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Genetics. 2004 Dec; 168(4): 2059–2065.
doi: 10.1534/genetics.104.032532
PMCID: PMC1448718
PMID: 15611175

The Drosophila U1-70K Protein Is Required for Viability, but Its Arginine-Rich Domain Is Dispensable

Helen K. Salz,*,1 Ricardo S. Y. Mancebo,†,2 Alexis A. Nagengast,* Olga Speck,‡,3 Mitchell Psotka,‡,4 and Stephen M. Mount‡
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

The conserved spliceosomal U1-70K protein is thought to play a key role in RNA splicing by linking the U1 snRNP particle to regulatory RNA-binding proteins. Although these protein interactions are mediated by repeating units rich in arginines and serines (RS domains) in vitro, tests of this domain's importance in intact multicellular organisms have not been carried out. Here we report a comprehensive genetic analysis of U1-70K function in Drosophila. Consistent with the idea that U1-70K is an essential splicing factor, we find that loss of U1-70K function results in lethality during embryogenesis. Surprisingly, and contrary to the current view of U1-70K function, animals carrying a mutant U1-70K protein lacking the arginine-rich domain, which includes two embedded sets of RS dipeptide repeats, have no discernible mutant phenotype. Through double-mutant studies, however, we show that the U1-70K RS domain deletion no longer supports viability when combined with a viable mutation in another U1 snRNP component. Together our studies demonstrate that while the protein interactions mediated by the U1-70K RS domain are not essential for viability, they nevertheless contribute to an essential U1 snRNP function.

IN metazoans, the majority of precursor messenger RNAs (pre-mRNAs) contain one or more introns that must be removed to generate functional RNA molecules. This processing event, known as RNA splicing, is carried out by the spliceosome, a catalytic RNA-protein machine composed of five snRNAs and ∼300 proteins (Jurica and Moore 2003). Assembly of the spliceosome on its pre-mRNA target occurs in several stages from the preassembled U1, U2, U4, U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) and a set of proteins that are loosely associated with the snRNPs. The U1 snRNP, which is recruited to the 5′ splice site early in the spliceosomal assembly pathway, contains the U1 snRNA, whose 5′ sequence is complementary to the 5′ splice site, a set of seven Sm proteins shared with the other spliceosomal U snRNPs and three U1-specific proteins: U1-70K, U1-A, and U1-C. Although in most cases the individual contributions of the U1-specific proteins are still poorly defined, studies from several different organisms have suggested that U1-70K plays a key role in targeting the U1 snRNP to the 5′ splice site through protein/protein interactions with regulatory splicing factors (Kohtz et al. 1994; Jamison et al. 1995; Cao and Garcia-Blanco 1998).

In extracts from cultured mammalian cells, U1-70K interacts with members of the SR protein family to mediate recruitment of the U1 snRNP to the 5′ splice site. One of the best-studied examples of this type of interaction is with the ASF/SF2 protein (Caceres et al. 1993; Eperon et al. 1993; Woppmann et al. 1993; Wu and Maniatis 1993; Kohtz et al. 1994; Jamison et al. 1995; Cao and Garcia-Blanco 1998). The interaction between these two proteins is mediated by arginine-/serine-rich (RS) domains located at the C termini of each protein. Because both U1-70K and at least one SR protein are essential for splicing in vitro, it was anticipated that the interaction between the two proteins would be essential for recruitment of the U1 snRNP to the 5′ splice site and splicing. However, recent studies have suggested that this is not universally true, as splicing of a number of substrates is not impaired when the RS domain of ASF/SF2 is deleted (Eperon et al. 2000; Zhu and Krainer 2000). Given that ASF/SF2 is but one member of the SR protein family and that U1-70K is capable of interacting with several of these splicing factors, it has been postulated that in vivo the other SR proteins are capable of providing the necessary connection to U1-70K.

While these and other studies point to a key role for the U1-70K RS domain in linking the U1 snRNP particle to regulatory RNA-binding proteins, studies directly testing its importance have not been carried out. Here we report that in Drosophila, the loss of U1-70K function leads to embryonic lethality, consistent with the view that U1-70K is an essential splicing factor. However, contrary to the current view of U1-70K function, we find that animals carrying a mutant U1-70K protein lacking its RS domain, including its two embedded sets of RS dipeptide repeats, have no mutant phenotype. Surprisingly, we find that when the U1-70K RS deletion allele is combined with a viable mutation in another U1 snRNP component, the double-mutant animal dies during embryogenesis. Our finding that the U1-70K RS domain becomes essential in a splicing-compromised background thus demonstrates that the protein links mediated by the U1-70K RS domain are part of a redundant network of interactions necessary to support viability. The unexpected redundancy revealed by these studies underscores the importance of examining protein function directly in the animal.

MATERIALS AND METHODS

Fly strains and the isolation of U1-70K mutant alleles:

U1-70K2 was isolated in a standard chemical mutagenesis screen where ∼5000 individual second chromosomes were isolated from males fed 0.025 m ethyl methanesulfonate (EMS) and tested for their ability to complement the lethality of the U1-70K1 allele. U1-70KON35 was isolated by transposase-induced male recombination according to the method of Preston and Engels (1996). Briefly, 27 recombinants between al and sp were collected from the progeny of U1-70K1/al px sp; ry506 Sb P{Δ2-3}/ry506 males. Ten recombinants were selected for molecular analysis because they failed to complement U1-70K1 and all were found to contain deletions extending from the original site of the P-element insertion. Of these, U1-70KON35 proved to be the most useful for this study. Descriptions of marker mutations and balancers not listed in the text are described on FlyBase (http://www.flybase.org). All crosses were carried on standard Drosophila medium at room temperature (22°).

P-element-mediated transformation and genetic rescue experiments:

The P{hs::U1-70K} rescue construct was generated by subcloning a natural EcoRI fragment into the pCaSpeRhs transformation vector (Thummel et al. 1988). This 1390-bp fragment extends from the transcription start site to a region that lies between the AATAAA polyadenylation signal and the polyadenylation site for the longer transcript described in Mancebo et al. (1990). The P{genomic}, P{HCterm}, and P{ΔRrichHCterm} transgenes were generated in the context of a 9-kb XhoI/NotI genomic fragment (which contains the U1-70K transcription unit and flanking sequences) and then inserted into the pCaSpeR4 transformation vector (Thummel and Pirrotta 1992). The P-element transformation vectors used in this study carry a white+ mini-gene to recognize and follow the transgene. Germline transformants were obtained by standard methods and multiple insertions were obtained for each transgene. Although initially multiple insertion lines were tested for their ability to rescue the U1-70K lethal phenotype, when the data for this article were collected, only one representative example of each transgene remained. Thus, the data presented here were all obtained with a single example of each transgene.

Western blot and immunoprecipitation experiments:

Immunoprecipitation, Western blot analysis, RNA isolation from the RNA-protein complexes, and Northern blot analysis were carried out as previously described (Stitzinger et al. 1999). The antibodies used in this study, anti-SNF (Flickinger and Salz 1994) and anti-70K (Nagengast et al. 2003), have been described previously.

RESULTS

The Drosophila U1-70K protein is U1 snRNP specific:

The Drosophila genome encodes a set of proteins that are clearly orthologous to each of the human U1 snRNP-specific proteins, except that the U1-A counterpart, encoded by the sans-fille (snf) gene, also fulfills the function of the U2 snRNP-specific protein, U2-B′′ (Mount and Salz 2000). Although the putative U1-70K ortholog was identified on the basis of sequence conservation over 14 years ago (Mancebo et al. 1990; Figure 1), its functional identity has been inferred only by the presence of an appropriately sized protein in purified U1 snRNPs (Labourier and Rio 2001). To establish that the Drosophila U1-70K protein is present in U1 snRNPs, we used antisera raised against amino acids 1–213 of the protein to ask whether U1 snRNAs can be co-immunoprecipitated from whole-cell extracts. As shown in previous studies, antibodies directed against SNF immunoprecipitate both U1 snRNAs and U2 snRNAs from whole-cell extracts (Figure 2). In contrast, the U1-70K specific antibody precipitates U1 snRNA without bringing down significant amounts of U2 snRNA. Thus, we conclude that in Drosophila melanogaster, as in other organisms, U1-70K is a U1 snRNP-specific protein.

An external file that holds a picture, illustration, etc. Object name is 2532f1.jpg

The U1-70K gene: sequence comparison with the human protein, gene structure, and location of mutations. (A) Schematic of the U1-70K transcription unit. Solid boxes represent exons and lines represent introns. The position of the P-element insertion in U1-70K1, the point mutation in U1-70K2, and the extent of the deletion in U1-70KON35 are indicated at the top of the diagram. As indicated below the diagram, each individual protein domain shares significant amino acid identity with the human U1-70K protein. (B) Alignment of the U1-70K protein from Drosophila and humans. Identical residues are boxed in green and conservative substitutions are boxed in gray. The positions of the different protein domains discussed in the text are indicated either above or below the sequence. An arrow indicates the position of the stop codon in the U1-70K2 mutation.

An external file that holds a picture, illustration, etc. Object name is 2532f2.jpg

U1-70K is a U1 snRNP-specific protein. snRNP incorporation was tested by immunoprecipitation of either U1-70K or SNF from extracts made from adult flies followed by Northern blotting to detect U1 and U2 snRNAs in the RNA extracted from the precipitated fractions.

U1-70K is essential for viability:

To identify mutations in the U1-70K gene, we searched the Drosophila databases for P-element insertions located in or near U1-70K and identified a recessive lethal called l(2)02107, which contained a P-element insertion in the 5′ UTR of the U1-70K transcription unit (Spradling et al. 1999). Homozygous mutant animals complete embryogenesis with no consistent cuticular phenotype, but never hatch (data not shown). The embryonic lethality is rescued by both a transgene carrying the U1-70K coding sequence under control of the hsp70 promoter and a genomic rescue construct that contains only the U1-70K transcription unit (data not shown). Together, these results demonstrate that the loss of U1-70K function is lethal and that U1-70K is an essential gene. We therefore refer to l(2)02107 as U1-70K1.

Additional U1-70K alleles were identified in two independent screens. Screening for EMS-induced mutations that failed to complement the lethality of U1-70K1 identified one new allele, U1-70K2. DNA sequencing revealed a single nucleotide substitution in the coding sequence of the U1-70K gene that results in conversion of W88 to a stop codon (UGG to UAG; Figure 1). We note that while some readthrough of Drosophila stop codons has been described (Chao et al. 2003), the circumstance most favorable for this (a C residue immediately after the stop) does not apply here.

A definitive null allele, U1-70KON35, was isolated by selecting for male-recombination events involving the P-element in U1-70K1. Sequencing of this allele shows that excision of the P-element generated a 1997-nt deletion of genomic DNA extending from the site of the U1-70K1 insertion in the 5′ UTR through the open reading frame, ending in the 3′ UTR (Figure 1B). As was observed for U1-70K1, homozygous U1-70KON35 mutant animals complete embryogenesis but do not hatch (data not shown). Similar late embryonic phenotypes are also observed with U1-70K2, indicating that all three alleles are genetic nulls.

Functional substitution of the fly C-terminal domain by the human C-terminal domain:

The structural organization of U1-70K is conserved between humans and flies, with sequence similarity extending over the entire length of the protein (Figure 1A). The amino-terminal half of U1-70K is highly conserved and consists of a 102-amino-acid N-terminal domain of unknown function (56% identical) followed by a 73-amino-acid RNA recognition motif (81% identical) and a 30-amino-acid glycine-rich region (G-rich, 84% identical). Following these highly conserved motifs is an arginine-rich region, which contains two embedded sets of RS dipeptide repeats called RS domains (Arg-rich, 31% identical) and a poorly conserved C-terminal domain (C-term, 13% identical). Interestingly, in Drosophila, this C-terminal region is expanded at the expense of the arginine-rich region when compared to the human protein.

To determine whether the expanded C-terminal domain is functionally significant, we modified the U1-70K wild-type genomic rescue construct by replacing the endogenous C-terminal domain (amino acids 353–448) with the human C-terminal domain (HC-term, amino acids 395–437; see Figure 3A). Transgenic lines carrying this construct (designated P{HCterm}) were generated and tested for function by genetic complementation. Specifically, the ability of the chimeric construct to rescue the lethal phenotype of the U1-70KON35/U1-70K2 trans-heterozygotes was compared to that of the wild-type genomic rescue construct P{genomic}. In this experiment, we used these trans-heterozygotes to avoid complications due to second-site mutations that may be carried on the parental chromosomes, but comparable rescue of U1-70KON35 homozygotes is also observed (data not shown). The results indicate that a single copy of P{HCterm} provides sufficient activity to fully complement the lethal phenotype of U1-70K mutants (Table 1). As described in more detail below, we found that in all of our assays, the rescuing activity of P{HCterm} is comparable to P{genomic}. Thus we conclude that the C-terminal domain of the human U1-70K protein can substitute for the fly C-terminal domain. These studies, however, do not address what the function of the C-terminal domain is, leaving open the possibility that the C-terminal domain is dispensable altogether.

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Structure and expression of wild-type and mutant U1-70K proteins. (A) Schematic of the wild-type and mutant constructs used in this study. (B) Expression of the transgenic proteins was assayed by Western blot analysis using a polyclonal antibody specific for U1-70K (top) and reprobed with an antibody specific for SNF as a loading control (bottom). Extracts were made from wild-type (WT) or from U1-70KON35/U1-70K2 animals that also carry a copy of the appropriate transgene. A caret indicates the position of the 25-kD molecular weight marker.

TABLE 1

Rescue activity ofU1-70K transgenes

Transgene (rescue activity)
GenotypeP{ΔRrich-HCterm}P{HCterm}P{genomic}
XX; U1-70KON35/U1-70K2; transgene  268 (80%)256 (100%) 83 (73%)
XY; U1-70KON35/U1-70K2; transgene  263 (81%)260 (100%) 92 (87%)
XX; U1-70K/CyO; transgene  670 454 226
XY; U1-70K/CyO; transgene  648 417 211

To assay for rescue of the U1-70KON35/U1-70K2 recessive lethal phenotype w/w; U1-70K2/CyO, virgin females were crossed to w; U1-70KON35/CyO males carrying a single copy of the appropriate transgene on the third chromosome, and the resulting progeny were scored. The transgene is recognized and followed by the expression of the white+ mini-gene included in each construct. Rescue activity was assessed by comparing the number of w/w; U1-70KON35/U1-70K2 animals carrying transgene to the number of “expected animals,” which is one-half of the number of same-sexed balanced progeny carrying the transgene. We note that we did not recover any w/w; U1-70KON35/U1-70K2 animals without the transgene, indicating that U1-70KON35/U1-70K2 is a tight lethal.

The arginine-rich domain, with its embedded RS dipeptide repeats, is dispensable:

A number of studies have led to the general conclusion that the U1-70K arginine-rich domain, with its embedded RS dipeptide repeats, is critical for linking the U1 snRNP to other splicing factors during the course of spliceosome assembly (see Introduction). We tested the importance of this domain in vivo by modifying the P{HCterm} construct such that the 138-amino-acid arginine-rich region (amino acids 215–352) is deleted (see Figure 3). Surprisingly, a single copy of the resulting P{ΔRrich-HCterm} transgene also provided sufficient activity to complement the lethal phenotype of U1-70K males and females (Table 1). The robust rescue of the lethal phenotype indicates that in an otherwise wild-type background the absence of the arginine-rich domain does not have a major effect on U1-70K function.

To allow a more rigorous interpretation of these genetic data, we assayed the expression of the endogenous and transgenic proteins in the U1-70KON35/U1-70K2; P{ΔRrich-HCterm} surviving animals by Western blot analysis. As expected, we did not detect any endogenous protein, confirming that both U1-70KON35 and U1-70K2 are protein null alleles. To our surprise, we found that the protein produced by P{ΔRrich-HCterm} and its progenitor P{HCterm} is not detectable on Western blots under conditions where the parental P{genomic} transgene could be detected easily (Figure 3B). It is not clear why the antibody, which is made against the N-terminal end of the protein, does not recognize the transgenic proteins. Clearly, the transgenes are functional, as judged by their ability to rescue the lethal phenotype. Perhaps the epitope is masked in the fusion proteins; or perhaps the level of expression changes dramatically during development and drops below the level of detection during adulthood.

The arginine-rich domain becomes essential in a snf mutant background:

Although our finding that U1-70KON35/U1-70K2; P{ΔRrich-HCterm} animals are wild type indicates that the loss of RS-mediated events does not disrupt U1 snRNP function sufficiently to impair viability, a subtle effect on U1 snRNP function cannot be ruled out. In an earlier study, we proposed a similar scenario to explain why SNF, the counterpart of the mammalian U1A protein, is also dispensable for U1 snRNP function (Nagengast et al. 2003). Although snf mutations that make no protein are lethal, one allele, snf148, is viable even though it encodes a protein that is not stably associated with the U1 snRNP. Despite the lack of phenotype, it nevertheless seemed likely that U1 snRNP function might be compromised in either of these mutants. If this is the case, then simultaneously mutating both components might have a significant effect on U1 snRNP function, perhaps resulting in lethality. To test this idea, we determined whether this snf mutation had a significant impact on the rescue activity of each transgene (Table 2). In control crosses, we found that the snf148 mutation did not have significant effect on the survival rate of U1-70KON35/U1-70K2; P{HCterm} or U1-70KON35/U1-70K2; P{genomic} animals. In contrast, snf148; U1-70KON35/U1-70K2; P{ΔRrich-HCterm} males were never recovered, even though their U1-70KON35/U1-70K2; P{ΔRrich-HCterm} siblings were viable. The finding that these two alleles have synergistic effects on viability demonstrates that the arginine-rich domain indeed contributes to U1-70K function.

TABLE 2

Rescue activity ofU1-70K transgenes in asnf mutant background

Transgene (rescue activity)
GenotypeP{ΔRrich-HCterm}P{HCterm}P{genomic}
snf148;U1-70KON35/U1-70K2; transgene  0139 (86%) 97 (66%)
snf148/+; U1-70KON35/U1-70K2; transgene203161 147

To assay for rescue in a snf mutant background, w snf148/FM6;U1-70K2/CyO virgin females were crossed to w; U1-70KON35/CyO males carrying a single copy of the appropriate transgene on the third chromosome, and the resulting progeny were scored. The transgene is recognized and followed by the expression of the white+ mini-gene included in each construct. Rescue activity was assessed by comparing the number of w snf148;U1-70KON35/U1-70K2 males carrying transgene to the number of “expected animals” as determined by the number of sibling w snf148/w +;U1-70KON35/U1-70K2 controls carrying the transgene.

DISCUSSION

Here, we provide evidence that U1-70K is an essential splicing factor by demonstrating that null mutations are embryonic lethal. Our finding that U1-70K is essential for development is in agreement with the conclusions drawn from RNA interference knock-down experiments in both Arabidopsis and Caenorhabditis elegans (Golovkin and Reddy 2003; MacMorris et al. 2003). While splicing assays have so far not established that the lethality associated with the loss of metazoan U1-70K function is caused by an accumulation of unspliced RNAs, genetic studies in both yeast and Drosophila have suggested that U1-70K is critical for proper splicing of at least some transcripts (Hilleren et al. 1995; Nagengast et al. 2003). Interestingly, the Saccharomyces cerevisiae U1-70K ortholog is not essential for viability, although its absence does cause a slow-growth phenotype (Hilleren et al. 1995). In yeast, U1-70K may be dispensable because the yeast U1 snRNP is more complex with seven more protein components than metazoan U1 snRNPs, leaving open the possibility that one or more of these proteins might provide a compensating function in its absence (Gottschalk et al. 1998; Rigaut et al. 1999; Mount and Salz 2000).

A striking outcome of our studies is the finding that U1-70K can accomplish its vital function in the absence of an RS domain. The failure to detect a phenotype in these mutant animals challenges the prevailing view that the U1-70K RS motif provides a vital link between splicing regulators and the U1 snRNP. We suggest instead that either the interactions detected in vitro are not essential in vivo or there are multiple means by which the U1 snRNP can interact with splicing regulators. Support for the latter view comes from our demonstration that U1 snRNP particles lacking both the U1-70K RS domain and SNF can no longer support viability. Synthetic lethality is attributable to the simultaneous loss of two functions that contribute to the same activity or pathway. Interestingly, in S. cerevisiae synthetic lethal interactions have been observed between nonlethal mutations in several different U1 snRNP components (Gottschalk et al. 1998). Thus, our in vivo results argue that while disruption of the U1-70K RS-mediated protein links has no detectable consequence to the living organism, the simultaneous disruption of multiple connections causes U1 snRNP function to fall below the level needed to support development and viability.

In an interesting parallel to our studies, Rio and colleagues demonstrated that disruption of the RS domain of one, but not both, of the U2AF heterodimer subunits does not result in lethality (Rudner et al. 1998a,b,c). The RS domains of both subunits of the U2AF heterodimer have RS domains that were believed, on the basis of in vitro studies, to interact with distinct proteins. Using a similar genetic approach to the one described here, the RS domain of the Drosophila large U2AF subunit ortholog was found to be dispensable for viability. Similarly, the RS domain of the small subunit ortholog was also dispensable. However, flies mutant for both RS deletions could not be recovered, indicating that either set of protein links provided by the U2AF heterodimer is sufficient for viability. Together, these studies highlight the importance of looking at the roles of splicing factors in their natural contexts to unambiguously identify which functions are truly essential and which functions are part of the redundant network necessary for accurate and efficient splicing.

Acknowledgments

We thank JoAnn Wise, Hua Lou, and Greg Matera for critical reading of the manuscript; Shane Stitzinger, Bonnie Sharp, and Shaohua Jin for their excellent technical assistance; and Don Rio and the Bloomington Stock Center for reagents. This research was supported by National Institutes of Health (NIH) grants GM61039 to H. Salz and GM37991 to S. Mount. A. Nagengast was partially supported by HD07104, a Developmental Biology Training grant from the NIH.

References

  • Caceres, J. F., T. Misteli, G. R. Screaton, D. L. Spector and A. R. Krainer, 1993. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 12: 4715–4726. [PMC free article] [PubMed] [Google Scholar]
  • Cao, W., and M. A. Garcia-Blanco, 1998. A serine/arginine-rich domain in the human U1–70K protein is necessary and sufficient for ASF/SF2 binding. J. Biol. Chem. 273: 20629–20635. [PubMed] [Google Scholar]
  • Chao, A. T., H. A. Dierick, T. M. Addy and A. Bejsovec, 2003. Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila. Genetics 165: 601–612. [PMC free article] [PubMed] [Google Scholar]
  • Eperon, I. C., D. C. Ireland, R. A. Smith, A. Mayeda and A. R. Krainer, 1993. Pathways for selection of 5′ splice sites by U1snRNPs and SF2/ASF. EMBO J. 12: 3607–3617. [PMC free article] [PubMed] [Google Scholar]
  • Eperon, I. C., O. V. Makarova, A. Mayeda, S. H. Munroe, J. F. Caceres et al., 2000. Selection of alternative 5′ splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF. Mol. Cell. Biol. 20: 8303–8318. [PMC free article] [PubMed] [Google Scholar]
  • Flickinger, T. W., and H. K. Salz, 1994. The Drosophila sex determination gene snf encodes a nuclear protein with sequence and functional similarity to the mammalian U1A snRNP protein. Genes Dev. 8: 914–925. [PubMed] [Google Scholar]
  • Golovkin, M., and A. S. N. Reddy, 2003. Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals. Plant Physiol. 132: 1884–1891. [PMC free article] [PubMed] [Google Scholar]
  • Gottschalk, A., J. Tang, O. Puig, J. Salgado, G. Neubauer et al., 1998. A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. RNA 4: 374–393. [PMC free article] [PubMed] [Google Scholar]
  • Hilleren, P. J., H.-Y. Kao and P. G. Siliciano, 1995. The amino-terminal domain of yeast U1–70K is necessary and sufficient for function. Mol. Cell. Biol. 15: 6341–6350. [PMC free article] [PubMed] [Google Scholar]
  • Jamison, S. F., Z. Pasman, J. Wang, C. Will, R. Luhrmann et al., 1995. U1 snRNP-ASF/SF2 interaction and 5′ splice site recognition: characterization of required elements. Nucleic Acids Res. 23: 3260–3267. [PMC free article] [PubMed] [Google Scholar]
  • Jurica, M. S., and M. J. Moore, 2003. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12: 5–14. [PubMed] [Google Scholar]
  • Kohtz, J. D., S. F. Jamison, C. L. Will, P. Zuo, R. Luhrmann et al., 1994. Protein-protein interactions and 5′ splice-site recognition in mammalian mRNA precursors. Nature 368: 119–124. [PubMed] [Google Scholar]
  • Labourier, E., and D. C. Rio, 2001. Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles. RNA 7: 457–470. [PMC free article] [PubMed] [Google Scholar]
  • MacMorris, M., C. Brocker and T. Blumenthal, 2003. UAP56 levels affect viability and mRNA export in Caenorhabditis elegans. RNA 9: 847–857. [PMC free article] [PubMed] [Google Scholar]
  • Mancebo, R., P. C. Lo and S. M. Mount, 1990. Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein. Mol. Cell. Biol. 10: 2492–2502. [PMC free article] [PubMed] [Google Scholar]
  • Mount, S. M., and H. K. Salz, 2000. Pre-messenger RNA processing factors in the Drosophila melanogaster genome. J. Cell Biol. 150: 37–43. [PMC free article] [PubMed] [Google Scholar]
  • Nagengast, A. A., S. M. Stitzinger, C.-H. Tseng, S. M. Mount and H. K. Salz, 2003. Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping. Development 130: 463–471. [PubMed] [Google Scholar]
  • Preston, C. R., and W. R. Engels, 1996. Flanking duplications and deletions associated with P-induced male recombination and gene conversion in Drosophila. Genetics 144: 1623–1638. [PMC free article] [PubMed] [Google Scholar]
  • Rigaut, G., A. Schevchenko, B. Rutz, M. Wilm, M. Mann et al., 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17: 1030–1032. [PubMed] [Google Scholar]
  • Rudner, D. Z., K. S. Breger, R. Kanaar, M. D. Adams and D. C. Rio, 1998. a RNA binding activity of heterodimeric splicing factor U2AF: at least one RS domain is required for high-affinity binding. Mol. Cell. Biol. 18: 4004–4011. [PMC free article] [PubMed] [Google Scholar]
  • Rudner, D. Z., K. S. Breger and D. C. Rio, 1998. b Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain in either the large or small Drosophila subunit is dispensable in vivo. Genes Dev. 12: 1010–1021. [PMC free article] [PubMed] [Google Scholar]
  • Rudner, D. Z., R. Kanaar, K. S. Breger and D. C. Rio, 1998. c Interaction between subunits of heterodimeric splicing factor U2AF is essential in vivo. Mol. Cell. Biol. 18: 1765–1773. [PMC free article] [PubMed] [Google Scholar]
  • Spradling, A. C., D. Stern, A. Beaton, E. J. Rhem, T. Laverty et al., 1999. The Berkeley Drosophila Genome Project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153: 135–177. [PMC free article] [PubMed] [Google Scholar]
  • Stitzinger, S., T. Conrad, A. Zachlin and H. K. Salz, 1999. Functional analysis of SNF, the Drosophila U1A/U2B′′ homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function. RNA 5: 1440–1450. [PMC free article] [PubMed] [Google Scholar]
  • Thummel, C. S., and V. Pirrotta, 1992. Technical notes: new pCasper P-element vectors. Dros. Inf. Serv. 71: 150. [Google Scholar]
  • Thummel, C. S., A. M. Boulet and H. D. Lipshitz, 1988. Vectors for P element-mediated transformation and tissue culture transformation. Gene 74: 445–456. [PubMed] [Google Scholar]
  • Woppmann, A., C. Will, U. Kornstadt, P. Zuo, J. Manley et al., 1993. Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors. Nucleic Acids Res. 21: 2815–2822. [PMC free article] [PubMed] [Google Scholar]
  • Wu, J. Y., and T. Maniatis, 1993. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75: 1061–1070. [PubMed] [Google Scholar]
  • Zhu, J., and A. R. Krainer, 2000. Pre-mRNA splicing in the absence of an SR protein SR domain. Genes Dev. 14: 3166–3178. [PMC free article] [PubMed] [Google Scholar]
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