A family of Drosophila genes encoding quaking-related maxi-KH domains

A Family of Drosophila Genes Encodingquaking-Related Maxi-KH Domains

Christine Fyrberg,1 Jodi Becker,1 Peter Barthmaier,1 James Mahaffey,2

and Eric Fyrberg1,3

Received 4 Nov. 1997Ð Final 16 Dec. 1997

We recently identi® ed a Drosophila gene, wings held out (who), that speci® es aSTAR (signal transduction and RNA activation) protein expressed within meso-derm and muscles. Genetic evidence suggests that WHO regulates muscledevelopment and function in response to steroid hormone titer. who is related tothe mouse quaking gene, essential for embryogenesis and neural myelination, andgld-1, a nematode tumor suppressor gene necessary for oocyte differentiation,both of which contain RNA binding `̀ maxi-KH’’ domains presumed to link RNAmetabolism to cell signaling. To initiate a broader study of Drosophila WHO-related proteins we used degenerate primers encoding peptides unique to maxi-KHdomains to amplify the corresponding genes. We recovered nine genes, allspecifying single maxi-KH domain proteins having tripartite regions of similaritythat extend over 200 amino acids. One is located within the 54D chromosomesubdivision, and one within 58C, while the remaining seven are within the 58Esubdivision. At least four of these STAR proteins are expressed in a generalmanner, suggesting that maxi-KH domains are employed widely in Drosophila.

KEY WORDS: Drosophila genetics; K homology domains; STAR proteins; nucleic acid-binding motifs.

INTRODUCTION

The K homology (KH) domain is present within a variety of proteins. Described

originally as a triple repeat within the hnRNP K protein primary sequence (Siomi

1 Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore,

Maryland 21218.2

Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695.3

To whom correspondence should be addressed. Fax: (410) 516-5213. e-mail: [email protected].

Biochemical Genetics, Vol. 36, Nos. 1/2, 1998

51

0006-2928/98/0200-00 51$15.00/0 r 1998 Plenum Publishing Corporation

et al., 1993a), single or multiple copies of the motif have been found in

polypeptides ranging from ribonuclease PNP of Escherichia coli (Regnier et al.,

1987) to human FMR1, encoded by the fragile-X mental retardation gene (Siomi

et al., 1993b). The common property of all proteins containing KH domains is

affinity for RNA or single-stranded DNA. For example, E. coli protein Nus A

binds to newly transcribed mRNA (Liu and Hanna, 1995), while the KH domain

of the prokaryotic ribosomal protein S3 is associated with ribosomally bound

mRNA (Urlaub et al., 1995). At least two single KH domain-containing proteins,

both having a preference for poly(rC), have been demonstrated to bind nucleic

acid in vitro (Ito et al., 1994; Leffers et al., 1995). Investigations of the

three-dimensional solution structure of KH domains, speci® cally of the sixth KH

domain of vigilin, an endoplasmic reticulum protein that contacts free ribosomes

(Schmidt et al., 1992; Musco et al., 1996), have established that the motif is

typically 80±100 residues in length and revealed its overall topology as b a a b b a ,

with RNA localized between the ® rst two a helices (Musco et al., 1996).

While it is reasonably well established that KH proteins typically bind RNA

or single-stranded DNA, neither the scope of their biochemical roles nor the

functions of individual proteins in particular cellular and developmental events

are known in satisfactory detail. In Drosophila melanogaster, several investiga-

tions have de® ned the functions of particular KH domain proteins in part. For

example, Drosophila protein PS1 represses the maturation of P element RNA

transcripts in somatic cells, preventing the removal of the third intron sequence by

attaching to the exon located immediately 58 to it (Siebel et al., 1995). Another ¯ y

protein, BICAUDAL C, expressed in the female germline, speci® es the anterior of

the oocyte (Mohler and Wieschaus, 1986; Schupbach and Wieschaus, 1991). KH

domain mutations of BICAUDAL C profoundly disrupt embryo polarity, causing

improper localization of follicle cells and faulty development of anterior struc-

tures (Mahone et al., 1995), although the mechanism by which disruption of RNA

binding creates these defects is not yet understood.

We and others recently discovered that a Drosophila KH domain protein

similar to those encoded by the mouse quaking and nematode gld-1 genes is

involved in muscle development (Baehrecke, 1997; Zaffran et al., 1997; Fyrberg

et al., 1997; Lo and Frasch, 1997). Transcripts of the gene, named wings held-out,

or who (aliases how, qkr93F, and struthio), accumulate in nuclei of muscles and

epithelial tendon cells, suggesting that WHO regulates gene activity in muscles as

well as in cells to which they attach. Synthesis of WHO is ecdysone dependent,

and mutations within the protein perturb muscle development and function,

suggesting that the gene coordinates myotube migration and patterning in

response to the titer of the steroid hormone ecdysone (Baehrecke, 1997). The

WHO KH domain closely resembles those of the SAM68 (Src-associated in

mitosis; 68-kD) family of proteins (Vernet and Artzt, 1997). The amino acid

52 Fyrberg, Becker, Barthmaier, Mahaffey, and Fyrberg

similarity of this protein family extends beyond the borders of the KH domain,

implying that the shared function may entail more than nucleic acid binding. The

prototype of this group, SAM68, is a phosphoprotei n target of SRC and FYN

signal transduction in mitotic cells (Taylor and Shalloway, 1994), hence the

suggestion that related proteins may also be part of signal transduction cascades

(Ebersole et al., 1996) and the recent naming of this protein family STAR (signal

transduction and activation of RNA (Vernet and Artzt, 1997).

As a step toward evaluating these ideas we have attempted to isolate

Drosophila genes encoding maxi-KH domain proteins using structural and

sequence information generated in studies of mouse quaking and Sam68 and

Drosophila who. Using degenerate PCR primers encoding peptides that are

unique to WHO and other members of the STAR protein family, we have

succeeded in isolating nine related Drosophila genes. Here we present their partial

characterization.

MATERIALS AND METHODS

Ampli® cation and Isolation of Related Genes

By comparing the primary sequences of the mouse quaking gene and several

relatives with that of the Drosophila wings held out (who), aliases held out wings

(how), and quaking-related gene at 93F (qkr93F) (Baehrecke, 1997; Zaffran et al.,

1997; Fyrberg et al., 1997), we were able to identify similar regions and construct

degenerate oligonucleotides for amplifying related Drosophila genes. The only

regions chosen for this endeavor were portions of the KH domain that are unique

to maxi-KH domain proteins. We utilized two degenerate overlapping forward

primers specifying the peptides DHPDFNFV (GGAATTCg/cAg/cc/tAc/t

CCXGAc/tTTc/tAAc/tTTc/tGT, where lowercase letters designate degenerate

nucleotides) and PDFNFVG, (GGAATTCCCXGAc/t TTc/tAAc/tTTc/tGTXGG),

respectively, and also encoding an EcoRI endonuclease site, in conjunction with

two degenerate reverse primers. The ® rst speci® es the peptide GKPNWEHL

(GGGGATCCg/aTGc/tTCCCAg/aTTXGGc/tTTXCC) and includes a BamHI site,

while the second speci® es SMRDKKK (GGGGATCCc/tTCc/tTTc/tTTc/tTTa/

gTCXCg/tCAT) and includes the same endonuclease site. We designed our

degenerate primers in accordance with codon bias characteristic of Drosophila.

After annealing primers to genomic Drosophila DNA, or reverse-transcribed

RNA at 45ÊC, we ampli® ed products for 30 cycles and selected those having the

expected electrophoretic mobilities. PCR products were subcloned into the

pUC19 vector and sequenced by the dideoxy-extension method (Sanger et al.,

1977).

quaking-Related KH Domains 53

Gene and cDNA Isolation

Fragments ampli® ed using degenerate primers in conjunction with the PCR were

used to screen either genomic libraries or cDNA populations representing various

stages of the Drosophila life cycle (Poole et al., 1985).

DNA Sequencing

All DNA sequences were generated using the dideoxy-extension method (Sanger

et al., 1977). In all instances we sequenced both strands of genes in an

overlapping manner.

Protein Sequence Comparisons

Sequence relatedness was analyzed and visualized using the TFASTA program

and aligned using the Pileup and Pretty programs of the UWGCG package

(Devereux et al., 1984). Highlighting was done using the SEQVU program

(Garvan Institute, Sydney, Australia).

Chromosomal Localization of KH Domain-Containing Genes

We prepared larval polytene chromosomes by the technique of Gall and Pardue

(1971). 3H-Labeled cRNA was synthesized using the protocol of Wensink et al.

(1974). Hybridization RNA and visualization of labeled regions were as described

by Fyrberg et al. (1980).

RNA Localization

Embryos for whole-mount in situ localization were dechorionated and ® xed

following the procedure of Tautz and Pfei¯ e (1989). In situ analysis utilized

ribonucleotide probes generated using an RNA transcription kit (Stratagene) and

DIG-11-UTP (Boehringer-Mannheim). cDNAs for each of the KH domain genes

were cloned in Bluescript vectors, and digoxygenin-labeled transcripts prepared

by incubating linearized template DNA with either T3 or T7 polymerase.

Hybridization was carried out using the method of Tautz and Pfei¯ e (1989) with

only minor modi® cations. Anti-DIG-AP (Boehringer-Mannheim) was used to

detect hybridization .

Northern Blotting

We extracted poly(A)-containing RNA from synchronously developing Dro-

sophila cultures using the SDS±phenol technique and oligo(d)T chromatography.


RNA preparations were separated by electrophoresis in 20 mM sodium acetate, 1

mM EDTA. For details of RNA transfer and hybridization , refer to Fyrberg and

colleagues (1983).

RESULTS

PCR Ampli® cation of quaking -Related Drosophila Genes

Our ® rst step in this investigation was to amplify Drosophila genes encoding

proteins closely related to quaking and other members of the SAM68 family. This

task was facilitated by recent sequence comparisons of maxi-KH domain-

containing proteins (see, e.g., Jones and Schedl, 1995; Ebersole et al., 1996;

Musco et al., 1996) and, in particular, by structural analysis of the sixth KH repeat

of human vigilin (Musco et al., 1996), which revealed that its topology is

b a a b b a . The protein sequence alignments, together with the structural character-

istics, demonstrate that mouse QK, nematode GLD-1, and other maxi-KH domain

proteins share two short sequences that are absent from other proteins of this type,

the ® rst being a seven-amino acid insertion preceding helix a 1, and the second

comprising a ¯ exible loop of 16±20 amino acids located between domain b 2 and

domain b 3. Utilizing this information, we were able to design two pairs of

oligonucleotide primers that would anneal uniquely to cDNAs encoding maxi-KH

domain proteins. The two forward primers chosen encode the overlapping

peptides DHPDFNFV (F1) and PDFNFVG (F2), both of which are part of the

seven amino acid insertion. The reverse primers specify the nonoverlapping

peptides GKPNWEHL (R1) and SMRDKKK (R2), both of which are part of the

¯ exible loop. Locations of these peptides relative to primary sequences of human

vigilin, Drosophila WHO, and additional STAR proteins are illustrated in Fig. 1.

Hybridization of the respective forward and reverse primer pairs to cDNA

representing several Drosophila developmental stages, together with the polymer-

ase chain reaction, allowed us to synthesize fragments having the expected 150-

and 200-nucleotide sizes.

To ascertain how many distinct cDNA sequences the ampli® ed fragments

represented, we ligated the population of molecules into the plasmid vector

pUC19, grew up individual transformants, prepared DNA from each, and deter-

mined their nucleotide sequences. After analyzing approximately 100 sequences

in this manner, we established that our PCR ampli® cation represented, in the

main, four distinct cDNAs and one genomic clone for which we were unable to

obtain a cDNA (and which will not be further described). We sequenced

fragments for each completely and used these as probes to isolate complete

genomic and cDNA sequences. Insofar as we are aware, expressed sequence tags

are not available for any of these genes.


Primary Sequence Comparisons

Alignment of the conceptual protein translations derived from full length cDNAs

for the four genes using the SEQVU program, illustrated in Fig. 2, demonstrated

that all of the primary sequences share similar tripartite regions extending for

approximately 200 amino acids, a hallmark of STAR proteins (Vernet and Artzt,

1997). The most similar portion of the conserved sequence encodes the KH

domain. Forty-® ve percent of the requisite 100 amino acids are identical in all

Fig. 1. Locations of peptides unique to Drosophila WHO and maxi-KH domain proteins. The® gure illustrates computer-generated alignments of KH domains from mammalian Vigilin,

SAM68, QUAKING, Drosophila WHO (alias QKR93F), and brine shrimp GRP33. Vigilincontains multiple `̀ conventional’ ’ KH domains (that illustrated is repeat number six, which is 68residues in length), while the latter four proteins contain single `̀ maxi’ ’ -KH domains of 100 or

102 residues. The length differences are accounted for by two peptide insertions. Locations withinthem of the sequences encoded by our degenerate PCR primers, designated forward1 (F1), F2,

reverse1 (R1), and R2, are indicated.

Fig. 2. Tripartite sequences encoded by four Drosophila quaking -related cDNAs. The ® gure illus-trates computer-generated alignments of sequences encoded by four Drosophila quaking -relatedcDNAs. The most similar portions specify the KH domains, indicated in dark gray. Also signi® cant is

the similarity of the QUA1 (residues 47±111 of Qkr58E-1) and QUA2 (residues 214±306 ofQkr58E-1) domains, indicated in light gray. The remainder of the protein sequences include only

limited similarity, indicated by unshaded boxes.



four proteins, and all four conform to the structural characteristic of `̀ maxi KH’ ’

domains.

Amino acid similarity was not con® ned to the RNA binding motifs, and we

noted additional regions of protein similarity located on either side of the KH

domain. Vernet and Artzt (1997) have named these ¯ anking domains QUA1 and

QUA2. These sequences, indicated in light gray (Fig. 2), display signi® cant

similarity, albeit far less than that characteristic of the KH domains. The QUA1

domains (residues 47±111 of QKR58E-1) have only 9 of 67 residues identical in

all four proteins, while we observe only 7 of 95 identical residues in the QUA2

domains (residues 214±306 of QKR58E-1). If our quali® er was three of four

identical residues, then we obtained `̀ matches’ ’ of 24 of 67 residues in QUA1 and

15 of 95 residues in QUA2. Outside of the tripartite domains we observed little

protein similarity, although two carboxy-terminal stretches collectively included

® ve positions wherein residues are identical in three of the four proteins. Finally,

we failed to note any compelling similarities of these proteins to those established

to be involved in signal transduction, for example, SAM68.

A Closely Related quaking -Related Gene Subfamily

In the course of screening genomic and cDNA libraries with qkr58E-3, we

discovered four additional maxi-KH domain genes, all of which are extremely

closely related to qkr58E-3. Figure 3 illustrates alignments of the KH domains of

all ® ve of these genes, demonstrating their similarity. Isolation and sequencing of

corresponding genomic fragments have demonstrated that these indeed represent

four genes distinct from one another and from qkr58E-3. Together with the ® ve

genes described above, this brings the total number of Drosophila maxi-KH

domain genes isolated in this study to nine.

Chromosome Locations

To facilitate genetic approaches to KH domain protein function, we determined

the chromosomal location of each of our genes. We synthesized tritium-labeled

cRNA probes representing each according to the protocol of Wensink et al. (1974)

and hybridized these to polytene chromosomes using the technique of Gall and

Pardue (1971). We found that all of the genes are located on chromosome 2. One,

qkr54B, is located within the 54B subdivision; one, qkr58C, is located within the

58C subdivision; and seven, qkr58E1-7, are distributed within the 58E subdivi-

sion. In only one instance have we delineated the spatial relationship of individual

58E subdivision genes. qkr58E-2 and qkr58E-3 are topologically organized as

shown in Fig. 4. The physical map of a third 58E region gene, qkr58E-1, is known

as well, but its relationship to the remaining six genes within the same subdivision


is not. Both the physical maps and the spatial arrangements of the remaining ® ve

58E subdivision genes remain to be derived.

Physical Maps

To re® ne further the ability to identify genetic mutations within the four quaking-

related genes described in Fig. 2, we have generated physical maps for the four

originally isolated. We used the respective full-length cDNAs as probes to screen

genomic libraries for overlapping fragments that contained complete gene copies.

By hybridizing these to the cDNA probes and sequencing the hybridizing

fragments, we were able to generate maps of the locations of all exons and introns.

These are summarized in Fig. 4. Using this information it will be possible to

screen extant Drosophila strains for corresponding mutations. We noted also, in

the course of comparing sequences and exon locations (refer to Table I), that some

intron positions are conserved between genes, strongly suggesting evolution from

an ancestral gene.

Gene Expression Patterns

To investigate temporal and spatial patterns of Drosophila quaking-related gene

expression, we synthesized digoxygenin-la beled probes for qkr54B and qkr58E-1,

Fig. 3. Comparisons of KH domains of Qkr58E-3 and four closely related genes. The ® gureillustrates alignment of KH domains encoded by these ® ve very closely related single maxi-KH

domain Drosophila genes. The marked similarity of the RNA binding motifs may imply that theproteins have similar roles.


-2, and -3. We hybridized these to embryo whole mounts prepared as described by

Tautz and Pfeifel (1989). We expected, on the basis of prior investigation of

mouse quaking, which is expressed speci® cally in the heart and nervous system,

and Drosophila who, expressed uniquely in muscles and their attachment points,

that a majority of these genes would be expressed in a tissue-speci® c manner.

However, on the basis of both whole mounts and RNA blotting experiments, the

Fig. 4. Physical maps of four Drosophila quaking -related genes. Theschematic diagrams illustrate the structures of the four quaking -relatedDrosophila genes that encode protein sequences summarized in Fig. 2.

Filled blocks represent translated exons, while open block representsuntranslated exons. Introns are represented by lines between blocks.

Table I. Codons Contained Within Particular Drosophila quaking -Related Gene Exonsa

Exon qkr58E-1 qkr58E-2 qkr58E-3 qkr54B

1 1±85 1±99 1±52 1±91

2 86±253 100±300 53±93 92±1333 254±288 301±333 94±215 134±284

4 289±348 334±372 216±245 285±3475 349±396 246±273 348±425

6 274±317

aThe contiguous codons, numbered according to Fig. 2, comprise each exon in the four quaking -re-

lated genes.


four novel genes appear to be expressed fairly generally. Relative to our control

preparations, we noted signi® cant hybridization in each case, but the intensity is

distributed fairly uniformly throughout diverse tissues and organs, rather than

being sharply localized, as in the case of Drosophila who (data not shown). This

observation was corroborated by hybridization of the same probes to RNA blots

representing embryonic, larval, pupal, and adult developmental stages. All four

probes hybridized at more or less the same intensity to all RNA preparations, a

result expected for genes expressed at low but signi® cant levels in a variety of

tissues (data not shown).

Cladogram

We ® nally examined the evolutionary relatedness of the proteins displayed in Fig.

2 to Drosophila WHO, mouse QK and SAM68, and brine shrimp GRP33

(Cruz-Alvarez and Pellicer, 1987). This work con® rms that Drosophila WHO

(alias QKR93F) is similar to mouse QUAKING in spite of their distinct develop-

mental roles (Fig. 5). The four novel Drosophila proteins described herein, while

conforming to the tripartite structure characteristic of both QUAKING and

SAM68, are more similar to one another than to SAM68, GRP33, QUAKING, or

GLD-1. This observation suggests that the number and diversity of proteins

Fig. 5. Evolutionary relatedness of predicted

proteins. The cladogram illustrated the evolution-ary distances between mouse QUAKING(QK), Drosophila WHO (QKR93F), C. el-egans GLD-1, mouse SAM68, brine shrimpGRP33, and the four novel Drosophila se-

quences reported in this paper. Note that thenew Drosophila proteins are not especiallyclosely related to any of the above listed

proteins, although all have considerable simi-larity.


containing single maxi-KH domains may be considerably greater than has been

documented previously.

DISCUSSION

In this paper we report the discovery of nine Drosophila genes that contain single

maxi-KH domains isolated using structural and sequence information derived

from Drosophila WHO, mouse QUAKING, and their relatives. These novel

maxi-KH domain-containing proteins, or STAR proteins (Ebersole et al., 1996;

Vernet and Artzt, 1997), warrant the interest of biologists for at least four reasons.

First, all are presumed to be capable of binding RNA and, as such, probably

modulate some aspect of temporal and spatially programmed gene expression

(Musco et al., 1996; Vernet and Artzt, 1997). Second, failures of muscles, oocytes,

and neurons to differentiate have been correlated with gene mutations in Dro-

sophila who, C. elegans gld-1, and mouse quaking, respectively (Baehrecke,

1997; Zaffran et al., 1997; Jones and Schedl, 1995; Ebersole et al., 1996). Third,

proteins speci® ed by all of the above-named genes are closely related to SAM68,

a phosphoprote in target for SRC and FYN that is believed to couple signal

transduction with cell cycle-mediated gene expression, and in that manner to

regulate mitosis. Fourth, and ® nally, in at least some cases particular STAR

protein sequences are conserved over large evolutionary distances. For example,

mammalian splicing factor1 (SF1) appears to be the homologue of S. cerevisiae

branch point bridging protein (Bbp1p), an essential splicing factor in yeast

(Vernet and Artzt, 1997).

In this work we sought to assess the size and diversity of Drosophila proteins

having single maxi-KH domains. To achieve this we designed degenerate oligo-

nucleotides that recognize gene regions that encode unique portions of maxi-KH

domains. Analyses of the resultant PCR products, and additional genomic and

cDNA screens, yielded a total of 9 new genes, which, added to who, give a total of

at least 10 Drosophila genes encoding maxi-KH domains. We have no evidence

that our biochemical screen is exhaustive, and design of additional oligonucleo-

tides may yield yet more genes. All that we are able to state de® nitively at this

time is that maxi-KH domain proteins are not rare in Drosophila.

The similar tripartite arrangements and extensive sequence conservation

suggest that all of the maxi-KH domain proteins are performing analogous

functions, although precisely what that role is for any such protein has yet to be

established. In every case where we have determined the complete sequence of

these maxi-KH domain proteins, we have found that they consist of the RNA

binding and two ¯ anking motifs. As yet, we have found no extensive similarities

between these novel Drosophila proteins and SAM68 that lead us to believe that

any are signal transduction proteins. Evidence that the functional roles may be

diverse is, however, suggested by the distinct patterns of gene expression. For


example, Drosophila WHO (alias QKR93F) is unique to muscles and the cells to

which they attach (Baehrecke, 1997; Zaffran et al., 1997; Fyrberg et al., 1997),

whereas most of the proteins described in this study appear to be expressed

ubiquitously, while mouse quaking is expressed only in the mouse heart and

nervous system (Ebersole et al., 1996). Resolution of this issue will come only

after more extensive studies of these proteins and the genes that specify them.

Perhaps the greatest progress in delineating the roles of maxi-KH domain

proteins has come through analyses of mutant alleles of quaking, gld-1, and who

(Ebersole et al., 1996; Jones and Schedl, 1995; Baehrecke, 1997; Zaffran et al.,

1997). It is our hope that the information provided in this paper, together with the

ability to utilize cell biological and genetic techniques in Drosophila, will

considerably improve opportunities for further de® ning the functions of these

proteins.

ACKNOWLEDGMENTS

This work was supported by grants from the NIH and MDA to E.A.F. and by a

grant from the NSF to J.W.M. We thank Drs. Corine Vernet, Karen Artzt, and Eric

Baehrecke for discussions and for communicating data prior to their publication.

Sequences described herein have been ® led in the Genbank Database under

accession numbers AF038581, AF038582, AF038583, and AF038584.

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