One can utilize the morpholinos to understand the function of lncRNA or circRNA by either targeting splice sites, functional or conserved regions of the lncRNA transcript and the back-spliced junction of the circRNA transcript [106]. Morpholinos provide an advantage in targeting lncRNA/circRNA originating from the antisense strand of protein-coding genes, as they only target the antisense-lncRNA/circRNA without disrupting the protein-coding gene. Morpholino is also an asset for specifically targeting the transcribed RNA without affecting any DNA elements present on the loci or the act of transcription [107, 108]. Also, circRNA biogenesis can solely be inhibited using morpholinos targeting its back-splice junctions. However, targeting lncRNAs/circRNA transcripts having single exons or originating from a repeat-rich region paves a greater technical complexity in designing and targeting the RNA [104, 105]. Hence, alternative approaches may be utilized for such transcripts. Recently, evidence of RNA targeting using a variant protein of Cas called Cas13d and Csm in the CRISPR-Cas system has been shown to be functional in zebrafish [107, 108]. CRISPR-Cas13d and CRISPR-StCsm system-mediated targeting of lncRNA/circRNA could help in understanding the transcript function as its off-target effect is negligible with no bystander phenotype, which is slightly observed during morpholinos mediated targeting. The back-spliced junctions in circRNA can also be targeted by generating an engineered CRISPR complex consisting of catalytic dead Cas13 (dCas13) protein conjugated with a splicing inhibitor (splicing factor with RS-EK-RS domains), which can hamper the circRNA biogenesis [109, 110]. Although it is a promising tool for RNA targeting in zebrafish, it has not been characterized for targeting lncRNA/circRNA [107, 108]. Hence, it needs to be further standardized to be used for lncRNA and circRNA targeting in zebrafish. Inhibition of lncRNA or circRNA origin (Transcription start site) can also be achieved by blocking the promoter in lncRNA. In CRISPRi, a complex of dead variants of Cas9 (dCas9) is linked with KRAB transcriptional represses [111]. This dCas9-KRAB complex blocks the RNA polymerase occupancy at the promoter site and inhibits the transcription of the downstream genes like lncRNAs. In zebrafish, CRISPRi has been previously used to understand the function of tie-1AS in cerebrovascular development [90, 111]. Although the use of CRISPRi is disadvantageous in cases where both the lncRNA and the protein-coding gene are in overlapping proximity and have a common promoter, as both the protein-coding and lncRNA transcript will be altered with no coherent readout. Another approach is to induce CRISPR-Cas9 mediated deletions that could be applied as a strategize to inactivate the lncRNA/circRNA in zebrafish [96]. Various approaches have been carried out utilizing the CRISPR-Cas9 mediated modifications on the targeted region. In the majority of the cases, the promoter is disrupted or insertion of premature poly-A in the lncRNA loci for its inactivation [96]. The deletion of small conserved regions in lncRNA ranging from 5 to 500 bp or large deletion of the entire or partial lncRNA locus (more than 2 kb) [112] is also possible using CRISPR-Cas9. These large deletions can sometimes be replaced with reporter genes like lacZ or fluorescent proteins [96]. The only caution needed while targeting lncRNA loci is that it should not be overlapping with any other protein-coding gene or be enriched with any DNA regulatory elements. Studies have also opposed the functional phenotype of lncRNA manifested during knockdown using morpholino in zebrafish because a similar phenotype could not be replicated upon knockout of that gene using CRISPR-Cas9 [113, 114]. This could be due to genetic compensation as well [115]. This type of discrepancy in the phenotype can be avoided if a functionally conserved-motif is targeted and appropriate genetic alteration approaches are utilized. Another commonly used approach is the overexpression of the transcripts to understand the gain of function. LncRNAs are in vitro transcribed and are injected into the zebrafish embryos, whereas circRNAs are in vitro transcribed and are circularized by using enzymes such as CircLigase to mimic the structural similarity of circRNAs [116]. Engineered CRISPR complex consisting of catalytic dead-Cas9 (dCas9) protein linked with transcription activator such as VP64 can also be used to overexpress lncRNA transcripts in vivo [117]. Physiological and molecular changes are further characterized after overexpression to understand the lncRNA/circRNA function. Functional characterization studies of lncRNA/circRNA have utilized various techniques to unravel their molecular mechanisms. To understand the localization of lncRNA/circRNA, high resolution whole-mount in situ hybridization (WISH) is commonly used in zebrafish [118]. Further sub-cellular localization is also analyzed either by extracting RNA from different cellular compartments (cytoplasm, nucleoplasm and chromatin) using single cells suspension of zebrafish and performing qRT-PCR using lncRNA-specific primers and divergent primers for circRNA or performing fluorescent in situ hybridization (FISH) using zebrafish primary cell culture or whole embryo [119]. While performing whole-mount in situ hybridization (WISH) or FISH for circRNA, one should always be cautious while designing the probes over the circRNA junction to provide specificity to the circular transcript. Ribonuclease protection assay (RPA) is performed to identify lncRNA–RNA interaction using the RNase-A enzyme [89, 120, 121]. Similarly, RNase-R is used to identify and validate circRNA as circular transcripts are resistant to RNase activity due to no open ends [54]. Predicting miRNA binding sites using in silico tools and further validating it using a reporter assay is commonly used to identify lncRNA/circRNA as a miRNA-sponge. More often RNA-protein interaction is important for various developmental processes. Molecular techniques like RNA-immunoprecipitation (RIP), RNA or antisense-RNA probe pull-down, electrophoretic mobility shift assay (EMSA), or chromatin isolation by RNA purification(ChiRP-Seq) are widely used to unravel the interacting partners of the lncRNA/circRNA [122–124]. These types of interaction could have a wide range of functionality including protein sponge or as a decoy for other proteins/enzymes. Sometimes lncRNA can also act as a scaffold mediator to join multiple proteins to make a functional complex. To understand DNA interacting lncRNAs chromatin immunoprecipitation (ChIP) assay could be performed to understand lncRNA/circRNA as a transcription regulator which can function by recruiting or hindering transcription factors/repressors to the given loci [125] (Figure 3). Figure 3 Open in new tabDownload slide Genetic manipulation approaches utilized in zebrafish to decipher the functions of lncRNA/circRNA. LncRNAs transcripts can be hampered to perform a function by using tools like Antisense-Oligos(AS)/Morpholino or CRISPR complexes with RNA targeting Cas9(RCas9)/Cas13d/Csm which directly targets the RNA. Also, CRISPR-Cas9 can be used to induce small/large deletion, and dCas9-KRAB can be used to repress the expression of lncRNA loci. Similarly, circRNA can also be inhibited to perform a function by targeting the back-spliced junction using ASO/morpholino, engineered CRISPR complex of dead Cas13 conjugated with splicing inhibitor or RNA targeting CRISPR-Cas9 (RCas9)/Cas13d/Csm complexes. CRISPR-Cas9 can also be used to disrupt the back- splice junction and inhibit circRNA formation. Figure 3 Open in new tabDownload slide Genetic manipulation approaches utilized in zebrafish to decipher the functions of lncRNA/circRNA. LncRNAs transcripts can be hampered to perform a function by using tools like Antisense-Oligos(AS)/Morpholino or CRISPR complexes with RNA targeting Cas9(RCas9)/Cas13d/Csm which directly targets the RNA. Also, CRISPR-Cas9 can be used to induce small/large deletion, and dCas9-KRAB can be used to repress the expression of lncRNA loci. Similarly, circRNA can also be inhibited to perform a function by targeting the back-spliced junction using ASO/morpholino, engineered CRISPR complex of dead Cas13 conjugated with splicing inhibitor or RNA targeting CRISPR-Cas9 (RCas9)/Cas13d/Csm complexes. CRISPR-Cas9 can also be used to disrupt the back- splice junction and inhibit circRNA formation. Identifying the human conserved counterpart of lncRNAs and circRNAs Unlike mRNAs, it is challenging to identify orthologs of lncRNAs across species. The sequence similarity of lncRNAs is just 20% between humans and mice and it drops further to 5% in zebrafish [17]. Therefore, different approaches have been adopted for the identification of functional orthologues in zebrafish. Sequence-based conservation Classically for identifying orthologues of mRNAs, one would check for similar sequences in other species, but lncRNAs have not been positively selected during evolution and are poorly conserved by sequence [126]. There are short stretches of nucleotide sequences (less than 5%) that are conserved between humans and zebrafish. It was noticed that of the 533 lncRNAs identified in zebrafish only 29 displayed sequence similarity in the human genome [25]. The study also showed the functional similarity of two zebrafish and human lncRNAs: Megamind and Cyrano, by rescuing zebrafish embryos lacking zebrafish specific lncRNAs and complementing them with conserved lncRNAs from human and mouse [25]. An orthologue of MALAT1 in zebrafish was identified on chromosome 14, a transcript named ZFLNCG08251. It is of similar length as its human counterpart, contains no intron, and has a noncanonical 3′ end with a short homology of 70 base pairs [25, 127]. A study by Chen et al. [127] employed BLASTN to identify sequence-based homology of known 21 128 Zebrafish lncRNA transcripts. They found homologs of 1258 lncRNA transcripts in humans and showed that they have a high correlation in their tissue-specific expression profile. UC.4, an intronic lncRNA identified in humans has an ultra-conserved region across different species. Its ortholog in zebrafish also performs a conserved role in the TGF-beta signaling pathway and regulates cardiac development in zebrafish [128]. Another lncRNA called THOR contains an ultra-conserved sequence across different species [102]. Its orthologs in humans and zebrafish have a similar expression in testes and shown to have an oncogenic role by interacting with the same IGF2BP1 protein in zebrafish and human cells [102]. On the other hand, circRNAs originating from the exons of protein-coding genes are highly conserved in sequence. It was observed that human circSLC45A4 shows greater conservation with mouse and X. tropicalis as it originates from the 1st exon of protein-coding SLC45A4 [129]. CircRNAs are also conserved if they have functional element sequences conserved across different species such as miRNA and protein-binding sites. Positional conservation During evolution, many stretches of genomes are positively selected and retained in species to perform certain biological functions. There are many genes that are co-localized in a particular arrangement across different species called syntenic blocks and have identical spatio-temporal expressions performing similar functions [130]. Across vertebrates, there are many examples of lncRNAs that do not have any sequence similarity but perform similar functions due to their conserved position in different species [17]. Similarly, in zebrafish, many lncRNAs have been identified based on synteny. A study by Kurian et al. identified three lncRNAs vital for cardiovascular functions in zebrafish. They demonstrated that TERMINATOR regulates stem cell specification, ALIEN controls cardiovascular development and PUNISHER maintains endothelial cell function in zebrafish. To identify their homologs in higher species they analyzed neighboring regions of these three lncRNAs in mice and humans and identified putative homologs. These were further functionally validated using complementation-based approaches, wherein lncRNA morphant zebrafish phenotype were rescued with human lncRNAs [93]. slincR lncRNA identified in proximity to sox9b loci in zebrafish has positionally conserved putative slincR orthologue in humans and mice. slincR transcript in humans has the same spatial arrangement, orientation, and conserved signature of AHR element in the promoter region of the transcript [131]. PU1-AS an antisense-lncRNA for PU1 mRNA was identified in human cells to play a vital role in adipogenesis [120]. Later, its orthologue was isolated in zebrafish by analyzing the same synteny. In zebrafish as well PU1-AS performed similar functions by mediating the levels of PU1 mRNA [101]. CircRNAs were also found to be conserved based on their origin from the syntenic region. CircRNAs produced by HIPK2 and HIPK3 were observed to be conserved in both humans and mice due to their abundance and loci of origin [30]. CircRNA having a similar locus of origin can also be conserved wherein if circRNA is made up of intron-exons, then its orthologue could also be having the same intron‑exon feature from a similar locus. Similarly, counterparts for zebrafish circRNAs in humans can also be probed by analyzing the syntenic loci of both the species. Structural conservation Structure-based conservation is another mode of conservation, in which there is a possibility of the presence of conserved secondary structural domain(s) with or without similar primary sequence. A computational study predicted that more than 500 lncRNAs have an evolutionary conserved secondary structure across vertebrates [135]. But there are very few well-characterized lncRNAs having conserved structural features across vertebrates. This could be due to a lack of prediction algorithms for detecting structural conservation between species at transcriptome levels. Y-lncRNA is the only so far known lncRNA that is conserved across zebrafish and humans. The Y-RNA has sequence and structure similarity in both zebrafish and humans. It is known to perform a similar function in initiating DNA replication in both the species [133]. Very recently JPX lncRNA, identified in mice and humans were found to have similar structure regions despite lacking any sequence similarity. Studies in mouse embryonic stem cells showed both human and mice JPX lncRNAs bind to CTCF via conserved domains and have overlapping functions [134]. Various chemical- and enzymatic-based biochemical approaches have been employed to identify the structure of lncRNAs in different species. Majorly, Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) method has been used in various studies to study structures of lncRNA such as SRA, XIST, HOTAIR, MALAT1 and NEAT1 [135–139]. Our group had used a different technique called parallel analysis of mRNA structure (PASR) to demonstrate the secondary structure of zebrafish Y-RNA and Tie1-AS lncRNA [140]. SHAPE or PASR methodologies should be utilized widely to predict and validate structurally conserved lncRNAs across species. Such techniques may also find use in cicRNA which also form small and irregular-hairpin structures and may be used for interacting with the binding proteins [140, 141]. These secondary structures can also be used to find orthologs in other species if they are similar and have the same protein binding partner. Conserved interacting molecules LncRNAs are known to interact with various biomolecules to perform their functions. These biomolecules including evolutionary well conserved proteins, RNA or DNA could be used as a handle/bait to identify interacting ncRNAs across species. As mentioned in the earlier section, different approaches can be adapted to identify interacting protein/RNA or DNA molecules for a particular lncRNA. lncRNAs govern their function by regulating their interacting partners. Now, different lncRNAs across species that might or might not have an obvious similar sequence/structure contain conserved functions by interacting with similar interaction partners. Human JPX and mouse Jpx lncRNA despite having sequence and structure divergence showed similar functions by interacting with common interacting protein CTCF [134]. Similarly, strategy could be adopted to identify orthologues of well-characterized zebrafish lncRNA in humans or other higher organisms, one can quest interactome of the same lncRNA interacting biomolecule in humans. CircRNA interaction with biomolecules can also be explored to identify conserved orthologous. CircRNA without any sequence conservation but have similar interacting proteins are said to be conserved as these proteins could bind to conserved structural domains present on the circRNA [134, 141]. Conclusion Zebrafish has always been an amenable vertebrate model organism to probe developmental genetics and disease pathophysiology associated with human’s protein coding genes. The same factors that led to the emergence of zebrafish as a prominent model for probing protein coding gene function is driving the utility of this model for elucidating function of ncRNAs. The increasing availability of deep transcriptome sequence data combined with advanced computational analysis tools and the ability to undertake precise manipulation of genetic loci have made zebrafish an ideal tool box for investigating regulatory functions of lncRNA/circRNAs. Key points Deep-sequencing of transcriptomes has identified many novel transcript isoforms including noncoding RNAs in zebrafish. Multiple RNA-sequencing and bioinformatic protocols may be utilized for identifying novel and known lncRNA and circRNA in zebrafish. Genetic manipulation tools like morpholino and CRISPRs may help in better understanding of the lncRNA/circRNA identified in zebrafish. Different models of conservation could be a way forward to identify zebrafish lncRNA/circRNA functional counterparts in humans. Acknowledgements PS acknowledges CSIR-SRF fellowship, DS acknowledges Intel India fellowship. The authors acknowledge help received from Ms. Anjali Bajaj for proofreading the manuscript. Funding The work was funded by the Council of Scientific and Industrial Research, India through grant MLP2001. Gyan Ranjan is an experimental biologist working on identification and characterization of functional lncRNAs in cardiovascular and blood biology using zebrafish as a model. He is a graduate student in Sridhar Sivasubbu’s lab at CSIR-IGIB Paras Sehgal is an experimental biologist working on identification and characterization of functional lncRNAs in vascular biology using zebrafish as a model. He is a graduate student in Sridhar Sivasubbu’s lab at CSIR-IGIB Disha Sharma is a computational biologist who works on identification and characterization of circular RNAs using model systems such as zebrafish and rats. Vinod Scaria is a clinician and computational biologist at CSIR-IGIB. His laboratory is interested in understanding the function, organization and regulation of vertebrate genomes, and how genomic variations could potentially impact them. 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Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Functional long non-coding and circular RNAs in zebrafish
JO - Briefings in Functional Genomics
DO - 10.1093/bfgp/elab014
DA - 2021-03-23
UR - https://www.deepdyve.com/lp/oxford-university-press/functional-long-non-coding-and-circular-rnas-in-zebrafish-tbo1mImIjf
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -