Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3′ Splice Site In Vivo

The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3′ splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3′ splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25°C. These suppressors differentially affected the recognition of the cryptic 3′ splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3′ splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3′ splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3′ splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans.


Introduction
Eukaryotic genes contain intervening introns that are spliced from transcribed pre-mRNAs to generate functional coding mRNAs [1,2]. Alternative splicing results in distinct mRNAs that encode proteins with distinct functions, increases the proteome size and is believed to be important to the biological complexity of metazoans [1,3,4]. In C. elegans, mRNA transcripts of at least 13% of predicted genes are alternatively spliced [5]. In humans, most genes are alternatively spliced [6,7]. A dramatic example of alternative splicing is provided by the Drosophila gene Dscam (Down syndrome cell adhesion molecule), which through alternative splicing could potentially generate over 30,000 isoforms [8], some of which have been shown to play important roles in immune responses [9] and neuronal arborization [10][11][12]. Mutations affecting the splicing process or splicing machinery cause numerous human diseases [13,14].
Pre-mRNA splicing involves five small nuclear ribonucleoprotein particles (snRNPs) and numerous associated factors [1,2,15]. The U1 snRNP recognizes the 59 splice donor site through basepairing between the U1 snRNA and the 59 splice site of the target intron [16]. The recognition of the 39 splice acceptor site is achieved by SF1/BBP (splicing factor one/branch-point binding protein) and the large and small subunits of U2AF (U2 auxiliary factor) [17][18][19][20][21][22][23]. In mammals, SF1/BBP binds a weak consensus branch-point sequence, the U2AF large subunit binds a long polypyrimidine sequence and the U2AF small subunit binds the 39 splice site YAG [19,21,[24][25][26]. The yeast Saccharomyces cerevisiae lacks a U2AF small subunit and a polypyrimidine sequence in its introns, and the recognition of a 39 splice site is achieved by binding of SF1/BBP to a highly conserved consensus branch-point sequence [17,24,27,28]. In the nematode Caenorhabditis elegans, there is no consensus branch-point sequence or long polypyrimidine sequence, and the recognition of a 39 splice site is achieved by the binding of the U2AF large and small subunits to a consensus UUUUCAGR sequence in which ''AG'' is the 39 splice site [23,29].
Splicing is also regulated by many Arginine-Serine-rich RNAbinding SR proteins [30][31][32][33] and hnRNP RNA-binding proteins [4]. These splicing factors recognize enhancer or silencer sequences in exons and introns to regulate the specificity and efficiency of splicing [4]. The genetic interactions among splicing factors and how signaling events regulate splicing efficiency and specificity are only partially understood.
C. elegans is a genetically tractable organism and has been used to study a broad variety of biological problems. Our laboratory has analyzed a set of genes, unc-93, sup-9 and sup-10, that encode components of a presumptive C. elegans two-pore domain K + channel complex and regulate muscle activity [34][35][36][37]. Rare gainof-function (gf) mutations in any of these three genes cause abnormal body-muscle contraction and are thought to activate the SUP-9 K + channel. The gf mutant animals are defective in egg laying, sluggish and exhibit a rubberband phenotype: when prodded on the head, the animal contracts and relaxes along its entire body without moving backwards. Complete loss-of-function (lf) mutations of unc-93, sup-9 and sup-10 do not cause any obvious abnormalities [35,36]. The SUP-9 protein is similar to the mammalian Two-pore Acid Sensitive K + channels TASK-1 and TASK-3 [34]. sup-10 encodes a novel single-transmembrane domain protein without identified mammalian homologs [34]. unc-93 encodes a multiple transmembrane-domain protein that defines a novel family of proteins conserved from C. elegans to mammals [34,37]. A mammalian UNC-93 homolog, UNC-93b, plays important roles in the innate immune response, probably by regulating signals mediated through Toll-like receptors [38][39][40][41][42].
Previous genetic screens for genes that affect the activities of unc-93, sup-9 and sup-10 genes were not designed to identify genes essential for fertility or animal survival. To seek such essential genes, we performed a clonal genetic screen for suppressors of the locomotion defect caused by the unc-93 gf mutation e1500. In this paper, we describe our studies of two suppressors identified from this screen and the establishment of a reporter system for in vivo analysis of RNA splicing in C. elegans. We suggest that the U2AF large subunit affects 39 splice site recognition and that some aspect of the function of the putative UNC-93/SUP-9/SUP-10 two-pore domain potassium channel complex depends on an unidentified gene the processing of which requires the functions of the U2AF large subunit and SF1/BBP.

Results
A clonal screen identified two new unc-93(e1500) suppressor genes essential for suvival Rare gf mutations in the C. elegans genes unc-93, sup-9 and sup-10 cause a rubberband Unc phenotype, while lf mutations in these genes result in a phenotypically wild-type phenotype [35,36,43]. Previous screens for suppressors of the rubberband Unc phenotype were not designed to identify genes essential for animal survival [35,36,43,44]. We performed a clonal genetic screen to seek new suppressors of the rubberband Unc phenotype caused by the unc-93(e1500) mutation, with the goal of identifying mutations that also cause sterility or lethality (see Materials and Methods). We screened about 10,000 F 1 progeny (about 20,000 mutagenized haploid genomes) of P 0 animals mutagenized with EMS (ethyl methanesulfonate) and isolated the suppressors n4588 and n4562. n4588 causes embryonic lethality at 25uC, and n4562 causes sterility at all temperatures. By mapping these mutations, we found that n4588 and n4562 are not alleles of any previously characterized suppressors of the rubberband Unc phenotype (see Materials and Methods).
n4588 is a missense mutation in uaf-1, which encodes the C. elegans ortholog of the U2AF large subunit splicing factor n4588 is a strong recessive suppressor of the locomotion defect and rubberband phenotype of unc-93(e1500) animals (Table 1). n4588 is or is closely linked to a mutation that causes a recessive temperature-sensitive (ts) lethal phenotype and results in embryonic lethality at 25uC (see Materials and Methods) (data not shown). At 20uC the lethal phenotype was incompletely penetrant. At 15uC n4588 animals appeared similar to wild-type animals. We mapped the ts-lethal phenotype of n4588 animals to an 80 kb region on the left arm of LG III (see Materials and Methods). By determining the sequences of the coding exons of four of eight genes located within this 80 kb interval, we found a point mutation in the third coding exon of the major isoform of the gene uaf-1 (uaf-1a in Figure 1A), changing codon 180 from ACT to ATT, a change predicted to replace a conserved threonine with an isoleucine. uaf-1a encodes the C. elegans ortholog of the highly conserved U2AF large subunit (U2AF, U2 auxiliary factor) [45]. In mammals, the U2AF large subunit binds a polypyrimidine sequence preceding the 39 splice site [25,46] to regulate pre-mRNA splicing. In C. elegans, together with the U2AF small subunit ortholog UAF-2, UAF-1 binds a consensus UUUUCAGR sequence, in which AG is the 39 splice site [23,29]. The U2AF large subunit contains an RS-rich (Arginine-Serine) domain ( Figure 1B, RS), a U2AF small subunit-interacting domain ( Figure 1B, W), two RRM (RNA recognition motif) domains ( Figure 1B, RRM) [26,47] and a C-terminal UHM (U2AF homology motif) domain that binds the splicing factor SF1/BBP [48]. The T180I change caused by the n4588 mutation lies between the U2AF small subunit-interacting domain and the first RRM domain of UAF-1a ( Figure 1B).
Expression of uaf-1a in body-wall muscles rescued the suppression of unc-93(e1500) by n4588 To test whether the point mutation found in the uaf-1a isoform caused the suppressor activity of n4588, we generated transgenic animals expressing a UAF-1a::GFP fusion protein under the control of a myo-3 myosin promoter, which drives transgene expression in body-wall muscle cells [49]. This uaf-1a cDNA, which encodes a predicted full-length UAF-1 protein, restored the Unc phenotype when expressed in uaf-1(n4588) unc-93(e1500) animals ( Figure 1B and Table 2). A predicted short uaf-1 isoform, uaf-1b, which contains only part of the second RRM domain and the C-terminal UHM domain, failed to restore the Unc phenotype ( Figure 1B and Table 2). Expression of these myo-3-driven transgenes (uaf-1a and uaf-1b) in wild-type animals did not cause a rubberband Unc phenotype or any other visible abnormality (data not shown). Introducing stop codons or the n4588 T180I

Author Summary
Eukaryotic genes contain intervening intronic sequences that must be removed from pre-mRNA transcripts by RNA splicing to generate functional messenger RNAs. While studying genes that encode and control a presumptive muscle potassium channel complex in the nematode Caenorhabditis elegans, we found that mutations in two splicing factors, the U2AF large subunit and SF1/BBP suppress the rubberband Unc phenotype caused by a rare missense mutation in the gene unc-93. Mutations affecting the U2AF large subunit caused the recognition of a cryptic 39 splice site generated by the unc-93 mutation, providing in vivo evidence that the U2AF large subunit can affect splice-site selection. By contrast, an SF1/BBP mutation that suppressed the rubberband Unc phenotype did not cause splicing using this cryptic 39 splice site. Our genetic studies identified a region of the U2AF large subunit important for its effect on 39 splice-site choice. Our mutagenesis analysis of in vivo transgene splicing identified a positional effect on weak 39 splice site selection and nucleotides of the endogenous 39 splice site important for recognition. The system we have defined should facilitate future in vivo analyses of pre-mRNA splicing.
mutation into the full-length uaf-1a cDNA abrogated its rescuing activity (Table 2). Heat-shock-driven expression of a transgene expressing the full-length uaf-1a cDNA under control of a heatshock promoter [50] partially rescued both the suppression of unc-93(e1500) by uaf-1(n4588) and the ts-lethality caused by uaf-1(n4588) ( Table 2 and data not shown), suggesting that the T180I mutation also caused the ts-lethal phenotype. Feeding unc-93(e1500) animals with uaf-1 RNAi-expressing bacteria ( Figure 1B and Materials and Methods) also partially suppressed the Unc phenotype ( Table 2), suggesting that normal expression of uaf-1 is required for the rubberband Unc phenotype caused by unc-93(e1500).
We isolated a uaf-1 deletion mutation, n5222D, which removes the fourth exon (encoding part of the first RRM and part of the second RRM of UAF-1a) of the uaf-1a isoform ( Figure 1A) and is predicted to cause a frameshift after amino acid 229 if the third Figure 1. uaf-1 gene and proteins. (A) Genomic structure of the uaf-1 isoforms uaf-1a and uaf-1b (adapted from Wormbase WS189) [45]. The locations of the n4588 missense mutation and the n5222 deletion allele are indicated. Black boxes: coding exons. Open box: 39 UTR. Positions of start (ATG) and stop codons (TAA) are indicated. SL1 and SL2, splice leaders associated with the uaf-1a transcript [45]. (B) Predicted UAF-1 protein domains encoded by uaf-1a and uaf-1b cDNAs, the position of the T180I change caused by the n4588 mutation and the domains affected by the n5222D deletion are shown. RNAi fragment: the portion of the uaf-1a cDNA used within a dsRNA-expressing plasmid for RNAi. RS: Arginine-Serine rich domain. W: U2AF small subunit-interacting domain. RRM: RNA recognition motif. UHM: U2AF homology motif. doi:10.1371/journal.pgen.1000708.g001 Table 1. Suppression of unc-93(e1500) and sup-10(n983) by uaf-1 and sfa-1 mutations. and fifth exons of the uaf-1a isoform are spliced together. uaf-1(n5222D)/+ animals grew and moved like the wild type, and uaf-1(n5222D)/+ did not suppress the rubberband Unc phenotype of unc-93(e1500) animals (data not shown). uaf-1(n5222D) homozygous mutants arrested and died at the late L1 to early L2 larval stages (based on body size), which precluded examination of the rubberband Unc behavior of n5222D homozygous animals (see Materials and Methods). uaf-1(n4588)/uaf-1(n5222D) suppressed the rubberband Unc phenotype of unc-93(e1500) animals as strongly as did homozygous uaf-1(n4588) ( Table 1). Similar to uaf-1(n4588) homozygotes, uaf-1(n4588)/uaf-1(n5222D) animals died embryonically at 25uC (data not shown). These results establish that n4588 is an allele of uaf-1 and that reducing the dosage of the uaf-1(n4588) allele by 50% does not affect the suppression of the rubberband Unc phenotype of unc-93(e1500) animals. These data suggest that uaf-1(n4588) causes either a reduction/loss of uaf-1 activity or an altered uaf-1 activity that is antagonized by the wildtype uaf-1 gene (see Discussion).
To determine if the expression of unc-93, sup-9, sup-10 or any of the other genes known to interact with these genes is reduced in uaf-1(n4588) animals, we examined unc-93, sup-10, sup-9, sup-18 [35] (I. Perez de la Cruz and H.R.H., unpublished results) and sup-11 [54] (E. Alvarez-Saavedra and H.R.H, unpublished results) mRNA levels. Like lf mutations in unc-93, sup-10 and sup-9, lf mutations in sup-18 and gf mutations in sup-11 can suppress the rubberband Unc phenotype caused by gf mutations in unc-93, sup-9 and sup-10. Using real-time qRT-PCR, we found no obvious reduction of the mRNA levels of these five genes ( Figure S1). We also examined the expression of UAF-1 protein using western blotting [45] and found no apparent difference in UAF-1 protein levels between wild-type and uaf-1(n4588) animals (data not shown), suggesting that the suppression of unc-93(e1500) by uaf-1(n4588) is not caused by a reduction of the level of the UAF-1 protein.
uaf-1(n4588) altered the splicing of unc-93(e1500) exon 9 by recognizing a cryptic 39 splice site generated by the unc-93(e1500) missense mutation We tested whether the splicing of unc-93 is altered by uaf-1(n4588). We examined the splicing of each exon of unc-93 in wild- Table 2. uaf-1a and sfa-1 transgenes rescued the suppression of the rubberband Unc phenotype of unc-93(e1500) animals by uaf-1(n4588) and sfa-1(n4562). type, uaf-1(n4588), unc-93(e1500), uaf-1(n4588) unc-93(e1500), unc-93(n200) and uaf-1(n4588) unc-93(n200) animals by RT-PCR ( Figure 2). Every exon other than exon 9 of the unc-93 gene was spliced similarly in all genotypes examined (Figure 2A and 2B). However, we had difficulty in consistently amplifying a cDNA band from uaf-1(n4588) unc-93(e1500) animals (data not shown) using the PCR primer pairs at the 39 end of exon 8 and the 59 end of exon 9 (indicated in black in Figure 2A). We therefore used a new pair of PCR primers that should amplify a larger region between exons 8 and 9 (Figure 2A, red arrows). With the new pair of PCR primers, we found that in unc-93(e1500) animals the region between exon 8 and 9 corresponded to a weak but consistent RT-PCR product of a reduced length ( Figure 2C, lane 3, lower arrow), and this RT-PCR product was seen only in samples from unc-93(e1500) mutant animals ( Figure 2C, lower arrow). In uaf-1(n4588) unc-93(e1500) animals, the RT-PCR product of reduced length was the most prominent product ( Figure 2C, lane 4, lower arrow). We determined the sequence of this RT-PCR product and found that it was a consequence of an alternative splicing event that utilized a cryptic 39 splice site in exon 9. This cryptic 39 splice site was generated by the unc-93(e1500) missense mutation, which has a G-to-A transition that changes amino acid 388 from Gly to Arg [37] ( Figure 2E). Quantification using Taqman RT-PCR (see Figure 4A for probe designs) indicated that the alternatively spliced exon 9 was about 1.3% of all spliced unc-93 exon 9 in unc-93(e1500) animals and 68% in uaf-1(n4588) unc-93(e1500) animals ( Figure 4B). Both non-quantitative ( Figure 2C) and quantitative RT-PCR ( Figure 4B) analyses failed to detect alternatively spliced (E) Partial genomic sequences of unc-93 intron 8 (lowercase letters) and exon 9 (uppercase letters) in the wild type (above) and in unc-93(e1500) mutants (below). The G-to-A nucleotide change of the e1500 mutation is indicated with an arrow. The AG sequence (red) forms a cryptic 39 splice site that is recognized by the splicing machinery in uaf-1(n4588) animals. doi:10.1371/journal.pgen.1000708.g002 exon 9 from wild-type, uaf-1(n4588), unc-93(n200) or uaf-1(n4588) unc-93(n200) animals, all of which lack the cryptic 39 splice site caused by the unc-93(e1500) mutation. The alternatively spliced unc-93 transcript is predicted to encode a truncated protein lacking 12 amino acids in one of the predicted transmembrane domains [37] (data not shown). To test whether the alternatively spliced unc-93 transcript in uaf-1(n4588) unc-93(e1500) animals encoded a functional UNC-93 protein, we expressed the cDNA in the body-wall muscles of sup-9(n1550); unc-93(lr12D) animals [34,44] and found that this transgene did not restore the rubberband Unc phenotype (Table S2). By contrast, expression of the wild-type unc-93 cDNA in these animals restored the severe rubberband Unc phenotype. These results suggested that the alternatively spliced unc-93 transcript encoded a lf UNC-93 protein or possibly a dominant-negative UNC-93 protein. To test the latter possibility, we expressed either unc-93 wild-type cDNA or the alternatively spliced unc-93 cDNA in the body-wall muscles of unc-93(e1500) animals (Table S3). Consistent with previous observations that unc-93(e1500)/+ animals have better locomotion than unc-93(e1500) animals [36,37], overexpression of wild-type unc-93 cDNA dramatically improved the locomotion of unc-93(e1500) animals (Table S3). If the alternatively spliced unc-93 transcript encoded an UNC-93 protein that could interfere with the endogenous UNC-93 function and cause the suppression of the rubberband Unc phenotype by uaf-1(n4588) (68% alternatively spliced unc-93 transcript), the transgene should also suppress the Unc phenotype of unc-93(e1500) animals. However, expression of the alternatively spliced unc-93 transcript in the body-wall muscles did not suppress the Unc phenotype of unc-93(e1500) animals (Table S3), suggesting that the alternatively spliced unc-93 transcript caused a loss of unc-93 function and did not interfere with endogenous unc-93 function.
To examine whether reducing UAF-1 expression, like the uaf-1(n4588) mutation, would alter the splicing of unc-93(e1500) exon 9, we fed animals with bacteria expressing dsRNA targeting uaf-1 and assessed unc-93 exon 9 splicing. As shown in Figure 2D and Figure 4B, reducing UAF-1 did not increase the relative level of alternatively spliced unc-93(e1500) exon 9. The RNAi treatment did significantly reduce the level of UAF-1 protein ( Figure S2). That reducing uaf-1 expression with RNAi did not cause altered splicing of unc-93(e1500) exon 9 similarly to that by the uaf-1(n4588) mutation is consistent with the hypothesis that uaf-1(n4588) does not reduce the function of UAF-1a but rather alters the function of UAF-1a, which leads to the recognition of the cryptic 39 splice site of unc-93(e1500) exon 9 (see Discussion). However, it is possible that uaf-1(n4588) reduces uaf-1 function and that uaf-1(RNAi) does not reduce uaf-1 function as much.
n4562 is a nonsense mutation in sfa-1, which encodes the C. elegans ortholog of the splicing factor SF1/BBP The mutation n4562 was also isolated from our clonal screen as a suppressor of the rubberband Unc phenotype of unc-93(e1500) animals. The suppressed phenotype was recessive, and n4562 caused a completely penetrant recessive sterility that was temperature independent and was tightly linked to its suppressor activity (see Materials and Methods). Like uaf-1(n4588), n4562 suppressed unc-93(e1500) and sup-10(n983) but did not suppress unc-93(n200) or sup-9(n1550) ( Table 1). Therefore, n4562 is also an allele-specific suppressor of unc-93 gf mutations but not a genespecific suppressor for the rubberband Unc genes.
We mapped n4562 to the right of LG IV (see Materials and Methods). No known suppressors of unc-93(e1500) are located in this region. The genes uaf-2, encoding the C. elegans U2AF small subunit ortholog [61] and Y116A8C.32, encoding the SF1/BBP (splicing factor 1/branch-point binding protein) ortholog [62], are located in this genomic region and are expressed from the same operon together with three other genes (Wormbase WS189) [61,62]. Orthologs of UAF-2 and SF1/BBP function with the ortholog of UAF-1 to regulate pre-mRNA splicing [2], leading us to consider these two genes as candidates for being mutated by n4562. We determined the DNA sequences of coding regions of uaf-2 and Y116A8C.32 from n4562 animals and identified a nonsense mutation in Y116A8C.32, which we named sfa-1 (sfa, splicing factor) ( Figure 3A). n4562 changed amino acid 458 from a Cys (TGT) to an opal stop (TGA) codon in a conserved C2HCtype zinc finger domain of the predicted SFA-1 protein ( Figure 3A and 3B). This mutation is predicted to cause the expression of a truncated SFA-1 protein. We rescued the suppression of unc-93(e1500) by sfa-1(n4562) by expressing in body-wall muscles an SFA-1::GFP fusion protein driven by the myo-3 promoter [49] ( Table 2). Feeding unc-93(e1500) animals with bacteria expressing dsRNA targeting sfa-1 partially suppressed the rubberband Unc phenotype (Table 2).
We isolated an sfa-1 deletion mutation, n5223D, which removes the third and fourth exons and a majority of the fifth exon ( Figure 3A). Together these regions are predicted to encode most (101 aa) of the U2AF large subunit-interacting domain (118 aa) of SFA-1 [62]. n5223D is predicted to cause a frameshift after amino acid 188 if the second exon and the residual fifth exon are spliced together. sfa-1(n5223D) caused recessive embryonic lethality, and sfa-1(n5223D)/+ did not suppress the rubberband Unc phenotype of unc-93(e1500) animals (data not shown). sfa-1(n4562)/(n5223D) similarly caused embryonic lethalilty (data not shown), suggesting that the lethal phenotype of sfa-1(n5223D) homozygotes is caused by the sfa-1(n5223D) mutation. The embryonic lethality caused by sfa-1(n5223D) and sfa-1(n4562)/sfa-1(n5223D) precluded the use of n5223D for an analysis of the rubberband Unc phenotype, because our behavioral assay is performed with young adults (see Materials and Methods).
We also examined sup-10 splicing in sfa-1(n4562) animals and failed to detect alternative splicing of the sup-10 transcript ( Figure  S3B).
Nucleotide substitutions at the intron 8 endogenous 39 splice site and the exon 9 cryptic 39 splice site alter the recognition of these two sites differently The alternative splicing between the intron 8 endogenous 39 splice site (I8) and the exon 9 cryptic 39 splice site (E9) in wild-type and uaf-1 mutant animals allows an analysis of the effects of different nucleotides on the in vivo recognition of these alternatively spliced sites. We constructed a transgene that fuses the genomic sequence between exon 8 and exon 10 of unc-93(e1500) and the GFP reporter gene ( Figure 6A) and placed the fusion transgene under the control of 2 kb of the promoter region of unc-93. We used a pair of PCR primers ( Figure 6A, red arrows) that recognize unc-93 exon 8 and the GFP sequences to specifically amplify transgene cDNAs in RT-PCR experiments. The Taqman probes shown in Figure 4A were used to quantify the wild-type and alternatively spliced isoforms ( Figure 6A). Because I8 and E9 have the same nucleotides at positions 23 to 21 (CAG), our mutagenesis analysis focused on nucleotides 27 to 24 ( Figure 6B), which are variable and are known to be critical for recognition and binding by the U2AF complex [29,[63][64][65]. We named each of 16 transgene constructs 1-16 ( Figure 6B-6E).
We replaced E9 with the sequence of I8 ( Figure 6B, No. 2). In both wild-type and uaf-1(n4588) animals, splicing occurred mostly at the new E9 (97% and 98%, respectively) ( Figure 6B and 6E). When I8 was replaced with the sequence of E9 ( Figure 6B, No. 3), splicing again occurred mostly at E9 in both wild-type and uaf-1(n4588) animals (97% and .99%, respectively) ( Figure 6B and 6E). These results suggest that the sequence that surrounds the original E9 is preferred by the splicing machinery in both wildtype and uaf-1(n4588) animals when two identical 39 splice sites are present. We next switched the positions of I8 and E9 ( Figure 6B, No. 4). In the wild type most splicing (.99%) occurred at the new E9 ( Figure 6B and 6E). Similarly, in uaf-1(n4588) animals, most splicing (80%) occurred at the new E9 ( Figure 6B and 6E, No.4). However, that a significant amount of splicing (20%) occurred at the new I8 in uaf-1(n4588) animals ( Figure 6B and 6E, No. 4) suggested that the mutant UAF-1 can efficiently recognize the original E9 sequence even at the I8 position, which is normally a less favorable position.
The pattern of alternative splicing in cell culture can depend on the promoter used [66]. unc-93 is expressed in body-wall muscles [34,37]. We tested whether a different muscle-specific promoter would alter the splicing pattern of transgene No. 1 by expressing the transgene under the control of a myo-3 promoter [49] ( Figure 6B and 6E, No. 5). We found almost identical splicing patterns of the transgene driven by the myo-3 promoter and the unc-93 promoter ( Figure 6B and 6E, compare No. 1 and No. 5), suggesting that the alternative splicing of unc-93(e1500) involves a mechanism that is not promoter-specific.
We examined the effects of base substitutions at I8. Replacing I8 with the C. elegans consensus 39 splice site TTTTcag [29,[63][64][65] caused splicing to occur exclusively (100%) at the new I8 in both wild-type and uaf-1(n4588) animals ( Figure 6C and 6E, No. 6). To identify the nucleotides required for the recognition of I8 in wildtype animals, we substituted each base from 27 to 24 of I8 with a G ( Figure 6C, No. 7 to No. 10). G is the least used nucleotide from 27 to 24 of identified 39 splice sites [29,64] and in previous studies substituting T with G at any of the four bases from 27 to 24 of the highly consensus TTTTcag site significantly compromised binding of the U2AF complex to this site [29]. A G substitution at 27 (No. 7), 25 (No. 9) and 24 (No. 10) of I8 all dramatically reduced splicing at the new I8 (to the level of 15%, 0%, 0%, respectively; Figure 6C and 6E) in wild-type animals, suggesting that these nucleotides are critical for the recognition by wild-type UAF-1. However, a G substitution at 26 ( Figure 6C and 6E, No. 8) did not cause a significant change of splicing at the new I8 (which is 96% compared to 98% of No. 1), suggesting this nucleotide is not essential for recognition by wild-type UAF-1. We also substituted the A at 26 with a C to generate an I8 more Table 5. uaf-1(n4588) and sfa-1(n4562) partially suppress the rubberband Unc phenotype caused by overexpression of unc-93(e1500) cDNA in body-wall muscles.  Figure 6C and 6E, No. 7, 8, 9, 10 and 11) in uaf-1(n4588) animals is similar to that of transgene No.1 ( Figure 6B and 6E), suggesting that none of the substitutions significantly increased the affinity of I8 for mutant UAF-1.

uaf-1 and sfa-1 represent a new class of suppressors of the rubberband phenotype
The known suppressors of gf mutations of unc-93, sup-9 and sup-10 are of three classes. First, lf mutations in any of these three genes are recessive suppressors of the rubberband Unc phenotypes caused by gf mutations in any of these three genes, because the functions of all three genes are necessary for expression of the Unc phenotype [35,36,43]. Second, rare gf mutations of sup-11 are strong dominant suppressors of unc-93(e1500) and unc-93(n200) and partial recessive suppressors of sup-9(n1550) and sup-10(n983) [54]. The mechanism of sup-11 suppression is unknown. Third, lf mutations of sup-18 are strong recessive suppressors of sup-10(n983) and weak recessive suppressors of unc-93(e1500), unc-93(n200) and sup-9(n1550) [35]. The mechanism of sup-18 suppression is also unknown. uaf-1(n4588) and sfa-1(n4562) define a new class of suppressors: they are recessive and allele-specific for unc-93 gf mutations (unc-93(e1500) was suppressed, but unc-93(n200) was not) but not gene-specific (sup-10(n983) was also suppressed). Previous genetic and molecular studies from our laboratory led to the hypothesis that UNC-93, SUP-9 and SUP-10 form a protein complex in the body-wall muscles [34][35][36][37]. The identification of multiple suppressors of the rubberband Unc phenotype with distinct suppression patterns suggests that the presumptive UNC-93/SUP-9/SUP-10 complex could have multiple in vivo functions regulated in different ways. As mentioned above, we propose that mutations in uaf-1 and sfa-1 affect the splicing of one or more unknown genes required for unc-93 and sup-10 activity. This unknown gene might be required specifically for the expression of the rubberband Unc phenotype caused by unc-93(e1500) or sup-10(n983) but have a negligible role in the expression of the rubberband Unc phenotypes caused by unc-93(n200) or sup-9(n1550). Similarly, sup-11 and sup-18 could affect functions of the UNC-93/SUP-9/SUP-10 complex distinct from that affected by uaf-1 and sfa-1.
uaf-1 and sfa-1 mutants provide new approaches for the analysis of the in vivo functions of the U2AF large subunit and SF1/BBP The SF1/BBP and U2AF proteins are critical splicing factors that regulate splicing by binding the branch-point sequence and the 39 splice sites [1,2], respectively. Mutations that affect the U2AF subunits and SF1/BBP in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the fruit fly Drosophila melanogaster have significantly facilitated the understanding of the in vivo function and regulation of these splicing factors [17,27,[67][68][69][70]. Studies of S. cerevisiae identified genetic and biochemical interactions between the U2AF large subunit and SF1/BBP [17,27], and studies of S. pombe provided in vivo evidence that the U2AF subunits are required for splicing [68,70]. In Drosophila null mutations of the U2AF large or small subunits cause lethality [67,69], hindering genetic analysis of these splicing factors. Similarly, in C. elegans, reducing the expression of uaf-1 or sfa-1 by RNAi causes lethality [61,62], suggesting that these genes are essential for animal survival. We identified mutations that affect uaf-1 and sfa-1 and allow the survival of animals in permissive conditions, such as at lower temperatures or when derived from heterozygous mothers. These mutations provide a valuable resource for analyzing the function and regulation of the U2AF large subunit and SF1/BBP genes in vivo in animals.

Mutations in UAF-1 alter the in vivo recognition of a 39 splice site
The recognition of 39 splice sites is achieved by interactions between SF1/BBP and the U2AF large and small subunits, which together bind specific intronic sequences [1,2]. However, it is not clear how these factors regulate the choice of the correct splice site when two or more potential 39 splice sites are proximal in vivo. Distinguishing different 39 splice sites is a critical aspect of alternative splicing.
The unc-93(e1500) missense mutation generates a new cryptic 39 splice site (AG) within exon 9 (ACTGcag). This site differs from the consensus 39 splice site for C. elegans (TTTTcag) [29] and is more rarely used by C. elegans than is TTTTcag or the intron 8 endogenous 39 splice site (AATTcag) (Table S4). Based on in vitro studies, this cryptic site should not be or be more weakly recognized by UAF-1 compared to TTTTcag and probably the intron 8 site AATTcag [29]. In a wild-type background, the choice between the wild-type 39 splice site of unc-93(e1500) intron 8 and the cryptic non-consensus site in unc-93(e1500) exon 9 followed this prediction, as only 1.3% of the splicing events utilized this cryptic 39 splice site. Strikingly, however, the n4588 missense mutation in uaf-1 shifted this specificity, causing splicing to occur mostly at the cryptic site, generating 68% of aberrantly spliced transcripts. This result suggests that UAF-1 might play an important role in determining the choice among alternative 39 splice sites in vivo (see discussion below).
The n4588 mutation did not cause an apparent change of UAF-1 protein level, and reducing UAF-1 using RNAi did not increase the relative amount of alternatively spliced exon 9, suggesting that uaf-1(n4588) might alter UAF-1 function. However, RNAitreatment did not abolish the expression of UAF-1 ( Figure S2), and we might have failed to detect an effect of UAF-1 on the splicing of unc-93(e1500) exon 9 because of residual UAF-1 protein in RNAi-treated animals. Thus, the altered splicing of unc-93(e1500) exon 9 in uaf-1(n4588) unc-93(e1500) mutants might reflect the consequence of the absence of UAF-1 activity. It is also possible that uaf-1(n4588) causes both a loss of function and an altered function of UAF-1, which cause the suppression of the rubberband Unc phenotype of the unc-93(e1500) animals and the altered splicing of unc-93(e1500) transcript, respectively.
The other 14 introns of the unc-93 transcript appeared to be spliced similarly in wild-type and uaf-1(n4588) animals, suggesting that uaf-1(n4588) did not alter the recognition of most wild-type 39 splice sites. We also found that uaf-1(n4588) did not suppress the Unc phenotype caused by the unc-52(e669) mutation, which can be suppressed by mutations in the splicing factor genes smu-1 and smu-2. We conclude that the uaf-1(n4588) mutation does not affect all cases in which alternative splicing is possible.
We isolated four intragenic suppressors of the temperaturesensitive lethality caused by uaf-1(n4588). Three of the suppressors (n4588 n5120, n4588 n5125 and n4588 n5127) carried both the original n4588 mutation and a second site mutation in uaf-1. These three new uaf-1 mutations reduced the alternative splicing of unc-93(e1500) exon 9 to levels intermediate between those of uaf-1(n4588) and wild-type animals. This finding supports the hypothesis that UAF-1 is important in 39 splice-site choice. The fourth intragenic suppressor, n5123, affected the same site as the original n4588 mutation by generating a phenylalanine codon different from both the wild-type codon (threonine) and the codon generated by the n4588 mutation (isoleucine). The uaf-1(n5123) allele behaves like the uaf-1(+) allele, suggesting that this mutation restored the normal specificity of UAF-1. The amino acids affected by these uaf-1 mutations (n4588, n5120, n5123, n5125 and n5127) are confined to a region between the U2AF small subunitinteracting domain [47] and the first RRM domain [26,45] ( Figure 5). We postulate that this region of UAF-1 defines a domain of UAF-1 important for 39 splice-site selection.
The sequences and positions of 39 splice sites together define the efficiency of splicing events In C. elegans, the first two nucleotides (22 to 21) of 39 splice sites are more highly conserved than nucleotides 27 to 23 [29,64], which affect the binding of the U2AF factors [29]. We sought to identify the nucleotides that affect the recognition of I8 and E9 by UAF-1 in vivo.
First, we conclude that the location of a 39 splice site affects its recognition. We found that the location of E9 was preferred to that of I8 by both wild-type and mutant UAF-1 when identical 39 splice sites were present at the two locations ( Figure 6B, No. 2 and 3). However, this positional effect was not absolute. When the highaffinity 39 splice site TTTTcag was placed at either of the two locations, the site with the TTTTcag was preferred by both wildtype and mutant UAF-1 ( Figure 6, No. 6 and No. 12). That splicing using I8 and E9 (both are likely weak 39 splice sites, since there are fewer such sites in C. elegans introns than there are copies of the strong 39 splice site sequence TTTTcag (Table S4)) was more affected by position than was splicing using the sequence TTTTcag suggests that weak 39 splice sites might be preferably used for alternative splicing, and, strong 39 splice sites such as TTTTcag might be generally used for constitutive splicing. If so, we might identify alternatively spliced genes by searching apparently weak 39 splice sites and then performing RT-PCR analyses. That TTTTcag is strongly recognized by mutant UAF-1 is consistent with our finding that the uaf-1(n4588) mutation does not appear to affect the splicing of most other introns of unc-93 ( Figure 2B), which have a 39 splice site identical or highly similar to TTTTcag (data not shown).
Second, we conclude that the nucleotides at 27, 25 and 24 were more important than the nucleotide at 26 for wild-type UAF-1 to recognize the sequence of I8 ( Figure 6, No. 7 to No. 10). The nucleotides at 24 and 25 appear to be more important than that at 27. That the nucleotide at 24 is more important than the nucleotide at 26 also appears to be the case for splicing at E9 by wild-type UAF-1 (No. 15 and No. 16, compared to No. 2, Figure 6), which indicates that nucleotide substitution at 24 (No. 16) dramatically reduced splicing and nucleotide substitution at 26 (No. 15) had a minimal effect at E9 in wild-type animals.
Third, substituting individual non-T nucleotides with T in E9 improved its recognition by wild-type UAF-1. The original I8 (AATTcag) and E9 (ACTGcag) are both rare 39 splice sites compared to TTTTcag, which is found in about 26% of the approximate 40000 introns analyzed, and is the most commonly used 39 splice site in C. elegans (Table S4) [29,64]. I8 appears more frequently in introns than does E9 (Table S4), suggesting that E9 has a lower affinity for the wild-type UAF-1 than does I8. That the wild-type UAF-1 rarely recognized E9 even in the more favored position ( Figure 6, No. 1) is consistent with this notion. We found that substituting any E9 non-T nucleotide with T could increase the recognition of E9 in wild-type animals ( Figure 6, No. 13 to 15), and a T substitution at 26 and 24 had a much stronger effect than one at 27.
Fourth, we conclude that the T180I(n4588) mutation caused UAF-1 to be more tolerant of a G nucleotide at 24 of E9. In transgenes No. 11 and No. 16, a G substitution at 24 of E9 dramatically reduced splicing at E9 in wild-type animals but did not or only moderately affected splicing at E9 in uaf-1(n4588) mutants ( Figure 6). The splicing of transgenes No. 1, No. 4 and No. 5 is consistent with this observation, implying that a G at 24 is more tolerated in uaf-1(n4588) animals than in wild-type animals. Based on these observations, we propose that the G nucleotide at position 24 of E9 is critical for its recognition by the mutant UAF-1.
In vivo functions of UAF-1 and SFA-1 probably remain to be discovered In vivo studies have suggested functions for the U2AF large subunit beyond regulating pre-mRNA splicing. For example, Drosophila mutants with a temperature-sensitive U2AF large subunit are defective in the nucleus-to-cytoplasm export of intronless mRNAs at elevated temperatures [71], suggesting that lack of U2AF large subunit function can affect mRNA export in addition to pre-mRNA splicing. Studies of SF1/BBP suggest that this splicing factor might not be essential for splicing in vitro or in vivo. Biochemical depletion of SF1/BBP in extracts from HeLa cells [72] and S. cerevisiae [73] or genetic depletion of SF1/BBP in extracts from S. cerevisiae [73] did not significantly affect splicing in vitro. Reducing SF1/BBP expression by RNAi in HeLa cells does not affect the splicing of several endogenous genes and a reporter gene [74]. That uaf-1(n4588) and sfa-1(n4562) suppressed the Unc phenotype of unc-93(e1500) but had different effects on the splicing of unc-93(e1500) mRNA at the cryptic 39 splice site suggests that uaf-1 and sfa-1 could have both distinct and shared in vivo functions in C. elegans. Specifically, the splicing of some genes might be affected similarly by uaf-1 and sfa-1, with other genes differentially affected. Alternatively, it is possible that uaf-1(n4588) has a stronger effect on the splicing of unc-93(e1500) exon 9, while sfa-1(n4562) has a weaker effect not detected in the experiments we performed.
The lack of conditionally viable mutants of the U2AF large subunit and SF1/BBP has impeded the analysis of the in vivo functions of these splicing factors in animals. The mutations we isolated affecting these two splicing factors should allow novel approaches for in vivo analyses of RNA splicing and of the functions of the U2AF large subunit and SF1/BBP in C. elegans. The transgene splicing system we developed provides an in vivo reporter assay for understanding the role of UAF-1 and possibly other splicing factors in regulating alternative 39 splice site recognition.
Clonal screen to identify unc-93(e1500) suppressor genes essential for animal survival Synchronized L4 unc-93(e1500) animals (P 0 ) were mutagenized with EMS (ethyl methanesulfonate) as described [55]. F 1 progeny from these animals were picked to single wells of 24-well culture plates with OP50 bacteria grown on NGM agar. F 2 progeny were observed using a dissecting microscope to identify animals with improved locomotion. From ,10,000 F 1 clones screened, 100 independent suppressed strains were isolated. 97 of the isolates, including two weak recessive sterile suppressors and 95 recessive fertile suppressors, were kept as frozen stocks for possible later study. Three stronger suppressors that caused or were closely linked to mutations that caused sterility (n4562) or ts-lethality (n4588 and n4564) were chosen for further analysis. The analysis of n4564 is ongoing.

Cloning of n4588 and n4562
We mapped n4588 to the left of dpy-1 on LGIII based on the suppression of unc-93(e1500) using standard methods. As the suppressor activity and ts-lethality were very closely linked, e.g., more than 500 n4588 unc-93(e1500)/+ unc-93(e1500) individuals failed to segregate Sup non-Let progeny, we then followed the phenotype of ts-lethality to further map n4588. We mapped n4588 to the right of nucleotide 186577 on BE0003N10 (cosmid BE0003N10 sequences refer to nucleotides of accession no. AC092690) using 10 Vab recombinants recovered after crossing vab-6(e697) n4588 hemaphrodites with males of the Hawaiian strain CB4856 [75] and to the left of nucleotide 13164 on Y92C3A (accession no. AC024874) using 37 Dpy recombinants recovered after crossing n4588 dpy-1(e1) hemaphrodites with males from the Hawaiian strain CB4856. We determined the coding sequences of four genes in this interval, uaf-1, rab-18, kbp-4 and par-2, and identified a missense mutation in the third exon of the uaf-1a isoform.
As the suppressor activity and sterility of n4562 were very closely linked, e.g., over 500 unc-93(e1500); n4562/+ individuals failed to segregate Sup non-Ste progeny, we followed the sterility phenotype to map n4562 to the right of dpy-4 on LG IV using standard methods. We next mapped n4562 to the right of nucleotide 37163 on Y43D4A (accession no. AL132846) using 234 Dpy recombinants recovered after crossing dpy-4(e1166) n4562 with males of the Hawaiian strain CB4856. The sequences of coding exons of sfa-1 and uaf-2, both located in this region, were determined, and a Cys458Opal (TGT-to-TGA) mutation was identified in sfa-1.

Isolation of deletion alleles
Genomic DNA pools from EMS-mutagenized animals were screened for deletions using PCR as described [77]. Deletion mutant animals were isolated from frozen stocks and backcrossed to the wild type at least three times. uaf-1(n5222D) removes nucleotides 9786 to 11082 of YAC Y92C3B. sfa-1(n5223D) removes nucleotides 207818 to 208925 of YAC Y116A8C.

Quantitative RT-PCR
Total RNA was prepared using Trizol according to the manufacture's instructions (Invitrogen), treated with RNase-Free DNase I (New England Biolabs) and followed by incubation at 75uC for 10 minutes to inactivate DNase I. First-strand cDNA was synthesized with random hexamer primers (New England Biolabs) using the Superscript II or III First-Strand Synthesis Kit (Invitrogen). Quantitative RT-PCR was performed using either a DNA Engine Opticon System (MJ Research) or a Mastercycler realplex system (Eppendorf). For the SYBR green-based assay (DNA Engine Opticon System), each 30 ml PCR reaction contained 1 to 10 ng RT template, 0.5 mM PCR primers and 15 ml 26 SYBR Green PCR Master Mix (Applied Biosystems). Three independent samples of synchronized wild-type (N2) and uaf-1(n4588) L1 animals were prepared, and levels of control genes (rpl-26, gpd-2, act-1) and tested genes (myo-3, unc-93, sup-9, sup-10, sup-11, sup-18) were quantified from each biological replicate. For the Taqman probe-based assay (Mastercycler realplex system), the probes ( Figure 4A) were labeled at their 59-ends with 6carboxyfluorescein (FAM) and at their 39-ends with Black Hole Quencher (BHQ-1) (Integrated DNA Technologies). Two independent samples of each genotype of animals of mixed stages were prepared, and levels of rpl-26 and unc-93 wild-type and alternatively spliced transcripts were quantified from each biological replicate. For RNAi-treated animals (see below), one sample for each assay was quantified. PCR primers and Taqman probes are listed in Table S5.

Screen for suppressors of uaf-1(n4588)
Synchronized uaf-1(n4588) animals (P 0 ) at the L4 larval stage grown at 15uC were mutagenized with EMS as described [55]. These P 0 animals were allowed to grow to young adults at 15uC in a mixed population and bleached, and F 1 progeny were synchronized at the early L1 stage by starvation in S medium [78]. The F 1 animals were placed on 50 Petri plates (,1000 animals/plate) with NGM agar seeded with OP50 and permitted to grow to young adults at 15uC and then moved to 25uC. After six days at 25uC, the animals were grown at 20uC for six days and examined each day for the presence of living F 2 animals. About 50,000 F 1 progeny from a mixture of more than 10,000 P 0 animals were screened. From the screen, we recovered 13 surviving F 2 animals from 13 different F 1 plates. Six of the 13 suppressors were intragenic suppressors representing four different mutations: one was n5120, one was n5123, three were identical to n5125 and one was n5127. It is possible that the three isolates containing the n5125 mutation were derived from the same P 0 animal, because all of the P 0 animals were in a mixed population when bleached to release eggs. The other seven suppressors were extragenic mutations, i.e., n4588/+; sup/+ animals segregated n4588-like progeny. Some or all of the extragenic isolates could have been derived from the same P 0 animal.

RNA interference
Young adult animals (wild-type or unc-93(e1500)) were fed HT115(DE3) bacteria containing plasmids directing the expression of dsRNAs targeting either uaf-1 or sfa-1 on NGM plates with 1 mM IPTG and 0.1 mg/ml Ampicillin [79]. Surviving F 1 progeny of the unc-93(e1500) animals (escapers) were examined for suppression of locomotion defects. Animals were washed from plates, rinsed three times with H 2 O, and resuspended in Trizol (Invitrogen) for preparation of total RNA or in 26 protein loading buffer (see Western blots, below) for SDS-PAGE analysis. We generated the DNA construct expressing dsRNA targeting uaf-1 (see below). The bacterial strain expressing dsRNA targeting sfa-1 was obtained from a whole-genome RNAi library [80], and the sequences of plasmids from single colonies of the strain were determined to confirm the presence of sfa-1 coding sequences.
Quantification of locomotion and the rubberband phenotype L4 animals were picked 16-24 hrs before assaying and were grown at 20uC. Young adults were then individually picked to Petri plates containing NGM agar seeded with OP50, and bodybends were counted for 30 seconds using a dissecting microscope as described [81]. The rubberband phenotype was scored as described [43].

Plasmids
To rescue the suppression of the Unc phenotype of unc-93(e1500) by uaf-1(n4588) or sfa-1(n4562), uaf-1 and sfa-1 cDNAs were subcloned to vector pPD93.97 using BamHI and AgeI restrictions sites. uaf-1b cDNA was amplified with PCR using uaf-1a cDNA as template and subcloned to pPD93.97 using BamHI and AgeI restrictions sites. uaf-1a cDNA was subcloned to pPD49.83 (for heatshock induced expression of uaf-1a cDNA) using BamHI and SacI restriction sites. An XhoI/SpeI fragment of uaf-1a cDNA subcloned in a pGEM-TA easy vector (Promega) was subcloned to pPD129.36 (for the uaf-1 RNAi construct) using XhoI and NheI sites. To test whether the truncated unc-93(D) cDNA caused by altered splicing of the unc-93(e1500) transcript in uaf-1(n4588) mutants encodes a functional UNC-93 protein, unc-93 cDNA and unc-93(D) cDNA were subcloned to pPD93.97 using BamHI(blunt) and AgeI sites. To test whether uaf-1(n4588) or sfa-1(n4562) mutations could suppress the Unc phenotype caused by ectopic expression of the unc-93(e1500) cDNA, unc-93(e1500) cDNA was subcloned to pPD93.97 using BamHI(blunt) and AgeI sites. To examine the effect of nucleotide substitutions on the recognition of the intron 8 endogenous 39 splice site and the exon 9 cryptic 39 splice site, we fused the genomic sequence between exon 8 and exon 10 of unc-93(e1500) in-frame with the GFP gene of pPD93.97 using BamHI and AgeI sites. We replaced the myo-3 promoter of pPD93.97 with a 2 kb promoter of unc-93 using PmlI and BamHI sites. Point mutations in uaf-1(stop codons), the unc-93 (e1500 mutation) or mutated transgenes were introduced using QuickChange II or III Site-Directed Mutagenesis Kit (Stratagene) with primers containing corresponding mutations. PCR was performed using Eppendorf Cyclers, and DNA products were resolved using agarose gels. DNA sequence determination was performed with an ABI Prism 3100 Genetic Analyzer. PCR primers are listed in Table S5.

Transgene experiments
Germline transgene experiments were performed as described [82]. Transgene mixtures generally contained 20 mg/ml 1 kb DNA ladder (Invitrogen), 20 mg/ml Arabidopsis genomic DNA and 10 mg/ml of the transgene of interest. When the transgene did not cause the expression of a GFP fusion protein, 10 mg/ml pPD95.86-GFP plasmid (expressing GFP in body-wall muscles) or 5 mg/ml p myo-2 dsRED (expressing RFP in pharynx) was added to the injection mixture as a visible fluorescence marker to identify animals carrying the transgene.

Table S3
The alternatively spliced unc-93 transcript likely does not encode a dominant-negative UNC-93 protein product. Tansgenes driving the expression of the unc-93 cDNA(D) did not suppress the rubberband Unc phenotype of unc-93(e1500) animals, while transgenes expressing a wild type unc-93 cDNA suppressed the Unc phenotype. As loss of function of unc-93 results in phenotypically wild-type animals, the lack of suppression of unc-93(e1500) by the unc-93(D) transgenes suggests that the function of unc-93 was not antagonized by unc-93(D), indicating that the unc-93(D) cDNA does not encode a dominant-negative UNC-93 protein.
Found at: doi:10.1371/journal.pgen.1000708.s007 (0.02 MB DOC) Table S4 Sequences and distributions of the three 39 splice sites we analyzed. Approximate 40,000 unique introns were analyzed, and the numbers and ratios of all types of 39 splice sites were calculated. The list here includes the three sites we analyzed in our mutagenesis experiments shown in Figure 6. TTTTcag is the most commonly used 39 splice site. Found at: doi:10.1371/journal.pgen.1000708.s008 (0.03 MB DOC)