Human NDUFS3 gene coding for the 30-kDa subunit of mitochondrial
Complex I: genomic organization and expression
Rene´ De Coo,
Laboratoire BECP—EA2943 UJF/LRA6V CEA—DBMS, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble, cedex 9, France
Ge´nome Express SA, 38054 Grenoble, France
Faculte´deMe´decine de la Timone, INSERM U 406, 13385 Marseille, France
Laboratoire de Biochimie, Hoˆpital Debrousse, 69322 Lyon, France
Service de Biologie, Hoˆpital de la Timone, 13385 Marseille, France
Division of Genetics, University of Maastricht, 6229 GR Maastricht, The Netherlands
Department of Neurology, University Hospital Rotterdam, 3015 GJ Rotterdam, The Netherlands
Received: 4 January 2000 / Accepted: 6 May 2000
Mitochondrial Complex I (or NADH-ubiquinone reductase,
E.C.126.96.36.199) catalyzes the oxidation of NADH, with the concomi-
tant reduction of ubiquinone and ejection of protons out of the
mitochondria. Mammalian Complex I is a multimeric enzyme
composed of at least 42 different subunits (Skehel et al. 1998), 35
of which are encoded by nuclear genes. Human Complex I defi-
ciencies are involved in a variety of heterogeneous diseases. Many
pathogenic mitochondrial DNA point mutations responsible for
Complex I deficiency have been reported (see Mitomap database).
Mutations were more recently identified in the nuclear genes cod-
ing for the 51-, the 23-, the 20- and the 18-kDa subunits, in patients
suffering from Leigh’s syndrome or multisystemic disorders (for
review see Loeffen et al. 2000). A missense mutation in the exon
coding for the mitochondrial import presequence of the 24-kDa
subunit was also reported as a factor predisposing to Parkinson’s
disease when present in a homozygous state (Hattori et al. 1998).
In addition, we recently established that pathological Complex I
inactivations may also result from mutations in unknown nuclear
genes coding for factors specifically involved in Complex I as-
sembly (Procaccio et al. 1999). Additional information concerning
all these data are available from our Complex I database (MitoPick
Clearly, understanding the pathological events linked to the
inability of Complex I-defective mitochondria to maintain cellular
energetic production is a process that requires in-depth character-
ization of all the genes involved in Complex I biosynthesis. So far,
the genomic organization of only 10 nuclear genes encoding hu-
man Complex I subunits has been reported (see MitoPick). In the
present report, we describe the genomic sequence and organization
of the human NDUFS3 gene. Transcription motifs were identified
in the upstream region of this gene, and promoter activity of this
region was demonstrated.
We screened human cDNA and genomic libraries and isolated
several cDNA fragments coding for the 30-kDa subunit and one
cosmid clone that was shown to hybridize to these cDNAs. Each
of these clones was extensively characterized. The cDNA se-
quence was found to contain an open reading frame that encodes
a 264-amino acid-long protein, with a molecular mass of 30.2 kDa,
as reported previously (Loeffen et al. 1998; Genbank AF067139).
Once trimmed of its mitochondrial import presequence, the mature
protein was expected to be 228 amino acids long, with a molecular
mass of 26.4-kDa and a theoretical pI of 5.51. These features
perfectly fit with the experimental data gathered (27 kDa and pI
5.5) from the position of the protein spot assigned to the 30-kDa
subunit, by peptide mass fingerprint analysis in 2D gels of human
mitochondrial proteins (see 2D map at MitoPick).
The NDUFS3 gene was found to consist of seven exons rang-
ing in size from 66 to 248 bp (Fig. 1). Intron sizes varied between
110 and 1845 bp. Exon 1 was found to contain the 64-bp 5Ј
untranslated region and the first 67 bp of the coding region. FISH
analysis and radioactive in situ hybridization were performed, and
hybridization signals appeared consistently associated with the
Chromosome (Chr) 11p11-p12 region. The NDUFS3 gene was
previously located to Chr 11p11.11 by radiation hybrid mapping
(Emahazion et al. 1998), and our localization by other technical
approaches confirmed this finding.
Examination of the genomic sequence of the NDUFS3 gene
revealed the presence of two CA repeats in introns. The first CA
repeat in intron 2 (at position 895–972 downstream from the trans-
lation start) was identical to the polymorphic satellite marker
D11S4109 (Genbank Z52649); the second was a discrete 18-CA
motif located at position 3952–3969 downstream from the trans-
lation start in intron 6 (Fig. 1). The presence of the D11S4109
marker inside the NDUFS3 gene makes this repeat a valuable
marker for linkage analysis of diseases that may be associated with
the 30-kDa subunit of Complex I.
The sequence of the promoter region of the NDUFS3 gene was
examined to find putative domains important for transcription ac-
tivity (Fig. 1). CCAAT and TATA boxes were absent from this
region, which contained five sites for transcription factor Sp1,
which is important for the basal expression level of numerous
genes (Kadonaga et al. 1986). These sites were located at nt −465,
−355, −351, −286 and −105 from the translation start, respectively.
We also located two other potential transcription factor-binding
sites, one for Ets, a factor with a general role in transcription
activation, at position −121, and one for the nuclear respiratory
transcription factor NRF-2, at nt −85. One binding site for the
transcriptional activator/repressor YY1 (Hyde-DeRuyscher et al.
1995) was found overlapping the translation start (position nt −4).
AP-2 and AP-1 motifs were identified at nt −461 and −8 respec-
tively. As shown in Fig. 1, the 5Ј region of the NDUFS3 gene
clearly appeared as a CpG-rich region compared with the rest of
the genomic DNA fragment. This CpG island extended approxi-
mately from nt −510 to +280 (i.e., upstream from exon 1 down to
the beginning of the second intron) and probably constitutes an
important domain for proper transcription of the NDUFS3 gene.
Correspondence to: J.-P.Issartel (CNRS); E-mail: Issartel@ellebore.
The nucleotide sequence data reported in this paper have been submitted to
Genbank and have been assigned the accession number AF200954.
Mammalian Genome 11, 808–810 (2000).
© Springer-Verlag New York Inc. 2000