3762–3769 Nucleic Acids Research, 1999, Vol. 27, No. 18 © 1999 Oxford University Press
Drosophila and human RecQ5 exist in different
isoforms generated by alternative splicing
Jeff J. Sekelsky1,2,*, Michael H. Brodsky3, Gerald M. Rubin3and R. Scott Hawley1
1Section of MCB, University of California, Davis, CA 95616, USA,2Department of Biology, University of North Carolina,
Chapel Hill, NC 27599, USA and3Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA
Received March 23, 1999; Revised and Accepted July 26, 1999DDBJ/EMBL/GenBank accession nos AF134239, AF135183
Members of the RecQ helicase superfamily have
been implicated in DNA repair, recombination and
replication. Although the genome of the budding
yeast Saccharomyces cerevisiae encodes only a single
member of this family, there are at least five human
RecQ-related genes: RecQL, BLM, WRN, RecQ4 and
RecQ5. Mutations in at least three of these are asso-
ciated with diseases involving a predisposition to
malignancies and a cellular phenotype that includes
increased chromosome instability. Metazoan RecQ
helicases are defined by a core region with character-
istic helicase motifs and sequence similarity to
Escherichia coli RecQ protein. This core region is
regions, of largely unknown function. The recently
reported human RecQ5, however, has only the core
RecQ-homologous region. We describe here the
identification of the Drosophila RecQ5 gene. We
recovered cDNAs corresponding to three alternative
splice forms of the RecQ5 transcript. Two of these
generate nearly identical 54 kDa proteins that, like
human RecQ5, consist of the helicase core only. The
third splice variant encodes a 121 kDa isoform that,
like other family members, has a C-terminal extension
rich in charged residues. A combination of RACE and
cDNA analysis of human RECQ5 demonstrates
extensive alternative splicing for this gene also,
including some forms lacking helicase motifs and
other conserved regions.
Genome instability is a significant factor in the development of
many cancers. Mutations in genes that function in DNA
metabolism frequently result in genome instability and, in
humans, cancer-associated syndromes (reviewed in 1). One
such class of genes is that encoding proteins related to
Escherichia coli RecQ helicase. Three of the five known
human members of this class have been shown to be associated
with hereditary diseases that include predisposition to cancers.
Mutations in BLM result in Bloom syndrome (BS), mutations
in WRN result in Werner syndrome (WS), and mutations in
RECQ4 result in Rothmund–Thomson syndrome (RTS) (2–4).
Each of these syndromes is a rare, autosomal recessive disorder
with unique clinical features. BS is characterized by dwarfism
and immune deficiency (reviewed in 5); WS is characterized
by the premature onset of a number of conditions associated
with aging (reviewed in 6,7); and RTS is characterized by skin
and skeleton abnormalities, but also includes features associated
with premature aging(reviewed in8). Eachof these syndromes
involves a greatlyincreased risk for manytypes ofmalignancy,
and each exhibits a cellular phenotype that includes genome
instability. In BS cells, genome instability is manifested as the
presence of chromosome aberrations and increased levels of
exchange between homologous chromosomes and between
sister chromatids (9,10). Chromosome instability in WS cells
is revealed as an increased rate of chromosome translocations
and deletions (11,12). RTS patients have been observed to
acquire somatic mosaicism for chromosome rearrangements
and changes in chromosome number (13,14).
Insights into the cellular functions of RecQ helicases have
come from studies in model organisms, especially microorgan-
isms. The protein family is named for E.coli RecQ, a 3' to 5'
DNA helicase that is required either to promote or to inhibit
recombination, depending upon the pathway (15–17). A single
RecQ helicase-encoding gene has been found in each of the
yeasts Saccharomyces cerevisiae and Schizosaccharomyces
pombe (18–20). Mutations in S.cerevisiae SGS1 result in
hyper-recombination and defects in both mitotic and meiotic
chromosome segregation (18,21). Sgs1p interacts with topo-
isomerases II and III in a two-hybrid assay, and sgs1 mutations
suppress the slow growth phenotype of top3 mutations (22).
These phenotypes suggest that Sgs1p is involved in both
replication and recombination.
One issue that studies in microorganisms cannot address is
the multiplication and divergence of the RecQ family in animals.
In addition to the BLM, WRN and RECQ4 loci described above,
two other human genes encoding RecQ-related proteins, RECQL
and RECQ5, have been reported (23–25). The genome of the
nematode Caenorhabditis elegans encodes at least four RecQ-
related proteins, and it is possible to define orthologous
relationships between these proteins and human WRN, BLM,
*To whom correspondence should be addressed at: Department of Biology, CB #3280, Fordham Hall, University of North Carolina, Chapel Hill,
NC 27599-3280, USA. Tel: +1 919 843 9400; Fax: +1 919 962 8472; Email: firstname.lastname@example.org
Nucleic Acids Research, 1999, Vol. 27, No. 18 3763
RecQL and RecQ5, based on sequence similarities within the
region conserved among family members. Thus, it appears that
this gene family underwent substantial divergence early in
metazoan evolution, with different family members presumably
acquiring different functions (26). Genetic characterization of
RecQ-related genes in model metazoans is likely to provide
important new insights into these functions.
We report here the identification of the Drosophila melano-
gaster RecQ5 gene. Characterization of cDNAs encoding
Drosophila RECQ5 show that alternative splicing produces
drastically different isoforms. We also found evidence that
human RECQ5 is alternatively spliced to produce different
MATERIALS AND METHODS
GenBank accession numbers
AF135183, human RECQ5 (for transcripts A and B).
DNA clones and sequences
We screened 105clones from a 0–4 h Drosophila embryo
cDNA library (27) and recovered 25 RecQ5 cDNAs. We initially
characterized four of these further. Two contained the entire
genomic sequence showed that three of these had the larger
One of the truncated cDNAs had the smaller intron (75 nt)
the remaining cDNAs by PCR and found that 19 had the larger
intron removed and two had the smaller intron removed. Two
RecQ5EST sequences were subsequently deposited into GenBank
(28). One of these, clone LD21474 (GenBank accession no.
AA735537), had the larger intron removed. The other, clone
GH01404 (GenBank accession no. AI062257), had no intron
removed at the alternative splice site, although it carries a poly-
adenylation tract at the 3' end and other introns were removed.
The original human RECQ5 cDNA in the EST database is
I.M.A.G.E. Consortium CloneID 590051 (GenBank accession
nos AA155835 and A155882) (29). Using this insert as a
probe, we screened 5 × 105plaques from each of two human
brain cDNA libraries and obtained a single positive clone. The
sequence of this cDNA and our RACE sequences has been
deposited into GenBank under accession number AF135183.
During the course of this work, several I.M.A.G.E. Consortium
cDNA cloneEST sequences were depositedintoGenBank.We
obtained and characterized the cDNAs that were available
(Table 1 and Fig.3).CloneID 2191126carries a 1292bpinsert.
The library from which this clone is derived has inserts cloned
directionally into the SalI (5') and NotI (3') sites of pCMV-
SPORT6. The SalI end appears to be intact. However, 162 bp
near this end matches the end of a 5' EST read from CloneID
380349, but in the opposite orientation. The last 512 bp of the
insert match RECQ5 sequence in a 3' to 5' direction. There is no
poly(A) tract at this end, and there is a fragment of rearranged
polylinker sequence between the NotI site and the insert. We
conclude that this clone carries a cDNA fragment with its 5'
end toward the NotI site and its 3' end (which does not include
the terminus) at the SalI end.
Sequence comparisons were performed with the Wisconsin
of the RecQ conserved amino acid sequences (corresponding
to human RecQ5 residues 1 through 406) were generated with
the PILEUP program. Protein sequence distances were determined
with the DISTANCES program (Kimura scoring method), and
Table 1. Human RECQ5 cDNAs
aSee text for definitions of transcript classes. Some cDNAs could not be assigned unambiguously to a single class due to
insufficient length or sequence information.
bCoordinates are relative to GenBank accession number AF135183. Numbers preceded by + denote sequences (of the indicated
length) not present in AF135183. Numbers in parentheses are approximations. Some cDNAs were unavailable for analysis;
these are marked with < to indicate that the insert continues in the 5' direction. Coordinates for these cDNAs are from the
sequence in the corresponding GenBank record.
GenBankI.M.A.G.E. Library tissue sourceClassa
AF135183– brain A11–1356; 1823–2178
AA155835590051umbilical vein endothelium A1985–1356; 1823–2178
AI686448 2267876serous papillary carcinomaA1 or B<1928–2176
AI6840412267435serous papillary carcinomaA2 or B<1961–2397
AI5378832189520 serous papillary carcinomaA31–1356; 1823–2828
N53539 284217multiple sclerosis lesions A3 or B 2198–2828
AI218469 1845920mixed fetal lung, testis, B-cellC1 (500)–1113; +332 bp
AI363275 2016056glioblastomaC2311–1113; +396 bp
AI3102291914385kidney clear cell tumorF1278–1034; +191 bp
AI6719402316345kidneyF2<866–1225; +47 bp
AI5670002191126serous papillary carcinomaF3846–1356; +780 bp
3764 Nucleic Acids Research, 1999, Vol. 27, No. 18
a phylogenetic tree was constructed with the GROWTREE
program (neighbor-joining method). Other methods gave
slightly different trees, especiallywith respect to the placement
of human RecQ4, but all methods generated essentially similar
orthologous relationships between the metazoan sequences
Searches for nuclear localization signals were done on the
PSORT server at http://psort.nibb.ac.jp
PCR and 3' RACE
Human RECQ5 introns were confirmed by PCR from genomic
DNA (Promega), used at a concentration of 1 ng/µl. Primers
used were Q12 (GCGCTTTGGCTTCTCTGA) and Q205a
(CAAACCCAAAGCCTTCTT), which amplify a 666 bp frag-
ment, including a 472 bp intron at 113; Q974 (GGGTGT-
GAACGCCAAGGCTT) and Q1061a (CAGGGACCTTCTC-
CTCCAT), which amplify a 626 bp fragment that includes a
538 bp intron at 1011; Q1090 (GGGAGTGGATAAA-
GCCAAT) and QC326 (GGGCACAGCACTAGGCAAT),
which amplify a 90 bp fragment across the 5' boundary of the
intron between motifs V and VI; Q1318 (GGCCTTTGAT-
GCCCTGGTGACC) and Q2081a (CCTCGATCTACCAT-
GAGCTT), which amplify a 765 bp fragment that spans the
467 nt intron position; Q1773 (GGGGAGTCATGTGCTTT-
GAA) and Q2111a (CTGAAAATCAGGAGACGGG), which
amplify a 339 bp fragment; and Q2458 (GGCCAAGTGTTC-
CTGTTCAT) and Q3157a (GCCCCGCCTCATTAGTTA),
which amplify a 700 bp fragment.
To determine whether there are alternative 3' ends on
RECQ5 transcripts, we used two gene-specific primers from the
1.7 kb cDNA: Q1173 (GGGCTGGCAGGATGGGAAGCCTT)
and Q1318. Amplification was done with the Marathon kit
(Clontech), on human placenta second-strand cDNA ligated to
adapters, according to the instructions. We obtained bands of
2.7 and 1.7 kb with primer 1173, and 2.5 and 1.5 kb with
primer 1318. Several smaller bands were also detected by
probing a blot of the RACE products, but these were not
characterized. Bands were gel-isolated and reamplified with
the same or internal primers for sequencing.
Radiation hybrid mapping
Radiation hybrid mapping was performed using the Stanford
G3 Radiation Hybrid Panel (20) obtained from Research
Genetics, according to the instructions. Primers used were
1852 (CTGAGGGCTGCTTGGTGTAGTCAGGTT; 3' end of
coding) and 2081a (above; 3' UTR). Raw scoring was submitted
to the RH server at http://www-shgc.stanford.edu/RH/index.
html . The result returned indicated linkage to AFMb054zf9 on
chromosome 17, with a LOD score of 21.7, and a distance of
Northern blot hybridization
(Rockville, MD). The 229 bp PCR amplimer from the radiation
hybrid mapping was32P-labeled using the Ready-to-Go DNA
labeling kit (Pharmacia), and the blot probed and washed
according to the manufacturer’s instructions. We also probed a
similar blot (Clontech) containing mRNA from leukocytes,
colon, small intestine, ovary, testis, prostate, thymus and
spleen, and obtained similar results to those in Figure 4 (data
Immunolocalization of Drosophila RECQ5
The protein-coding region of a RecQ5 cDNA encoding the
121 kDa isoform was fused in-frame downstream of a segment
encoding the FLAG epitope DYKDDDDK. This was placed
under the control of the Ubiquitin promotor through the initiating
methionine codon. 5 × 106Schneider S2 cells were transiently
transfected with 5 µg DNA and 10 µl Superfect (Qiagen). Two
days after transfection, these cells were stained with a 1:1000
RecQ5 is conserved between Drosophila and humans
We discovered a D.melanogaster gene encoding a novel
member of the RecQ helicase superfamily while cloning an
unrelated cell cycle checkpoint gene. Database searches using
the deduced protein sequence revealed putative orthologues in
C.elegans (E03A3.2) and humans. A sequence of the human
orthologwas recently published with the nameRECQ5 (25), so
we designate the Drosophila gene RecQ5. The predicted human
RecQ5andDrosophila RECQ5proteins are 49% identical to one
another in the RecQ-conserved region. Sequence comparisons
between family members using this region place human
RecQ5, Drosophila RECQ5 and C.elegans E03A3.2 onto a
distinct branch (Fig. 1), suggesting that these genes share a
conserved function in DNA metabolism.
We mapped the Drosophila RecQ5 gene to interval 70E1-4 by
in situ hybridization to polytene chromosomes. We determined
the genomic position of human RECQ5 by radiation hybrid
mappingusingthe StanfordG3panel (Materials andMethods).
Our results place the gene in 17q23–25 near the marker
Drosophila RECQ5 isoforms generated by alternative
Previouslydescribedmembers ofthe eukaryotic RecQ helicase
family range in size from 75 kDa for human RecQL to 162 kDa
for human WRN. In most family members, the highly conserved
helicase region is flanked by extensive sequences that show
similarity to one another primarily in amino acid composition,
being rich in charged residues. The reported human RECQ5
sequence encodes a 46 kDa protein that consists of the core
region conserved among RecQ family members. Analysis of
Drosophila RecQ5 cDNAs reveals predicted isoforms of 54
and 121 kDa (Fig. 2). The smaller isoforms are very similar in
structure to human RecQ5, consisting entirely of the region
encompassing the seven motifs common to known helicases
(30), together with flanking sequences conserved in all RecQ
family members. The larger isoform has in addition an extensive
C-terminal region abundant in charged residues. Among the
588 residues in this region, 98 (16.7%) are acidic, and 126
(21.4%) are basic. The charged residues are primarily glutamic
between this region and other RecQ family members, other
than in composition.
The different Drosophila RECQ5 isoforms are encoded by
transcripts that differ by alternative splicing (Fig. 2). The
region encoding the helicase motifs is contained entirely on the
second exon, which can be joined to either of two alternative
third exon start sites. Exon 3a, following removal of a 327 nt
Nucleic Acids Research, 1999, Vol. 27, No. 18 3765
intron,maintains an open readingframe intosubsequent exons,
resulting in a 121 kDa isoform (RECQ5a). Exon 3b, following
removal of a 75 nt intron, has an immediate stop codon at the
5' end, resulting in the truncated 54 kDa isoform (RECQ5b).
We also found a cDNA in which no intron was removed at this
position. Other introns hadbeen removed andthere was a poly-
adenylation tract at the 3' end, indicating that this cDNA is
derived from a processed transcript. This cDNA encodes a 54
kDa isoform (RECQ5c) almost identical to RECQ5b.
Human RECQ5 isoforms generated by alternative splicing
We wanted to know whether alternative splicing is a feature
conserved in human RECQ5. Human RECQ5 was originally
defined by EST sequences from a single cDNA (Table 1, line
2). We obtained and sequenced this cDNA and found it to be
incomplete at the 5' end. Using this clone as a probe, we
screened human cDNA libraries, and recovered one RECQ5
clone from a brain cDNA library. The 1710 bp sequence of this
cDNA is identical tothe sequence of theESTclone,except that
it is longer at the 5' end and it contains a complete open reading
frame. Both cDNAs have a polyadenylation tract 275 bp 3' to
the termination codon. The corresponding mRNA encodes a
49 kDa polypeptide, slightly longer than the 46 kDa polypeptide
predicted by the published RECQ5 coding sequence (25).
Comparison of the published nucleotide sequence to our
sequences showed that they are identical except at the extreme
3' end of the open reading frame, at which point the published
sequence has a stop codon not found in our sequences.
To investigate the differences between our cDNAs and the
published sequence, we performed 3' RACE on human placenta
mRNA, using two gene-specific primers to sequences near the
end of the protein-coding region (Materials and Methods).
With each primer we amplified a product of the size predicted
by our cDNA sequences (data not shown). In addition, we
obtained two largerproducts foreach primer, which we refer to
as RACE products 1 and 2.
Sequencing of these larger RACE products showed that each
contains an insertion of 467 bp near the end of the coding
region (Fig. 3). The insertion begins with a GT and ends with
an AG, so we considered that it might correspond to an intron,
and that RACE products 1 and 2 represent amplification of
transcripts in which this intron had not been removed. We
designed primer pairs that span either the 5' or 3' boundary of
the insertion, and that span the entire insertion (Materials and
Methods). When we used these primers in PCR reactions using
human genomic DNA as a template, products of the predicted
sizes were specifically amplified (data not shown), confirming
that this insertion is indeed derived from an unspliced intron.
Figure 1. The eukaryotic RecQ helicase family. (A) Dendrogram depicting relationships between RecQ helicase family members from E.coli (Ec), S.cerevisiae
(Sc), S.pombe (Sp), C.elegans (Ce) and humans (Hs), along with Drosophila RECQ5. The relationship of human RecQ4 to the other sequences is unclear
(Materials and Methods), but the other metazoan proteins fall into four branches. Another Drosophila RecQ family member, DmBLM, the ortholog of human
BLM, was recently reported(26), but is not indicated here. (B)Schematics ofthe corresponding protein structures. The shaded region represents the RecQ helicase-
related conserved region that was used in sequence comparisons. Black boxes indicate the seven conserved helicase motifs. Arrows in Drosophila and human
RecQ5 indicate termination sites of alternative isoforms (see Figs 2 and 3).
3766 Nucleic Acids Research, 1999, Vol. 27, No. 18
One possible explanation is that RACE products 1 and 2
were from unprocessed primary transcripts. Several observations
suggest that this is not the case. First, the template inthe RACE
reactions was double-stranded cDNA made from polyadenylated
mRNA, and both products carry a polyadenylation tract at their
3' ends. Second, the 3' end of product 1 is identical to that of
two 3' EST sequences in the database (see below). Third, the 5'
end of the unspliced intron has an in-frame stop codon, resulting
in a sequence that is identical to the published RECQ5 protein-
coding sequence (25). We conclude that this 467 nt intron is
removed from some transcripts but not from others. As is the
case in Drosophila, failure to remove this intron results in a
truncation of the polypeptide, inthis case removing 25 residues
to yield a 46 kDa isoform.
RACE products 1 and 2 also differ at their 3' ends from our
cDNA. Both RACE products contain the entire 3' end of tran-
script A, followed by additional sequences. Product 1 has an
additional 1118 bp, and product 2 has this same sequence
followed by an additional 718 bp, ending in an Alu sequence.
There is a putative polyadenylation signal near the 3' end of the
Product 1 sequence, and database searches revealed two ESTs
that this is a bona fide polyadenylation site.
During the course of this work, additional human RECQ5
EST sequences were deposited into GenBank (Table 1). Analysis
of these cDNAs reveals additional polyadenylation sites, splicing
patterns and predicted protein isoforms (Fig. 3). We have desig-
nated each transcript class according to predicted protein
product (distinguished by different letters) and polyadenylation
site (distinguished by different numbers). Transcripts in class
A encode the 49 kDa isoform. There are at least three different
polyadenylation sites for class A transcripts. Transcripts in
class B encode the 46 kDa isoform, due tofailure toremove the
467 bp intron. This class is presently represented only by our
RACE products and the coding sequence reported by Kitao et al.
A third class, designated C, is represented by I.M.A.G.E.
Consortium CloneIDs 2016056 and 1845920. The inserts in
these cDNAs match our RECQ5 cDNA sequence from their 5'
Figure 2. Alternative splicing at Drosophila RecQ5. (A) Schematic of three
alternative RecQ5 transcripts. Each box represents one exon. Protein-coding
regions are shaded; black boxes indicate the seven helicase motifs. Transcript
A encodes a 121 kDa isoform (RECQ5a); transcripts B and C encode nearly
identical 54 kDa isoforms (RECQ5b and RECQ5c). Positions of potential
nuclear localization signals (KRPKK, HRRKR and PYYKRKI) are indicated
with arrows. (B) DNA sequence corresponding to the region of the alternative
splice (indicated by a bracket in 3a), determined by sequencing cDNAs and
genomic DNA. The sequence begins near the endofthe common second exon.
The sequence corresponding to the larger version of the intron is in lower case.
Predicted amino acid sequences are shown, with asterisks indicating stops.
Figure 3. Transcripts and partial genomic structure of human RECQ5. (A) The structures of representative cDNAs carrying RECQ5 sequences are shown. Protein-coding
regions are shaded; black boxes indicate the seven helicase motifs. Known intron positions are indicated, but other intron positions were not determined. The third
intron depicted is ~2.1 kb. The fourth intron depicted is the one not removed in class B transcripts. The uppermost schematic represents our 1710 bp cDNA, and
others represent I.M.A.G.E. Consortium cDNAs (Table 1). A vertical line indicates that the cDNA was probably incomplete at that end. A sigmoid line indicates
that the cDNA continues for an undetermined distance (these clones were not yet available for analysis). The lower three cDNAs appear to be fusions between
RECQ5 at and the 3' end of another gene (hatched boxes).
Nucleic Acids Research, 1999, Vol. 27, No. 18 3767
ends to bp 1113 (of our sequence). At this junction, novel
sequence begins, extending 332 and 396 bp, respectively, to a
polyadenylation tract. The novel sequence begins with GT,
suggesting that these cDNAs might correspond to transcripts in
which termination and polyadenylation occurred within an
intron. PCR from human genomic DNA using primer pairs
spanning the junction point yielded the fragment predicted
from these two EST sequences, indicating that the sequences
across the junction point are contiguous in the genome. Hence,
there is an intron of ~2 kb between the regions encoding helicase
motifs V and VI; transcripts in class C terminate within this
intron. These transcripts encode a 36 kDa protein that lacks
motif VI as well as additional sequences conserved among
RecQ family members.
Transcript class D is represented by I.M.A.G.E. Consortium
CloneIDs 1914385, 2316345 and 2191126 (Table 1 and Fig. 3).
These transcripts appear to be hybrids between RECQ5 and
another gene. They each have RECQ5 sequences at their 5'
ends and another sequence at their 3' ends. CloneID 1914385
has RECQ5 sequence through 1034 (of RECQ5), followed by
191 bp of sequence that is identical to the 3' ends of a group of
19 other EST sequences. These other EST sequence reads,
which are up to 601 bp in length, are identical to one another
along their entire lengths, and none of this sequence is from
RECQ5, even in cases where 5' EST reads are available. Thus,
these cDNAs are probably derived from an independent gene,
which we will refer to as gene D.
point is at 1229 in RECQ5, and there are only 51 bp of the 3'
end of gene D. The insert in 2191126 also appears to be a
fusion between RECQ5and gene D. This insert is in a rearranged
orientation (Materials and Methods) and is lacking a polyadenyl-
ation tract at the 3' end, so it likely represents an internal
fragment of a cDNA. The 3'-most sequences from this insert
overlap the 5' end of CloneID 380349, whose 3' end identifies
it as a cDNA from gene D. Thus, 2191126 appears to identify
a fusion point more 5' in gene D.
RECQ5 is expressed in many tissues
To determine the relative abundances and tissue distribution of
the different RECQ5 transcripts, we probed blots of human
mRNA. We detected a 1.7 kilonucleotide (knt) transcript at a
low level in all tissues examined (Fig. 4). If our RACE products
correspond to transcripts identical at the 5' end to the 1710 bp
cDNA we sequenced, as is suggested by the published RECQ5
sequence, these transcripts wouldbe 2828and 3646nt (excluding
polyadenylation tracts). However, we were unable to detect
these larger transcripts using either probes within the region in
common to all transcripts or probes specific to the larger tran-
scripts. This was true even for placenta mRNA, which was the
source of our RACE products. In contrast, Kitao et al. (25)
detected transcript sizes of 3.6 and 3.8 knt for RECQ5, also
present in all tissues examined. However, these authors did not
report any signals corresponding to smaller transcripts.
Drosophila RECQ5 localizes to the nucleus
Saccharomyces cerevisiae Sgs1p and human WRN are both
found to be concentrated in the nucleolus (31,32). To determine
whether Drosophila RECQ5 shows a similar pattern of sub-
tagged with an N-terminal FLAG epitope, in Drosophila
Schneider2 cells. When cells were stained with anti-FLAG
antibody to detect FLAG-RECQ5, we detected localization to
thenucleus,but nosub-nuclearlocalizationwas apparent (Fig.5).
We have described a new D.melanogaster RecQ helicase family
member that shares a high degree of amino acid identity with
human RecQ5. A unique feature of the Drosophila gene is the
existence of isoforms that differ by the presence or absence of
a highly charged C-terminus. Most other eukaryotic family
members have regions of a similar size on one or both sides of
the helicase region, but the functions of these regions are
largely unknown. Perhaps the best understood case is WRN,
which has a recognizable exonuclease motif at its N-terminus
(33). This region has been demonstrated to possess a 3' to 5'
exonuclease activity in vitro, and this activity is separable from
the helicase activity (34). The region N-terminal to the helicase
region of Sgs1p has been shown to interact with the C-terminal
Figure 4. Expression of human RECQ5. A blot containing human mRNA
(OriGene Technologies) was probed for RECQ5 using sequences near the 3'
end of transcript A. The probe used should detect transcripts A and B, but not
C and D. RNA size markers are indicated to the left. The sources of mRNA are
indicated above each lane.
Figure 5. Immunolocalization of Drosophila RECQ5 to the nucleus. (A) DAPI
stainingofafieldofcells transfectedwiththeUbiq::FLAG-RecQ5 construct.Four
nuclei can be seen in their entirety. (B) The same field stained with anti-FLAG
antibody. Two cells can be seen to express FLAG-RecQ5. In both cells the protein
is strongly localized to the nucleus.
3768 Nucleic Acids Research, 1999, Vol. 27, No. 18
portion of topoisomerase II in a yeast two-hybrid assay (18). It
is possible that the highly charged, C-terminal region specific
to the 121 kDa isoform of Drosophila RECQ5 interacts with
other proteins, and that the difference between the isoforms is
a way of regulating these interactions. The existence of different
Drosophila RecQ5 isoforms provides a unique opportunity to
study the in vivo functions of these sequences outside the core
RecQ-related region. Such dramatically different isoforms
have not been reported for any other family members. It is
interesting to note that most family members have a dibasic
(RR) sequence at the C-terminus of the RecQ-conserved
region, suggesting the possibility of proteolytic processing at
Alternative splicing also generates different isoforms of
human RecQ5. In one case, the isoform produced depends on
whether or not an intron is removed, as was observed for the
Drosophila gene. The size difference between the 49 and
46 kDa RecQ5 isoforms, however, is much less than the differ-
ence between the Drosophila isoforms. These two human
RecQ5 isoforms carry all of the sequences conserved among
RecQ family members, and therefore both may have helicase
activity. Nonetheless, it is possible that these different isoforms
possess different biochemical activities or different capacities
to interact with other cellular proteins that may regulate activity.
Another possibility is that the different C-termini regulate
protein stability or sub-cellular localization. The gene encoding
murine RecQL is alternatively spliced in the testis to remove a
short region believed to contain a nuclear localization signal
(35). The only recognizable nuclear localization signal in human
RecQ5 is the sequence PERRVRS, which begins at residue 12.
All predicted isoforms of Drosophila RECQ5 have at least one
potential nuclear localization sequence (Fig. 2A, arrows). Con-
sistent with this observation is our finding that at least the
larger isoform localizes to the nucleus when expressed in a
Drosophila cell line. It seems unlikely that the function of
Drosophila RECQ5 alternative splicing is to regulate nuclear
localization, but it is possible that this is the case for human
A third human RecQ5 isoform is produced by transcripts in
class C, in which polyadenylation occurs 5' to the sequences
encoding the last helicase motif. This 36 kDa protein lacks
both helicase motif VI and additional sequences conserved
among RecQ helicases, and likely has no catalytic function.
Transcripts in this class may be important for regulating
Class D transcripts are somewhat perplexing. They appear to
be fusions between RECQ5 and another gene. Such fusions
could have occurred in the DNA (e.g. by chromosome rearrange-
ment), the RNA (e.g. by aberrant splicing), or the cDNA (e.g. by
cloning artifacts).The three fusioncDNAs came from different
libraries, andtheyhavedifferent fusionpoints inbothgenes,so
it seems unlikely that these represent cloning artifacts. It is
possible that these transcripts result from aberrant splicing.
The fusion point in 2191126 is at a RECQ5 exon boundary.
However, the fusionpoint of 1914385 is not at anexonboundary.
Either chromosome rearrangements or aberrant splicing could
therefore account for these transcripts. Conceptual translation
of available sequences from this second gene does not reveal
any similarities to other proteins in the databases.
We used radiation hybrid mapping to map human RECQ5 to
of Kitao et al. Loss of heterozygosity in this region has been
associated with ovarian cancer and familial breast carcinomas
(36–40). Given the phenotypes associated with mutations in
other RecQ helicase family members, human RECQ5 may be a
good candidate for a tumor suppressor gene.
We mapped Drosophila RecQ5 to polytene region 70E1–4.
We were unable to identify probable candidates for mutations
in RecQ5 among known mutations in the region. Efforts are
now underway to generate mutations in this gene. Preliminary
results suggest that Drosophila RecQ5 is not an essential gene.
Analysis of these mutations will provide valuable insights into
the function of this gene in Drosophila, and perhaps other
organisms, and into the functional significance of the different
We thank Ken Burtis and Sarah Wayson for helpful suggestions,
Elaine Kwan for assistance with cell culture experiments, and
Todd Laverty for the in situ hybridization to polytene chromo-
somes. J.J.S. was supported in part by a fellowship from the
Cancer Research Fund of the DamonRunyon,Walter Winchell
Foundation, DRG 1355.
1. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and
Mutagenesis. American Society for Microbiology, Washington, DC.
2. Ellis,N.A., Groden,J., Ye,T.-Y., Straughen,J., Lennon,D.J., Ciocci,S.,
Proytcheva,M. and German,J. (1995) Cell, 83, 655–666.
3. Yu,C.-E., Oshima,J., Fu,Y.-H., Wijsman,E.M., Hisama,F., Alisch,R.,
Metthews,S., Nakura,J., Miki,T., Ouais,S., Martin,G.M., Mulligan,J. and
Schellenberg,G.D. (1996) Science, 272, 258–262.
4. Kitao,S., Shimamoto,A., Goto,M., Miller,R.W., Smithson,W.A.,
Lindor,N.M. and Furuichi,Y. (1999) Nature Genet., 22, 82–84.
5. German,J. (1993) Medicine, 72, 393–406.
6. Goto,M., Horiuchi,Y., Tanimoto,K., Ishii,T. and Nakashima,H. (1978)
J. Am. Geriatr. Soc., 26, 341–347.
7. Goto,M., Tanimoto,K., Horiuchi,Y. and Sasazuki,T. (1981) Clin. Genet.,
8. Vennos,E.M.,Collins,M. andJames,W.D.(1992)J.Am.Acad. Dermatol.,
9. Chaganti,R.S., Schonberg,S. and German,J. (1974) Proc. Natl Acad. Sci.
USA, 71, 4508–4512.
10. German,J., Schonberg,S., Louie,E. and Chaganti,R.S. (1977) Am. J. Hum.
Genet., 29, 248–255.
(1984) Am. J. Hum. Genet., 36, 387–397.
12. Scappaticci,S., Forabosco,A., Borroni,G., Orecchia,G. and Fraccaro,M.
(1990) Ann. Genet., 33, 5–8.
13. Lindor,N.M., Devries,E.M., Michels,V.V., Schad,C.R., Jalal,S.M.,
Donovan,K.M., Smithson,W.A., Kvols,L.K., Thibodeau,S.N. and
Dewald,G.W. (1996) Clin. Genet., 49, 124–129.
14. Miozzo,M., Castorina,P., Riva,P., Dalpra,L., Fuhrman Conti,A.M.,
Volpi,L., Hoe,T.S., Khoo,A., Wiegant,J., Rosenberg,C. and Larizza,L.
(1998) Int. J. Cancer, 77, 504–510.
15. Hanada,K., Ukita,T., Kohno,Y., Saito,K., Kato,J.-I. and Ikeda,H. (1997)
Proc. Natl Acad. Sci. USA, 94, 3860–3865.
16. Harmon,F.G. and Kowalczykowski,S.C. (1998) Genes Dev., 12,
17. Lovett,S.T. and Sutera,V.A.J. (1995) Genetics, 140, 27–45.
18. Watt,P.M., Louis,E.J., Borts,R.H. and Hickson,I.D. (1995) Cell, 81,
19. Murray,J.M., Lindsay,H.D., Munday,C.A. and Carr,A.M. (1997)
Mol. Cell. Biol., 17, 6868–6875.
20. Stewart,E., Chapman,C.R., Al-Khodairy,F., Carr,A.M. and Enoch,T.
(1997) EMBO J., 16, 2682–2692.
Nucleic Acids Research, 1999, Vol. 27, No. 18 3769
21. Watt,P.M., Hickson,I.D., Borts,R.H. and Louis,E.J. (1996) Genetics, 144,
22. Gangloff,S., McDonald,J.P., Bendixen,C., Arthur,L. and Rothstein,R.
(1994) Mol. Cell. Biol., 14, 8391–8398.
Ozawa,K., Eki,T., Nogami,M., Okumura,K., Taguchi,H., Hanaoka,F. and
Enomoto,T. (1994) Nucleic Acids Res., 22, 4566–4573.
24. Puranam,K.L. and Blackshear,P.J. (1994) J. Biol. Chem., 269,
(1998) Genomics, 54, 443–452.
26. Kusano,K., Berres,M.E. and Engels,W.R. (1999) Genetics, 151,
27. Brown,N.H. and Kafatos,F.C. (1988) J. Mol. Biol., 203, 425–437.
28. Harvey,D., Hong,L., Evans-Holm,M., Pendleton,J., Su,C., Brokstein,P.,
Lewis,S. and Rubin,G.M. (1997) The BDGP/HHMI Drosophila EST
29. Lennon,G.G., Auffray,C., Polymeropoulos,M. and Soares,M.B. (1996)
Genomics, 33, 151–152.
30. Gorbalenya,A.E., Koonin,E.V., Donchenko,A.P. and Blinov,V.M. (1989)
Nucleic Acids Res., 17, 4713–4730.
31. Sinclair,D.A., Mills,K. and Guarente,L. (1997) Science, 277, 1313–1316.
32. Marciniak,R.A., Lombard,D.B., Johnson,F.B. and Guarente,L. (1998)
Proc. Natl Acad. Sci. USA, 95, 6887–6892.
33. Mushegian,A.R., Bassett,D.E., Boguski,M.S., Bork,P. and Koonin,E.V.
(1997) Proc. Natl Acad. Sci. USA, 94, 5831–5836.
34. Huang,S., Li,B., Gray,M.D., Oshima,J., Mian,I.S. and Campisi,J. (1998)
Nature Genet., 20, 114–116.
35. Wang,W.S., Seki,M., Yamaoka,T., Seki,T., Tada,S., Katada,T.,
Fujimoto,H. and Enomoto,T. (1998) Biochim. Biophys. Acta, 1443,
36. Eccles,D.M., Russell,S.E.H., Haites,N.E., Atkinson,R., Bell,D.W.,
Gruber,L., Hickey,I., Kelly,K., Kitchener,H. and Leonard,R. (1992)
Oncogene, 7, 2069–2072.
37. Lindblom,A., Skoog,L., Andersen,T.I., Rotstein,S., Nordenskjold,M. and
Larsson,C. (1993) Hum. Genet., 91, 6–12.
38. Cornelis,R.S., Devilee,P., van Vliet,M., Kuipers-Deijkshoorn,N.,
Kersenmaeker,A., Bardoel,A., Meera Khan,P. and Cornelisse,C.J.
(1993) Oncogene, 8, 781–785.
39. Cropp,C.S., Champeme,M.-H., Lidereau,R. and Callahan,R. (1993)
Cancer Res., 53, 5617–5619.
40. Plummer,S.J., Paris,M.J., Myles,J., Tubbs,R., Crowe,J. and Casey,G.
(1997) Cancer Genet. Cytogenet., 20, 354–362.