MBE Advance Access originally published online on August 20, 2008
Molecular Biology and Evolution 2008 25(11):2431-2437; doi:10.1093/molbev/msn181
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Research Articles |
Alternative Splicing and the Steady-State Ratios of mRNA Isoforms Generated by It Are under Strong Stabilizing Selection in Caenorhabditis elegans
Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz
E-mail: zahler{at}biology.ucsc.edu.
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Abstract |
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Evolutionary studies indicate that a high proportion of alternative splicing (AS) events are species-specific; just 28% of minor-form alternatively spliced exons are conserved between mice and humans. We employed a splicing-sensitive microarray to study the evolution of allele-specific AS in nematodes. We compared splicing levels among five distinct Caenorhabditis elegans lines. Our results indicate that AS is less variable between natural isolates (NIs) from England, Hawaii, and Australia than when compared with mutation accumulation lines (6% vs. 21%, respectively, vary compared with N2). This suggests that strong stabilizing selection shapes the evolution of the ratios of isoforms generated by AS in C. elegans. When we analyzed some of the splicing changes between the NIs, we found examples of changes in both cis and trans that lead to alterations in gene-specific AS. This indicates that both these mechanisms for changing AS are employed along the path toward speciation in nematodes.
Key Words: alternative splicing • evolution • microarrays
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Alternative splicing (AS) is a common process for generating multiple proteins from a single gene. There is increasing evidence to show that this process may play an important role in the evolution of diversity by removing the negative selection pressure created by the accumulation of mutations in alternatively spliced regions (Modrek and Lee 2003
; Sorek et al. 2004; Zhuo et al. 2007). There is also evidence that low-abundance splice variants provide a "test bed" to evolve new or modified gene functions without interrupting the original function of the gene (Blencowe 2006). Indeed, AS has been linked to an increase in exon creation and/or loss (Modrek and Lee 2003). By using different comparative approaches, it has been shown that AS events are poorly conserved in mammals. Modrek and Lee (2003) showed that close to 72% of human and mouse minor-form alternatively spliced exons were created after their evolutionary split 75–110 MYA. It has been suggested that differences in the levels of AS between lower and higher eukaryotes might be important for the higher level of protein diversity found in the latter (Maniatis and Tasic 2002; Kim et al. 2007). Studies in both mammals and insects concluded that AS has an intrinsically low rate of conservation (Modrek and Lee 2003; Malko et al. 2006). These studies even proposed that the low conservation of AS might be "universal" (Malko et al. 2006).
Different studies locate Caenorhabditis elegans near the root of the AS expansion in metazoans. A lower percentage of C. elegans genes undergo AS, predicted to be 
10%, relative to other animals (60% in humans, 55% in mice, 20% in flies) (Kim et al. 2007). It is therefore important for understanding the mechanisms of both evolution and AS to recognize the forces that shape the development of AS in a species. Using comparative genomics, it was found that between different Caenorhabditis species there is a rate of intron gain/loss that is at least 25 times higher than between mammals at a similar evolutionary distance (Kent and Zahler 2000; Roy et al. 2003; Ratsch et al. 2005). This is a striking rate considering that C. elegans genes undergo much less AS than mammalian genes. In comparison to mammals and insects, multiple reports show that in Caenorhabditis, the levels of conservation of alternatively spliced exons, their cis elements, splice sites and in some cases even the regulation of isoform ratios are highly conserved even after 100 Myr of evolution (Kent and Zahler 2000; Kabat et al. 2006; Rukov et al. 2007; Irimia et al. 2008). The underlying force that creates this dramatic difference in AS evolution between nematodes and mammals is not currently known. By studying the variability of splicing among different genetic isolates of the same species (C. elegans), we will gain further understanding of the forces responsible for this dramatic conservation of AS.
The regulation of an AS event involves interactions between cis elements on the pre-mRNA and trans-acting protein factors present in the nucleus (for a review, see Wang and Burge 2008). Splicing factors are a diverse family of proteins with RNA-binding motifs that recognize different varieties of cis elements. These RNA–protein interactions are integrated into both positive and negative splicing regulatory decisions, and specific splicing factors can regulate the AS of multiple genes. The splicing regulation of splicing factors themselves has been found to be of extreme importance for their function. Proof of this can be seen in the presence of ultraconserved elements in mammals in the alternatively spliced regions of several splicing factors (Lareau et al. 2007; Ni et al. 2007).
We recently developed a DNA microarray that is capable of detecting changes in AS isoform ratios for 352 C. elegans cassette exons (Barberan-Soler and Zahler 2008). We used this assay to look for differences in AS for five C. elegans lines. We found that AS is much less variable (6%) between natural isolates (NIs), which were under natural selection in the environment, than between mutation accumulation (MA) lines (21%), which were developed with the minimum selection possible in the laboratory in which they were generated. Our results suggest that a strong stabilizing selection shapes the evolution of both the AS and the ratios of isoforms generated by this process in C. elegans.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Strains and RNA Samples
NIs were obtained from the Caenorhabditis Genetics Center. MA lines (Vassilieva et al. 2000
) were kindly provided by Kevin Simonelic and Michael Lynch. RNA samples were collected as previously described from a synchronized population of embryos (Barberan-Soler and Zahler 2008).
Splicing-Sensitive Microarray
We previously reported 449 examples of genes with strong cDNA evidence for alternative spliced cassette exons in the C. elegans genome (Kabat et al. 2006). These, together with the 50 predictions made with the recognition of alternatively spliced exons in C. elegans algorithm, were used for the array (Ratsch et al. 2005). For this work, we designed microarray probes for all the cassette exons where it was possible to design a junction probe that will be specific for the skip isoforms (i.e., single cassette exons). We found 352 alternative cassette exons that pass these criteria. For details about probe and microarray design, see Barberan-Soler and Zahler (2008).
Hybridizations
In all, 20 µg of purified embryonic RNA per channel were labeled with Alexa Fluor dyes (555 and 647) using the SuperScript Indirect Labeling System (Invitrogen Carlsbad CA) according to the manufacturer's recommendations for each of the five strains used. Hybridizations were done in triplicate. Labeled samples were hybridized to slides for 14–16 h in 20% formamide, 5x standard saline citrate, 0.1% sodium dodecyl sulfate, and 0.1 mg/ml sheared salmon sperm DNA. Following hybridization, the slides were washed and dried prior to scanning with an Axon Instruments 4000 series scanners.
Data Analysis
Data were normalized and further processed using R and Bioconductor (Dudoit et al. 2003). Specifically, Limma "rma" background correction was used to avoid blow out of M values at low intensities. Median normalization was done prior to differential expression analysis using lmFit (Smyth and Speed 2003; Smyth 2004). AS ratios were calculated as described previously (Barberan-Soler and Zahler 2008).
Reverse Transcriptase–Polymerase Chain Reaction
Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed using SuperScriptIII One-Step RT-PCR Kit (Invitrogen) following the manufacturer's recommendations. Polymerase chain reaction (PCR) products were analyzed using ethidium bromide–stained agarose gels and an Agilent Bioanalyzer 2100 with the Agilent DNA 1000 kit. AS ratios and inclusion proportions were calculated from the molar concentrations of each isoform as reported by the Bioanalyzer 2100 software (Agilent Santa Clara CA).
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Stabilizing Selection in AS evolution in C. elegans
Two previous studies suggest a high rate of conservation of AS events between C. elegans and Caenorhabditis briggsae, two nematode species separated by 100 Myr of evolutionary divergence (Stein et al. 2003
). One study of 21 alternatively spliced cassette exons in C. elegans genes showed that 19 of these genes were also alternatively spliced in the same way in C. briggsae and that their regulation during development was also conserved (Rukov et al. 2007). A study from our group looked at 449 alternatively spliced cassette exons in C. elegans. A total of 147 of these cassette exons, when compared with C. briggsae, showed the presence of evolutionarily conserved sequence elements in the introns that flank them (Kabat et al. 2006). For these alternative cassette exons, we found that there was 81.8% nucleotide identity in the exons and maintenance of the open reading frame was conserved for 91.3% of the cassette exons. The 5' and 3' splice sites were also highly conserved. This high conservation of nucleotide and amino acid sequence, along with the maintenance of open reading frame, is consistent with a high rate of evolutionary conservation of AS between these species and seemed to suggest a difference in evolutionary rates of changes in AS relative to the rates in mammals. These observations led us to design experiments to measure the changes in AS during the process of speciation.
To measure the intraspecies level of variability in AS for C. elegans, we compared the isoform ratios of three NI lines: Bristol N2, CB4856, and AB1. The CB4856 or "Hawaiian" strain is commonly used for genetic mapping and is known to have many phenotypic differences from the reference strain N2 (Hodgkin and Doniach 1997). AB1, isolated in Australia, is a peculiar strain that is believed to be the only strain that originated by mating between strains from different C. elegans clades (Denver et al. 2003). Many genomic differences between the strains are known. For example, by using oligonucleotide array–based comparative genomic hybridization and employing a conservative approach to detect large deletions spanning several probes, Maydan et al. (2007) showed that CB4856 contains at least 141 deletions that represent 1.54 Mb (1.54% of the genome). These deletions were reported to remove 483 predicted genes.
Our splicing-sensitive microarray has been described previously (Barberan-Soler and Zahler 2008). In the present study, we used N2 embryos as the reference sample for all hybridizations. Labeled embryonic cDNAs from the reference and each of the NI lines were hybridized in triplicate to a splicing-sensitive microarray. After analyzing the data as described in Materials and Methods and Barberan-Soler and Zahler (2008), we detected that a small percentage of the splicing events have significant variability between N2 and either CB4856 or AB1. In pairwise comparisons, we found 15 events (4.3% of those tested) that significantly change (isoform ratio change >4-fold) between N2 and CB4856 (table 1) and 6 (1.7% of those tested) between N2 and AB1. For comparison, in a previous study, we showed that 18% of AS events tested showed isoform ratio changes >4-fold during the course of C. elegans development (Barberan-Soler and Zahler 2008). The differences in AS between CB4856 and AB1 against N2 correlate with the previously reported phylogeny of these three strains based on nuclear sequences (Denver et al. 2003) in which AB1 is much closer to N2 than CB4856. The isoform ratios for all 352 alternative splicing events detected in the microarray experiments for the AB1 and CB4856 lines are shown in supplementary table 1.
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To elucidate the consequences of mutation and natural selection in C. elegans transcription, Denver et al. (2005)
previously reported the differences in expression between NI and MA lines. To detect the rate at which AS can evolve when the selective pressure has been reduced to a minimum, we measured the levels of AS between N2 and two MA lines (MA41 and MA99). After applying the same experimental design as described for the NI lines, we found significantly more differences in AS regulation for the MA lines than for the NI lines (fig. 1). A total of 71 events (21% of those tested) for MA41 and 43 events (12% of those tested) for MA99 showed splicing isoform changes >4-fold when compared with the parental N2 strain from which they were derived.
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The MA lines were created starting with a single hermaphrodite N2 worm. This animal was allowed to self-fertilize, a single F1 hermaphrodite progeny was picked randomly, and this process of single hermaphrodite progeny self-fertilization was repeated for 280 consecutive generations. No selection was applied to the choice of the single hermaphrodite, and this allowed for free accumulation of mutations except for the most deleterious (Denver et al. 2005
). Denver et al. (2004) estimated that MA lines have a haploid genomic mutation rate of 2.1 mutations per genome per generation; after 280 generations, there would be 588 mutations in the genome. For CB4856, there is an average density of 2.7 variable sites/kilobase compared with N2 (Denver et al. 2003); if we extrapolate that number to the whole genome (100 Mb), then there are 270,000 variable sites in the Hawaiian strain. When comparing changes in AS between the NI and MA lines, we found that the ratios of AS events were altered for a higher proportion of genes in the MA lines; up to 71 genes in MA41 (21%), whereas only 15 genes (4.3%) had significant changes in CB4856. Given that there are predicted to be 500 times as many mutations in CB4856 than MA41 when compared with N2, this result allows us to postulate that although random accumulation of mutations can have a strong effect on AS, a strong stabilizing selection shapes the evolution of the ratios of isoforms generated by AS in C. elegans.
One of the main problems in employing microarrays to detect differences in gene expression and/or splicing during evolution is that changes in the sequence of a particular exon might interfere with probe binding. As explained in Materials and Methods and previously (Barberan-Soler and Zahler 2008), our calculation of AS variability uses the ratio of intensities for two types of probes. In this study comparing animals undergoing speciation, it is possible that ratios obtained for one of the probes will be biased toward the N2 sample due to point mutations and/or indels that interfere with the probe in the other samples. Given this, the small number of changes we detect in NIs are likely the maximum that can be detected, and if some primer-binding sites are indeed changed which we interpret as changes in AS ratios, the number of genes whose AS actually changed would go even lower. To overcome this problem, we limited the AS changes that we deem highly significant to those events where both probe ratios (inclusion or skipping) were greater than 0.5 (log scale). We found that just 25% of the changes detected for CB4856 passed this criterion, whereas for MA lines 60% of the events passed. An interesting result is that for AB1, where we just detected six changes in AS that were >2.0 (log scale), five of them pass this significance criterion. This suggests that AB1 might contain more real changes in AS than CB4856 because in CB4856 more sequence changes have collected, potentially leading to altered ability to detect the splice isoforms using the microarray. This prompted us to further analyze changes in AB1. To do this, we considered all the splicing events where the ratio of both the inclusion and skipping probes was greater than 0.5 (log scale), even when the combined isoform ratio is less than 2.0 (log scale). We found 18 events that pass this criterion (table 2).
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Changes in cis: Deletions Shape the Evolution of Isoforms
To search for mechanisms that created the variability in isoform ratios that we detected between the strains, we studied a few individual examples by RT-PCR followed by sequencing. Variability in splicing between strains can be due to cis mutations that change the splice sites or any silencer/enhancer sequences in introns or exons. Alternatively, the source of AS changes may lie in mutations in trans to the alternatively spliced gene that modify the function of splicing factors or genes required for nonsense-mediated decay (NMD) that may have an effect on the steady-state level of the spliced isoforms produced by an AS event. In C. elegans, the seven smg genes (suppressors with morphogenetic effects on genetalia) are required for NMD (Pulak and Anderson 1993
; Cali et al. 1999). In NMD, mRNAs with premature termination codons generated by mutation or AS undergo targeted degradation (for a review, see Mango 2001).
Y69H2.3 belongs to a family of protease inhibitors that have been shown to have a male-specific function. According to the microarray, CB4856 has a different isoform proportion for this gene when compared with N2 worms. Although this gene does not pass the criterion of having both inclusion and skipping probe changes having a log ratio >0.5, we found it to be a good example of how AS evolves in C. elegans. RT-PCR analysis shows that although N2 embryos have five different isoforms (fig. 2A), CB4856 embryos either skip all exons (isoform F) or include exon seven exclusively (isoform E). To further investigate this difference in AS, we sequenced the region covering all three alternatively spliced exons and found that there is a deletion of 477 bp in the CB4856 genome that removes exons 5 and 6. As shown in a PCR amplification from genomic DNA for six different NIs (fig. 2C), this deletion is specific for CB4856 and is not present in any of the other six NI lines tested. It will be interesting to perform further experiments to determine whether this deletion in a putative sperm-specific gene is responsible for any of the known phenotypic differences between CB4856 and N2 males including the formation of a copulatory plug, the endurance of the fertility period, and the number of sperm produced (Hodgkin and Doniach 1997).
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Changes in trans: Splicing versus mRNA Stability
Interestingly among the genes with changes in the isoform ratios in AB1 is swp-1, a splicing factor that we previously showed is regulated by AS coupled to NMD (Barberan-Soler and Zahler 2008
). RT-PCR analysis indicates that in N2 worms, the skipping isoform represents on average 4% of the steady-state transcripts, whereas in AB1, this proportion goes up to 33%. By sequencing the alternatively spliced region of swp-1 in AB1 and CB4856, we found no differences with respect to N2 worms in the AB1 genome; we did find however a small insertion (3 bp in intron 2) for CB4856. This leads us to propose that the differences in AS for swp-1 between AB1 and N2 are not due to mutations in the gene itself but are likely due to changes in trans.
One of the caveats to studying AS regulation by using isoform-specific microarrays is that the platform is not able to distinguish between changes at the level of splice site choice and changes in the stability of the mRNA isoforms generated by AS as we only measure changes in the relative steady-state levels of the mRNA isoforms in the worms. The changes in swp-1 that we detect in AB1 embryos were similar to the changes we observed previously in mutant N2 animals in which the NMD pathway had been inactivated. To determine whether the changes detected in swp-1 are due to a change in NMD for AB1, we analyzed by RT-PCR two other splicing factors that are known to be regulated by AS coupled to NMD (AS-NMD), hrpf-1 and rsp-4 (Morrison et al. 1997; Barberan-Soler and Zahler 2008). For both genes, we were able to detect an increase in the isoform that is targeted to NMD. hrpf-1b skips exon 5 and contains a premature termination codon that targets this isoform to NMD (fig. 3 and Barberan-Soler and Zahler 2008). Figure 3B shows how this isoform represents 31% of hrpf-1 messages in N2 embryos and 52% in AB1 embryos. AS of rsp-4 has been shown previously to be linked to NMD (Morrison et al. 1997). Although we did not detect a dramatic increase in the NMD isoform of rsp-4, we were able to detect the NMD isoform of rsp-4 in AB1 embryos at 1%, whereas this isoform is not detectable at all in N2 embryos. Even in NMD defective worms (fig. 3C), rsp-4b represents a minor isoform at only 19% of the steady-state transcripts. This is very different when compared with the levels of NMD isoforms for swp-1 and hrpf-1 (88% and 90% of the total isoforms, respectively). These percentages let us conclude that the NMD isoform for rsp-4 is a minor isoform whose level increases, but not dramatically, in AB1. On the other hand, the swp-1 and hrpf-1 NMD isoforms are major isoforms that do show clear increases in the AB1 strain consistent with a potential defect in NMD.
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The correlation between AB1 and NMD defective worms for two different splicing factors prompted us to compare the isoform ratios for all the splicing events detected with the microarray for these two samples. We used our previously reported data (smg-2(e2008) vs. N2) and compared that with the AB1 versus N2 experiment described here. We found a low correlation when we compared these two different microarray results (AB1 vs. smg-2(e2008), r = 0.201), whereas when we compared two different mutants of the NMD pathway we obtained a strong correlation (smg-1(r861) vs. smg-2(e2008), r = 0.695). This causes us to speculate that even if there is a defect in NMD in AB1 worms, this pathway is for the most part still functional and that the defect leads to only a mild effect on a small subset of genes. It would be interesting to further characterize this difference to determine whether changes in isoform ratios in AB1 are at the level of message stability or AS. If these changes are at the level of isoform stability, this strain would become a useful tool for understanding the way by which the NMD machinery is directed to specific mRNAs in worms as the rules for targeting messages for NMD in worms appear to be different from those in mammals (Longman et al. 2007
). To further characterize the importance of mRNA stability in the evolution of the ratios of isoforms created by AS, we compared the microarray results from the MA lines against the NMD defective strain smg-2(e2008). We found that there is a moderate correlation (r = 0.522 for MA41 and r = 0.446 for MA99) between MA lines and smg-2(e2008). From the changes in MA41 with more than >4-fold difference with N2, there are 13/71 that also have >4-fold difference between smg-2(e2008) and N2. It is then possible that the changes in ratios of alternatively spliced products of this subset of genes are not due to changes in AS but to changes in mRNA stability of the alternative isoforms. This allows us to conclude that both the AS of genes and the steady-state mRNA levels of the isoforms created by this process are under strong stabilizing selection and that in the MA lines these levels are altered by both changes in AS and in the stability of the mRNAs due to changes in NMD.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We demonstrate that NIs of C. elegans, which have a high number of variable sites, have low variability in the steady-state mRNA levels of AS isoforms. In contrast, the MA lines, with a much lower number of variable sites, achieve a much higher variability. This allows us to conclude that the ratios of isoforms generated by AS in C. elegans are under a strong stabilizing selection. This conclusion indicates that nematodes in the Caenorhabditis genus have a much lower rate of AS evolution than insects or mammals. Populations of C. elegans have been shown to have a low genetic diversity that is influenced by a surprisingly small effective population size (Ne) that is comparable to the one found in humans (Barriere and Felix 2005
; Tenesa et al. 2007). This low genetic diversity can affect the evolution of processes like AS and gene expression. In particular, several differences in the splicing machinery between worms and mammals may be the source of the difference in evolutionary conservation of AS found in the present study. It is known that the splice signals in worms are particularly well conserved and represent strong consensus sequences, whereas in mammals, the consensus sequences at the splice sites for many exons are weaker and require the presence of splicing enhancers in order to be recognized by the splicing machinery (Fahey and Higgins 2007; Wang and Burge 2008). We previously showed that the introns flanking alternatively spliced exons often contain evolutionarily conserved sequences important for AS regulation in nematodes (Kabat et al. 2006). Caenorhabditis elegans possesses much smaller introns relative to vertebrates (Kim et al. 2007). This smaller size makes the worm introns richer in splicing regulatory information at the nucleotide level relative to vertebrate intron sequences. This difference may make worms much less tolerant of insertions and deletions in introns, restricting the possibilities for the evolution of changes in AS relative to organisms with much larger intron size.
Sorek et al. (2002) showed that close to 5% of the human alternatively spliced exons are alu-derived. Working in C. elegans, Carr and Anderson (1994) reported that the excision of Tc1 transposons from a gene leaves a residual sequence that is similar to a 5’ splice site. Anderson's group also reported that several Tc1 transposons can be removed by splicing so that their sequences do not get incorporated into the mature mRNA, thus allowing them to escape the negative selection that comes from mutating their host genomes (Rushforth and Anderson 1996). Therefore, we can think of this as Tc1-intronization, in contrast to alu-exonization. It is possible that differences of this type are responsible for a completely distinct evolution of AS in worms and mammals. Whereas alu elements allow the evolution of new functions for alternatively spliced exons in humans, in C. elegans, the creation of new alternatively spliced exons might start with the regulated exclusion of constitutive exons.
The higher conservation of AS in C. elegans can also help explain the different phenotypes found in NMD mutants between worms and other species (Metzstein and Krasnow 2006). In a species with a lower percentage of aberrant AS-like worms, the need for a surveillance pathway is not as important as in other species such as humans where alu element insertion can lead to the formation of new exons that may cause alterations to the reading frame and lead to nonsense mRNAs. These differences may help to explain why a mutation in the NMD pathway in C. elegans will still produce viable worms, whereas mutations in other species are lethal (Rehwinkel et al. 2005). One can even propose that in C. elegans, the NMD pathway is used more as a gene regulation mechanism and not as a surveillance pathway to degrade toxic mRNAs produced in genes that are testing potential new AS.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary table 1 is available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We are grateful to Lily Shiue and Manny Ares for establishing the University of California, Santa Cruz microarray facility and for assistance with its usage. Many thanks to Lily Shiue, Manny Ares, Grant Hartzog, Susan Strome, and all the Zahler laboratory members for helpful discussions. Thanks to the C. elegans Genetics Center, Kevin Simonelic, and Michael Lynch for kindly providing strains. This research is supported by a grant from the National Institutes of Health (R01-GM61646) to A.M.Z. S.B.S. is supported by a UC Mexus-CONACYT Doctoral Fellowship and the Miguel Velez Scholarship.
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Footnotes |
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Douglas Crawford, Associate Editor
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