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International Journal of Cell Biology
Volume 2013 (2013), Article ID 636050, 10 pages
Alternative Splicing Regulation of Cancer-Related Pathways in Caenorhabditis elegans: An In Vivo Model System with a Powerful Reverse Genetics Toolbox
1SomaGenics, Inc., Santa Cruz, CA 95060, USA
2Department of Molecular, Cell and Developmental Biology, The Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, CA 95064, USA
Received 17 June 2013; Accepted 29 July 2013
Academic Editor: Claudio Sette
Copyright © 2013 Sergio Barberán-Soler and James Matthew Ragle. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Alternative splicing allows for the generation of protein diversity and fine-tunes gene expression. Several model systems have been used for the in vivo study of alternative splicing. Here we review the use of the nematode Caenorhabditis elegans to study splicing regulation in vivo. Recent studies have shown that close to 25% of genes in the worm genome undergo alternative splicing. A big proportion of these events are functional, conserved, and under strict regulation either across development or other conditions. Several techniques like genome-wide RNAi screens and bichromatic reporters are available for the study of alternative splicing in worms. In this review, we focus, first, on the main studies that have been performed to dissect alternative splicing in this system and later on examples from genes that have human homologs that are implicated in cancer. The significant advancement towards understanding the regulation of alternative splicing and cancer that the C. elegans system has offered is discussed.
Since the early 1960s with the efforts of Sydney Brenner, the nematode Caenorhabditis elegans has been established as a popular model organism in developmental biology and neurobiology. There are many biological advantages that make it an attractive system for several fields of research. The adult worm contains 959 somatic cells, making its anatomy relatively simple. Experiments in the late 1970s showed that it has an invariant cell lineage during establishment of the somatic tissues . It has two sexes, a self-fertilizing hermaphrodite and males, allowing genetic crosses to be performed. C. elegans has a short life cycle of less than three days and each hermaphrodite produces about 300 progeny by self-fertilization or up to 1000 progeny from cross progeny with males. The gonad is a relatively large organ in this animal, allowing for studies of organogenesis, cell proliferation, meiosis, and embryogenesis. During its development, the hermaphrodite worms produce sperm at one stage of their life cycle before switching to produce oocytes. The molecular pathways of this sperm to oocyte transition have been studied extensively . Its complete genome, sequenced in 1998, was the first sequenced genome from a multicellular organism. It has a genome size of 97 megabases containing close to 19,000 protein coding genes . Genome-wide alignments with other five related nematodes are now available for any comparative genomics approach . The modENCODE project systematically generated genome-wide data from transcriptome profiling, transcription factor-binging sites, and maps of chromatin organization to improve genome annotation . When comparing the C. elegans genome to higher eukaryotes, it was found that close to 40% of the genes that have been associated with diseases in humans have worm orthologues, and cancer is not the exception .
2. Alternative Splicing Prevalence
In 1990, the first report of an alternative splicing event in the C. elegans genome was published. Interestingly the event corresponds to the PKA mRNA, a kinase implicated in the onset and progression of several cancers . Since then, several groups have used different approaches to predict the percentage of genes in the C. elegans genome that undergo alternative splicing. Initial estimates using a limited number of Expressed Sequence Tags (ESTs) predicted fewer than 1000 genes to be alternatively spliced . By normalizing the occurrence of alternative splicing, taking into account the coverage of ESTs, it was estimated that close to 10% of the C. elegans genome undergoes alternative splicing . More recent analysis using next-generation sequencing identified 8,651 putative novel splicing events, suggesting that up to 25% of genes have an alternative splicing event (Figure 1(a)) . While this percentage is far from the >90% reported for the human genome, it does show that alternative splicing in C. elegans is not an uncommon mechanism to generate protein diversity.
3. Effects of Genome Compaction on AS
The worm genome appears to be under selective pressure to promote a reduction in genome size . This natural selection towards a small genome can be seen in features like short intergenic regions, short UTRs, and small introns [8, 12, 13]. For example, in humans, the median size of introns in the coding sequences is 1,334 bases, while in worms the average intron is just 65 bases (Figure 1(b)) [4, 14]. More than 20 years ago, the small size of C. elegans introns was already the subject of study. It was demonstrated that a short 53 nucleotide worm intron could not be efficiently spliced in human extracts, while an expansion of this intron with 31 extra nucleotides allowed for efficient splicing . In spite of this reduction in intron size, the worm spliceosome is still capable of removing big introns (144 introns in the C. elegans genome are bigger than 10 kb (Figure 1(c)) ). Larger intron size has been correlated with alternative splicing in several species . Worms also have complex patterns of alternative splicing where multiple exons in the same gene are alternatively spliced to generate multiple isoforms (Figure 1(d)). This means that the information content of a worm intron is on average greater than in higher eukaryotes (higher density of functional elements in introns). This makes the molecular dissection of worm introns easier to achieve. By using genomic alignments between two Caenorhabditis species, the identification of novel intronic elements important for alternative splicing regulation has been described . Other groups have also used comparative genomics together with UV cross-linking and Electrophoretic Mobility Shift Assays (EMSA) to identify cis-elements important for alternative splicing regulation .
4. Evidence That AS in Nematodes Is under Stabilizing Selection
Several studies comparing alternative splicing events in mammals and insects concluded that a high percentage of the events are not conserved and are species specific [19, 20]. This high variability in alternative splicing makes it necessary to validate the functionality of the events studied. To test whether the smaller percentage of genes with alternative splicing in worms also follows these patterns of high variability during evolution, several groups have measured the levels of conservation between different C. elegans populations or between related species [21, 22]. In comparison to the findings in higher eukaryotes, the regulation of alternative splicing in natural populations of nematodes appears to be under strong stabilizing selection with low intra- an interspecies variability. These results point to an essential intrinsic characteristic of alternative splicing in worms: its functionality. The detection of alternative splicing in a C. elegans transcript has a higher probability of being a functional and regulated event than in other systems.
5. Alternative Splicing Regulation
A proxy for the functionality of an alternative splicing event that has been used for other systems is its regulation. If a particular event is detected as regulated across different conditions or during development then the possibility that the isoforms have specific functions is higher. In the last five years C. elegans joined other species in terms of the detection of changes in alternative splicing at genome-wide levels. Initially with the use of splicing-sensitive microarrays and later with the use of next-generation sequencing the regulation of alternative splicing in worms has been studied in detail across different conditions and mutations [10, 23, 24]. Initial measurement of changes in splicing during development demonstrated that up to 40% of the events detected are regulated (>2 fold) between different stages of worms (Figure 1(a)) . This result was further validated with tiling arrays and next-generation sequencing . Several examples of tissue-specific splicing are known. The alternative splicing regulation of a neuron-specific exon of unc-32, the worm a subunit of V0 complex of vacuolar-type H+-ATPases, was recently characterized . A male-specific isoform of unc-55, a transcription factor, has also been reported . While individual examples of tissue-specific splicing are known, a genome-wide analysis of tissue-specific splicing is still missing. With the increased sensitivity of next-generation sequencing techniques, together with the availability of manual or molecular dissections that allow the isolation of tissue-specific mRNA (mRNA-tagging ) a complete catalog of tissue-specific splicing should be possible in the near future.
While a big set of splicing events produce two protein isoforms, another important group introduces premature termination codons (PTC) to one of the isoforms. It is known that the introduction of a PTC to one isoform targets it to nonsense-mediated decay (NMD) (reviewed in ). NMD mutants were first described in C. elegans almost 25 years ago . Contrary to other systems where mutations in NMD factors are lethal, in worms, null mutations for all the seven core factors of NMD are viable (smg-1 to smg-7). This has facilitated the study of AS coupled to NMD in worms. Some of the first events of wild-type transcripts that were shown to be regulated by NMD are splicing factors in the C. elegans genome . An important discovery concerning the regulation of alternative splicing in worms is that between 20–35% of the events in the genome appear to be targets of NMD (Figure 1(a)) [24, 31]. The conclusion from these studies is, then, that alternative splicing in C. elegans is not just a generator of protein diversity but also an important regulator that fine-tunes gene expression levels by targeting specific isoforms for degradation. Furthermore, it has been proposed that NMD in worms can also be regulated with the potential to stabilize particular NMD targets allowing them to be translated into truncated proteins with putative dominant negative functions .
6. Powerful Reverse Genetics to Dissect Alternative Splicing
One of the advantages of C. elegans as a model system is the availability of powerful tools for reverse genetics. Any laboratory can obtain stable mutants for many genes from the Caenorhabditis Genetics Center (CGC) at The University of Minnesota. The National Bioresource Project in Japan runs a program where mutants for a gene of interest can be requested and they are obtained at the facility by a protocol involving random mutagenesis with TMP/UV. These two centers allow for any group to obtain mutant strains for the gene of interest in an inexpensive and expedited manner. Recently, a more ambitious project to create a million different mutants has been performed by the Moerman and Waterston labs . The aim of this project was to use next-generation sequencing and a collection of 2000 mutagenized strains to identify multiple mutations in virtually all the genes in the worm genome. This project will likely allow the study of mutant worms that have mutations in isoform-specific regions. All these resources allow for the use of stable mutants to characterize the roles of either putative splicing factors or more specifically of particular isoforms. This approach to screen the effects that mutations in different splicing factors have on a particular splicing event has been used before . The conclusion from this work was that the coregulation of alternative splicing by diverse factors is a common phenomenon in worms.
Another great resource for the worm community is the availability of genome-wide RNAi libraries . Researchers aiming to characterize genetic pathways have used these libraries for genome-wide screens. Some of them have found that several splicing-related components have interesting phenotypes when targeted by RNAi . More focused screens with a subset of clones from these libraries are used to characterize a specific group of genes. For example, Kerins et al. used an RNAi screen of all the predicted C. elegans splicing factors and found that many of them showed an overproliferation phenotype in a sensitized germline background .
7. In Vivo Alternative Splicing Reporters
One of the most advantageous tools for the study of alternative splicing in C. elegans is the use of bichromatic alternative splicing reporters . This transgenic reporter system allows the visualization of splicing events by tagging each one of the isoforms with a different fluorescent protein. A worm population with differences in splicing regulation can then be sorted using FACS cytometry and worms with a particular splicing ratio obtained. This together with the use of mutagenesis or RNAi allows performing an in vivo screen for regulators of any splicing event of interest. The advantage of these reporters in worms is that the characterization of an alternative splicing event can be performed in vivo. Different events that have been studied with this technology are egl-15, let-2, unc-60, and unc-32 [25, 37–40]. This technology has allowed the identification of splicing factors that regulate these events, as well as the cis-elements that are necessary for its regulation.
8. C. elegans AS Regulation of Cancer-Related Pathways
Given the diversity of tools available to perform studies in C. elegans, researchers have set out to understand details about cancer that were long a mystery. Questions surrounding cell proliferation and cell-cell communication as well as the cellular and molecular components that make up a stem cell niche often must be answered in a multicellular context. Furthermore, the fact that biological processes and factors active in splicing and human cancers are almost wholly homologous with those in C. elegans makes the move to worm studies rewarding. Disrupted cancer pathways in C. elegans, for the most part, have clear, simple, and observable phenotypes that extend beyond just cell death or proliferation. Using these pathways as tools and read-outs, splicing factors and splicing events have been identified, in C. elegans, to interact with, regulate, and cooperate with homologous pathways that lead to cancer in humans (Figure 2).
8.1. Extensive Splicing of Ras/let-60 Pathway Components Contributes to Cell Fate Determination and Proliferation
Overwhelming evidence links cell signaling and gene expression changes in the promotion of human cancers. Alternative splicing plays a major role in defining the activity of signal transduction pathways such as Ras/let-60. Strikingly, of the 50–60 genes that are known components of, regulate or, are directly regulated by the Ras/let-60 pathway in C. elegans , approximately half of them produce multiple isoforms differing by at least one alternative exon . This highlights the importance of proper gene expression through post-transcriptional regulation (Table 1).
The ligand-dependent Ras pathway in C. elegans stimulates the induction of vulval precursor cells (VPCs) in the hypodermis to divide and differentiate into a functional vulva during larval development [42, 43]. Increased signaling from the Ras pathway leads to a multivulval (Muv) phenotype, while decreased signaling causes a vulvaless (Vul) phenotype . These simple and observable phenotypes provide an excellent system to ascertain the contributions of alternative splicing to Ras signaling activity. The C. elegans homolog of the Ras-MAPK pathway stimulant Epidermal Growth Factor, lin-3, produces several isoforms with unique characteristics. LIN-3 isoform specialization of function has previously been observed in pathways aside from Ras-mediated cell signaling and has been found to differentially mediate growth rates, feeding behavior, and cellular quiescence . In VPCs, the activity of the LIN-3L isoform is dependent on interaction with the C. elegans Rhomboid protease homolog, ROM-1, while the activity of LIN-3S is not . LIN-3S is expressed in a specialized Anchor Cell (AC) responsible for inducing a primary set of VPCs during larval development, and LIN-3L expression and possible secretion by primary VPCs themselves act as a ligand stimulant of the Ras pathway in secondary VPCs [42, 43]. This stimulation may cause the secondary VPCs to switch their cell fate from hypodermis to vulva . It is unknown if coordinated expression of these isoforms from distinct cell types establishes a gradient through Ras signaling that induces nearby VPCs to proceed through necessary cell divisions to develop into a proper vulva.
RasGAPs are GTPase-activating proteins that specifically promote the hydrolysis of Ras-bound GTP molecules, thereby inactivating the Ras molecule and its signaling . gap-2, in C. elegans, is similar to the p120 Ras-GAP family . The gene contains 25 exons that use alternative splicing and transcription start sites to produce 9 mRNA products . These isoforms were identified by a rapid amplification of cDNA end (5′RACE) technique using primers specific to a sequence common to many mature mRNAs in C. elegans, the SL1 trans-splice leader. This 22nt sequence is naturally spliced onto 5′ ends of transcripts from ~70% of genes . In this case, they served as 5′ primer binding sites in reverse transcription reactions followed by PCR to identify isoforms with alternative 5′ ends and promoters . Differential expression of gap-2 isoforms in several tissue types was revealed by expression of transgenes containing these alternative gap-2 promoters fused to GFP in C. elegans. The function of RasGAP proteins from various genes in other organisms  may be relegated to multiple isoforms from a small number of RasGAP genes producing many isoforms in C. elegans. While the effects of this dynamic expression pattern of isoforms on molecular signaling pathways such as the Ras pathway are difficult to tease apart, target sites for PKC, PKA, and protein tyrosine kinases present or absent in each isoform  may provide clues to individual isoform activities.
8.2. Redundant Retinoblastoma/lin-35 Pathways Are Populated by Splicing Factors
Homozygous mutations of the retinoblastoma (Rb) gene have been shown to promote retinal cancer, small-cell lung carcinomas, and osteosarcomas . In worms, a single homolog of Rb, lin-35, represses the multivulval phenotype observed in Ras pathway hyper-signaling mutants . Two redundant pathways, termed synthetic multivulval (synmuv) class A and class B, are active in the VPCs and induce changes in expression of genes that lead to a multivulval fate through histone modification and chromatin remodeling [53, 54]. To induce a phenotype, disruptive mutations in single genes within each class must be present. Interestingly, mutations in the lin-15 locus created alleles with Muv, class A synmuv, and class B synmuv phenotypes, leading to speculation that two genes existed at the locus with distinct functions in each pathway. In RNAi screens for interactors of the class B synmuv pathway in C. elegans, 57 genes were found to enhance the Muv phenotype observed in lin-35 or other synmuv class B mutant lines [35, 55]. Ten of these potential interactors (rsr-2, lsm-2/gut-2, lsm-4, snr-1–7) have roles in splicing. Their synmuv B mutant phenotypes are dependent on Ras activity and lin-3 ligand binding. While the activities, interactions, and mutant phenotypes of most of these splicing-related Rb enhancers are not well understood, RNAi of some of the snr genes (snr-1, snr-2, snr-4, snr-5, snr-6, and snr-7) led to embryonic lethality and nuclear pore organization disruption . The question of whether these phenotypes arise from disrupted functions that have no role in splicing regulation is still unanswered, but evidence is mounting supporting the idea that constitutive splicing and alternative splicing are major contributors in C. elegans to pathways that, when altered, lead to cancer in human tissues. Undoubtedly, future studies in C. elegans will enrich current knowledge concerning the interwoven activities of splicing and the Rb/lin-35 pathway.
8.3. Protein Kinase A Isoform Diversity Replaces the Need for Multiple Genetic Loci
Protein kinase A (PK-A) is involved in many cellular processes and has been implicated in several cancers . The mammalian PK-A is composed of two distinct subunits, the regulatory and catalytic, that are each interchangeable with protein subunits from several genetic loci. The two subunits of PK-A are conserved in C. elegans  but derived from possibly only three genes, kin-1, kin-2, and F47F2.1. Interestingly, the modularity of the subunits that make up PK-A in C. elegans has been conserved through alternative usage of exons. The catalytic subunit (C-subunit) alone is thought to undergo alternative splicing at both N- and C-termini to create at least 12 different isoforms . These isoforms are conserved in C. briggsae, a relative of C. elegans, and show differential expression during development, suggesting that the diversity of potential subunits itself has functional importance . An isoform of the C-subunit containing the N′1 exon harbors a myristoylation site and is highly expressed in eggs, where an alternative exon N′4 lacking the myristoylation site is highly expressed in adult worms . The presence of N′1 protein isoform expression and myristoylation was found to affect substrate targeting of PK-A but not the catalytic activity of the enzyme itself . Similarly, isoforms of the regulatory (R-subunit) have recently been identified and found to contain or lack domains necessary for docking to A-kinase anchor proteins and the C-subunit of the PK-A holoenzyme . A second gene on the X chromosome of C. elegans, F47F2.1, has homology to murine Cα subunits . Like the kin-1 N′4 isoform, expression of the longer F47F2.1b isoform is low in eggs but high in mixed populations of worms . A truncated isoform, F47F2.1a, lacks amino acids important for ATP-binding, and knockdown of N′4 kin-1 isoform has no obvious phenotype leading to speculation that some of these isoforms may be redundant or unnecessary [59, 62].
8.4. Topoisomerase-1 Isoforms Have Differential Expression and Potential SR Kinase Activity
Isoforms from the same genetic locus often have distinct expression patterns and functions that are seemingly unrelated. C.elegans DNA topoisomerase I (top-1) produces two isoforms that vary in temporal and spatial expression patterns. Cellular and subcellular localization differences between the isoforms lead to speculation they may function in different processes. The TOP-1β isoform skips exon 2 and is more ubiquitously expressed throughout C. elegans development, being found in multiple cell types and all stages from embryo through adulthood. Conversely, the inclusion isoform, TOP-1α, is detectably present in embryos and, interestingly, in neurons at the comma stage, then decreases in abundance as worms enter larval stages . Isoform-specific immuno-histochemical localization assays in germline cells identified TOP-1β concentration in nucleoli and TOP-1α concentration at centrosomes and on chromosomes [64, 65]. Expression of a GFP transgene driven by the topoisomerase promoter was also detected strongly in the distal tip cell of L3/L4 worms indicating that somatic gonad sheath development and/or germline stem cell proliferation may be regulated by TOP-1 activity. RNAi of C. elegans top-1 reduced the number of germ cells in the mature germline by anywhere from 50–100% . It is currently unknown how top-1 isoforms contribute to the maintenance of stem cell proliferation or exactly which isoform would be performing such functions. Localization of the inclusion isoform, TOP-1α, to centrosomes and chromosomes suggests it could be involved in chromosome segregation, in regulation of transcription, or possibly in posttranscriptional regulation. DNA topoisomerase I was identified as an SR protein kinase in HeLa cell extracts . It was found to phosphorylate splicing factors involved in cell cycle regulation, such as SF2/ASF [67, 68]. It may be possible that these isoforms are working more closely than originally thought if one or both of the top-1 isoforms affect the cell cycle and carry the same SR kinase activity as shown in HeLa cells. The activity of one or both of the TOP-1 isoforms as SR protein kinases suggests that top-1 alternative splicing may be autoregulated. Kinase activity of TOP-1 and its autoregulation have yet to be determined in worms.
8.5. Splicing Factors as Regulators of Cell Proliferation and Differentiation
Several genes involved in splicing regulation have been implicated in the proliferation/differentiation decision in the C. elegans germline. glp-1(ar202gf) worms were used to perform an RNAi screen searching for genes that led to increased cell proliferation in the germline. The resulting genes included factors involved in every step of the splicing process (spliceosome construction initiation, reorganization of the snRNP complex and removal of the lariat; ). prp-17, for example, encodes an ortholog of yeast CDC40 and human prp17 and is involved in the 2nd catalytic step of intron removal in the spliceosome [36, 69]. RNAi of prp-17 in the rrf-1(pk1417);glp-1(oz264) background enhanced germline tumors, suggesting that it is involved in the proliferation versus differentiation decision. Similarly, the mog genes represent C. elegans homologs of core yeast splicing factors that regulate the pathway downstream of glp-1 to promote differentiation and the oocyte fate of maturing germ cells [70–72]. The U2 snRNP-associated splicing complex SF3b is essential for splicing [73, 74]. TEG-4 is the worm homolog of human SF3b subunit 3 (aka SAP130) that increases excessive cell proliferation in the glp-1(ar202gf) background . Interestingly, epistasis experiments attempting to determine the genetic relationship between teg-4 and the glp-1/Notch signaling pathway in various cell types were inconclusive. Human CD2BP2 was suggested to regulate splicing via U4_U5_U6 tri-snRNP formation . TEG-1, the C. elegans homolog of CD2BP2 has been found to bind UAF-1, the worm U2AF large subunit homolog, suggesting that it could work in two important steps in the mechanism of pre-mRNA splicing . teg-1(oz230), in combination with a glp-1 mild gain-of-function mutant background, produces a tumorous germline phenotype as well. To what extent splicing factors, themselves, have individual genetic interactions within the proliferation versus differentiation pathways or if global splicing regulation is more indirectly influential is still unknown.
8.6. CED-4 Isoforms Promote and Inhibit Systematic Apoptosis during Development
Disruption of alternative splicing patterns may affect the onset of programmed cell death so intricately regulated in C. elegans. The worm homolog of APAF1, ced-4, was first identified as a core factor in the developmental induction of neuronal programmed cell death . ced-4 physically links ced-3, a member of a caspase family of proteases to ced-9, a cell death suppressor [79–81]. As a regulatory switch, ced-4 is a core determinant of the decision of a cell to be systematically culled or not. ced-4 encodes two protein isoforms, CED-4L and CED-4S, differing by 24 amino acids at the 5′ end of exon 4. While CED-4S normally promotes programmed cell death, overexpression of the CED-4L isoform led to ectopic cell growth and rescue of lethal phenotypes seen in ced-9 loss-of-function mutants [82, 83]. It is CED-4L association with CED-3 that surprisingly inhibits cell death in a dominant negative manner. The presence of the longer P-loop in the long isoform still allows for association of CED-4L with the protease domain of CED-3 but disrupts the association of CED-4L with the prodomain of CED-3, which normally contributes to its activation . Studies in mammalian cells have similarly identified ced-3 and ced-4 homologous genes that produce isoforms with opposing cell death functions [85, 86]. An imbalance in the concentrations of these isoforms or in the abundance or activities of specific SR or hnRNP proteins often leads to one developmental cell fate over the other . Complex regulation of alternative splicing factors, therefore, may direct developmental apoptosis programs. Loss-of-function of an SR protein kinase, spk-1, in a ced-4 partial loss-of-function background, as well as mutant alleles of several SR proteins themselves, leads to an increase in apoptosis , suggesting that control of alternative splicing factor activity may play a direct or indirect role in the regulation of developmentally programmed apoptosis. In vitro studies have shown that SPK-1 can bind and phosphorylate alternative splicing factors like SF2 and RNAi-mediated knockdown of spk-1 in C. elegans causes embryonic lethality and germline development defects [89, 90]. Experiments aimed at understanding the complex code that dictates what genetic material is included or skipped in mature mRNA and subsequent protein products will undoubtedly clarify further the impact that alternative splicing has in the mediation of programmed cell death through factors such as ced-4.
8.7. Research Focusing on the Links between Splicing and Cancer in C. elegans Is Bright
Great strides have been taken in research to understand the involvement of splicing factors in biological processes such as cell proliferation, differentiation, migration, communication, and death (as discussed previoussly). RNAi screens have identified splicing factors that promote or inhibit cell proliferation. Transgenic assays have revealed cellular and subcellular localization of specific isoforms that may be involved in apoptotic or cell cycle regulation. Transparent cuticles and eggs make worms ideal specimens for the use of fluorescent reporters. Lineage tracing experiments have not only identified every cell in an adult worm but also precursors and cell behavior throughout development. Furthermore, only in a multicellular organism, such as C. elegans, can the effects of cell-to-cell signaling on processes such as differentiation or cell proliferation become apparent. High-throughput sequencing and bioinformatic analyses are opening a new chapter on comparisons in isoform usage between genetic backgrounds. The modEncode project seeks to identify functional elements within the Drosophila and Caenorhabditis genomes by providing access to high-quality gene expression datasets. Furthermore, the million-mutation project in C. elegans will allow researchers to obtain gene and even isoform-specific mutant strains for further study. Given the speed and depth of discoveries using methods such as RNAi in worms, this project should make C. elegans a core model to study alternative splicing and its link to genes homologous to those tied to cancer in humans.
James Matthew Ragle is funded by a Grant from the National Science Foundation to Alan Zahler, University of California, Santa Cruz (MCB1121290). Funding for open access is provided by the UC Santa Cruz Open Access Fund.
- J. E. Sulston and H. R. Horvitz, “Post embryonic cell lineages of the nematode. Caenorhabditis elegans,” Developmental Biology, vol. 56, no. 1, pp. 110–156, 1977.
- S. Zanetti, S. Grinschgl, M. Meola, et al., “The sperm-oocyte switch in the C. elegans hermaphrodite is controlled through steady-state levels of the fem-3 mRNA,” RNA, vol. 18, no. 7, pp. 1385–1394, 2012.
- C elegans Sequencing Consortium, “Genome sequence of the nematode C. elegans: a platform for investigating biology,” Science, vol. 282, no. 5396, pp. 2012–2018, 1998.
- K. Yook, T.W. Harris, T. Bieri, et al., “WormBase 2012: more genomes, more data, new website,” Nucleic Acids Research, vol. 40, pp. D735–D741, 2012.
- M. B. Gerstein, Z. J. Lu, E. L. Van Nostrand, et al., “Integrative analysis of the Caenorhabditis elegans genome by the modENCODE Project,” Science, vol. 330, no. 6012, pp. 1775–1787, 2011.
- E. Culetto and D. B. Sattelle, “A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes,” Human Molecular Genetics, vol. 9, no. 6, pp. 869–877, 2000.
- R. E. Gross, S. Bagchi, X. Lu, and C. S. Rubin, “Cloning, characterization, and expression of the gene for the catalytic subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. Identification of highly conserved and unique isoforms generated by alternative splicing,” Journal of Biological Chemistry, vol. 265, no. 12, pp. 6896–6907, 1990.
- W. J. Kent and A. M. Zahler, “The intronerator: exploring introns and alternative splicing in Caenorhabditis elegans,” Nucleic Acids Research, vol. 28, no. 1, pp. 91–93, 2000.
- D. Brett, H. Pospisil, J. Valcárcel, J. Reich, and P. Bork, “Alternative splicing and genome complexity,” Nature Genetics, vol. 30, no. 1, pp. 29–30, 2002.
- A. K. Ramani, J. A. Calarco, Q. Pan et al., “Genome-wide analysis of alternative splicing in Caenorhabditis elegans,” Genome Research, vol. 21, no. 2, pp. 342–348, 2011.
- D. R. Denver, K. Morris, M. Lynch, and W. K. Thomas, “High mutation rate and predominance of insertions in the Caenohabditis elegans nuclear genome,” Nature, vol. 430, no. 7000, pp. 679–682, 2004.
- C. H. Jan, R. C. Friedman, J. G. Ruby, and D. P. Bartel, “Formation, regulation and evolution of Caenorhabditis elegans 3'UTRs,” Nature, vol. 469, no. 7328, pp. 97–103, 2011.
- L. W. Hillier, A. Coulson, J. I. Murray, Z. Bao, J. E. Sulston, and R. H. Waterston, “Genomics in C. elegans: so many genes, such a little worm,” Genome Research, vol. 15, no. 12, pp. 1651–1660, 2005.
- X. Hong, D. G. Scofield, and M. Lynch, “Intron size, abundance, and distribution within untranslated regions of genes,” Molecular Biology and Evolution, vol. 23, no. 12, pp. 2392–2404, 2006.
- S. C. Ogg, P. Anderson, and M. P. Wickens, “Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract,” Nucleic Acids Research, vol. 18, no. 1, pp. 143–149, 1990.
- N. P. Kandul and M. A. F. Noor, “Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3,” BMC Genetics, vol. 10, article 1471, p. 67, 2009.
- J. L. Kabat, S. Barberan-Soler, P. McKenna, H. Clawson, T. Farrer, and A. M. Zahler, “Intronic alternative splicing regulators identified by comparative genomics in nematodes,” PLoS Computational Biology, vol. 2, no. 7, article e86, 2006.
- H. Kuroyanagi, G. Ohno, S. Mitani, and M. Hagiwara, “The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo,” Molecular and Cellular Biology, vol. 27, no. 24, pp. 8612–8621, 2007.
- B. Modrek and C. J. Lee, “Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss,” Nature Genetics, vol. 34, no. 2, pp. 177–180, 2003.
- D. B. Malko, V. J. Makeev, A. A. Mironov, and M. S. Gelfand, “Evolution of exon-intron structure and alternative splicing in fruit flies and malarial mosquito genomes,” Genome Research, vol. 16, no. 4, pp. 505–509, 2006.
- S. Barberan-Soler and A. M. Zahler, “Alternative splicing and the steady-state ratios of mRNA isoforms generated by it are under strong stabilizing selection in Caenorhabditis elegans,” Molecular Biology and Evolution, vol. 25, no. 11, pp. 2431–2437, 2008.
- M. Irimia, J. L. Rukov, D. Penny, J. Garcia-Fernandez, J. Vinther, and S. W. Roy, “Widespread evolutionary conservation of alternatively spliced exons in Caenorhabditis,” Molecular Biology and Evolution, vol. 25, no. 2, pp. 375–382, 2008.
- S. Barberan-Soler and A. M. Zahler, “Alternative splicing regulation during C. elegans development: splicing factors as regulated targets,” PLoS Genetics, vol. 4, no. 2, Article ID e1000001, 2008.
- A. K. Ramani, A. C. Nelson, P. Kapranov, I. Bell, T. R. Gingeras, and A. G. Fraser, “High resolution transcriptome maps for wild-type and nonsense-mediated decay-defective Caenorhabditis elegans,” Genome Biology, vol. 10, no. 9, article R101, 2009.
- H. Kuroyanagi, Y. Watanabe, and M. Hagiwara, “CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans,” PLoS Genetics, vol. 9, no. 2, Article ID e1003337, 2013.
- G. Shan and W. W. Walthall, “Copulation in C. elegans males requires a nuclear hormone receptor,” Developmental Biology, vol. 322, no. 1, pp. 11–20, 2008.
- F. Pauli, Y. Liu, Y. A. Kim, P.-J. Chen, and S. K. Kim, “Chromosomal clustering and GATA transcriptional regulation of intestine-expressed genes in C. elegans,” Development, vol. 133, no. 2, pp. 287–295, 2006.
- S. Kervestin and A. Jacobson, “NMD: a multifaceted response to premature translational termination,” Nature Reviews Molecular Cell Biology, vol. 13, no. 11, pp. 700–712, 2012.
- J. Hodgkin, A. Papp, R. Pulak, V. Ambros, and P. Anderson, “A new kind of informational suppression in the nematode Caenorhabditis elegans,” Genetics, vol. 123, no. 2, pp. 301–313, 1989.
- M. Morrison, K. S. Harris, and M. B. Roth, “smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 18, pp. 9782–9785, 1997.
- S. Barberan-Soler, N. J. Lambert, and A. M. Zahler, “Global analysis of alternative splicing uncovers developmental regulation of nonsense-mediated decay in C. elegans,” RNA, vol. 15, no. 9, pp. 1652–1660, 2009.
- O. Thompson, M. Edgley, P. Strasbourger, et al., “The Million Mutation Project: a new approach to genetics in Caenorhabditis elegans,” Genome Research, 2013.
- S. Barberan-Soler, P. Medina, J. Estella, J. Williams, and A. M. Zahler, “Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans,” Nucleic Acids Research, vol. 39, no. 2, pp. 666–674, 2011.
- R. S. Kamath, A. G. Fraser, Y. Dong et al., “Systematic functional analysis of the Caenorhabditis elegans genome using RNAi,” Nature, vol. 421, no. 6920, pp. 231–237, 2003.
- J. Ceron, J.-F. Rual, A. Chandra, D. Dupuy, M. Vidal, and S. Van Den Heuvel, “Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity,” BMC Developmental Biology, vol. 7, article 30, 2007.
- J. A. Kerins, M. Hanazawa, M. Dorsett, and T. Schedl, “PRP-17 and the pre-mRNA splicing pathway are preferentially required for the proliferation versus meiotic development decision and germline sex determination in Caenorhabditis elegans,” Developmental Dynamics, vol. 239, no. 5, pp. 1555–1572, 2010.
- H. Kuroyanagi, T. Kobayashi, S. Mitani, and M. Hagiwara, “Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo,” Nature Methods, vol. 3, no. 11, pp. 909–915, 2006.
- G. Ohno, M. Hagiwara, and H. Kuroyanagi, “STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo,” Genes and Development, vol. 22, no. 3, pp. 360–374, 2008.
- G. Ohno, K. Ono, M. Togo, et al., “Muscle-specific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans,” PLoS Genetics, vol. 8, no. 10, Article ID e1002991, 2012.
- H. Kuroyanagi, H. Kuroyanagi, Y. Watanabe, Y. Suzuki, and M. Hagiwara, “Position-dependent and neuron-specific splicing regulation by the CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans,” Nucleic Acids Research, vol. 41, no. 7, pp. 4015–4025, 2013.
- M. V. Sundaram, “RTK/Ras/MAPK signaling,” WormBook, pp. 1–19, 2006.
- J. Kimble, “Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans,” Developmental Biology, vol. 87, no. 2, pp. 286–300, 1981.
- P. W. Sternberg and H. R. Horvitz, “Pattern formation during vulval development in C. elegans,” Cell, vol. 44, no. 5, pp. 761–772, 1986.
- M. Han and P. W. Sternberg, “let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein,” Cell, vol. 63, no. 5, pp. 921–931, 1990.
- C. Van Buskirk and P. W. Sternberg, “Epidermal growth factor signaling induces behavioral quiescence in Caenorhabditis elegans,” Nature Neuroscience, vol. 10, no. 10, pp. 1300–1307, 2007.
- A. Dutt, S. Canevascini, E. Froehli-Hoier, and A. Hajnal, “EGF signal propagation during C. elegans vulval development mediated by ROM-1 Rhomboid,” PLoS Biology, vol. 2, no. 11, article e334, 2004.
- M. Trahey and F. McCormick, “A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants,” Science, vol. 238, no. 4826, pp. 542–545, 1987.
- M. Trahey, G. Wong, R. Halenbeck et al., “Molecular cloning of two types of GAP complementary DNA from human placenta,” Science, vol. 242, no. 4886, pp. 1697–1700, 1988.
- S. Hayashizaki, Y. Iino, and M. Yamamoto, “Characterization of the C. elegans gap-2 gene encoding a novel Ras-GTpase activating protein and its possible role in larval development,” Genes to Cells, vol. 3, no. 3, pp. 189–202, 1998.
- M. A. Allen, L. W. Hillier, R. H. Waterston, and T. Blumenthal, “A global analysis of C. elegans trans-splicing,” Genome Research, vol. 21, no. 2, pp. 255–264, 2011.
- S. Iwashita and S.-Y. Song, “RasGAPs: a crucial regulator of extracellular stimuli for homeostasis of cellular functions,” Molecular BioSystems, vol. 4, no. 3, pp. 213–222, 2008.
- A. G. Knudson Jr., “Retinoblastoma: a prototypic hereditary neoplasm,” Seminars in Oncology, vol. 5, no. 1, pp. 57–60, 1978.
- X. Lu and H. R. Horvitz, “Lin-35 and lin-53, two genes that antagonize a C. elegans Ras pathway, encode proteins similar to Rb and its binding protein RbAp48,” Cell, vol. 95, no. 7, pp. 981–991, 1998.
- C. J. Ceol and H. R. Horvitz, “dpl-1 DP and efl-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development,” Molecular Cell, vol. 7, no. 3, pp. 461–473, 2001.
- E. L. Ferguson and H. R. Horvitz, “The multivulva phenotype of certain Caenorhabditis elegans mutants results from defects in two functionally redundant pathways,” Genetics, vol. 123, no. 1, pp. 109–121, 1989.
- D. Joseph-Strauss, M. Gorjánácz, R. Santarella-Mellwig, E. Voronina, A. Audhya, and O. Cohen-Fix, “Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos,” Developmental Biology, vol. 365, no. 2, pp. 445–457, 2012.
- D. Merkle and R. Hoffmann, “Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: cross-talk with the androgen receptor,” Cellular Signalling, vol. 23, no. 3, pp. 507–515, 2011.
- M. Tabish, R. A. Clegg, H. H. Rees, and M. J. Fisher, “Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1),” Biochemical Journal, vol. 339, no. 1, pp. 209–216, 1999.
- L. C. Bowen, A. V. Bicknell, M. Tabish, R. A. Clegg, H. H. Rees, and M. J. Fisher, “Expression of multiple isoforms of the cAMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans,” Cellular Signalling, vol. 18, no. 12, pp. 2230–2237, 2006.
- R. A. Clegg, L. C. Bowen, A. V. Bicknell et al., “Characterisation of the N'1 isoform of the cyclic AMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans,” Archives of Biochemistry and Biophysics, vol. 519, no. 1, pp. 38–45, 2012.
- M. W. Pastok, M. C. Prescott, C. Dart, et al., “Structural diversity of the cAMP-dependent protein kinase regulatory subunit in Caenorhabditis elegans,” Cell Signaling, vol. 25, no. 1, pp. 168–177, 2013.
- P. Murray, R. A. Clegg, H. H. Rees, and M. J. Fisher, “siRNA-mediated knockdown of a splice variant of the PK-A catalytic subunit gene causes adult-onset paralysis in C. elegans,” Gene, vol. 408, no. 1-2, pp. 157–163, 2008.
- M. H. Lee, Y. J. Jang, and H.-S. Koo, “Alternative splicing in the Caenorhabditis elegans DNA topoisomerase I gene,” Biochimica et Biophysica Acta, vol. 1396, no. 2, pp. 207–214, 1998.
- M. H. Lee, H. Park, G. Shim, J. Lee, and H.-S. Koo, “Regulation of gene expression, cellular localization, and in vivo function of Caenorhabditis elegans DNA topoisomerase I,” Genes to Cells, vol. 6, no. 4, pp. 303–312, 2001.
- D. S. Cha, S. E. Hollis, U. S. Datla et al., “Differential subcellular localization of DNA topoisomerase-1 isoforms and their roles during Caenorhabditis elegans development,” Gene Expression Patterns, vol. 12, no. 5, pp. 189–195, 2012.
- F. Rossi, E. Labourier, T. Forné et al., “Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I,” Nature, vol. 381, no. 6577, pp. 80–82, 1996.
- E. Labourier, F. Rossi, I.-E. Gallouzi, E. Allemand, G. Divita, and J. Tazi, “Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor,” Nucleic Acids Research, vol. 26, no. 12, pp. 2955–2962, 1998.
- T. D. Dubatolova, E. I. Volkova, and L. V. Omelyanchuk, “Drosophila splicing factor SF2 knock-down mutant shows altered cell-cycle in vivo,” Cell Biology International, vol. 37, no. 2, pp. 187–190, 2013.
- Y. Kassir and G. Simchen, “Meiotic recombination and DNA synthesis in a new cell cycle mutant of Saccharomyces cerevisiae,” Genetics, vol. 90, no. 1, pp. 49–68, 1978.
- P. L. Graham, T. Schedl, and J. Kimble, “More mog genes that influence the switch from spermatogenesis to oogenesis in the hermaphrodite germ line of Caenorhabditis elegans,” Developmental Genetics, vol. 14, no. 6, pp. 471–484, 1993.
- A. Puoti and J. Kimble, “The Caenorhabditis elegans sex determination gene mog-1 encodes a member of the DEAH-box protein family,” Molecular and Cellular Biology, vol. 19, no. 3, pp. 2189–2197, 1999.
- A. Puoti and J. Kimble, “The hermaphrodite sperm/oocyte switch requires the Caenorhabditis elegans homologs of PRP2 and PRP22,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 7, pp. 3276–3281, 2000.
- F. Caspary, A. Shevchenko, M. Wilm, and B. Séraphin, “Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly,” EMBO Journal, vol. 18, no. 12, pp. 3463–3474, 1999.
- A. Dziembowski, A.-P. Ventura, B. Rutz et al., “Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing,” EMBO Journal, vol. 23, no. 24, pp. 4847–4856, 2004.
- P. Mantina, L. MacDonald, A. Kulaga, L. Zhao, and D. Hansen, “A mutation in teg-4, which encodes a protein homologous to the SAP130 pre-mRNA splicing factor, disrupts the balance between proliferation and differentiation in the C. elegans germ line,” Mechanisms of Development, vol. 126, no. 5-6, pp. 417–429, 2009.
- B. Laggerbauer, S. Liu, E. Makarov et al., “The human U5 snRNP 52K protein (CD2BP2) interacts with U5-102K (hPrp6), a U4/U6.U5 tri-snRNP bridging protein, but dissociates upon tri-snRNP formation,” RNA, vol. 11, no. 5, pp. 598–608, 2005.
- C. Wang, L. Wilson-Berry, T. Schedl, and D. Hansen, “TEG-1 CD2BP2 regulates stem cell proliferation and sex determination in the C. elegans germ line and physically interacts with the UAF-1 U2AF65 splicing factor,” Developmental Dynamics, vol. 241, no. 3, pp. 505–521, 2012.
- H. M. Ellis and H. R. Horvitz, “Genetic control of programmed cell death in the nematode C. elegans,” Cell, vol. 44, no. 6, pp. 817–829, 1986.
- D. Wu, H. D. Wallen, N. Inohara, and G. Nuñez, “Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9,” Journal of Biological Chemistry, vol. 272, no. 34, pp. 21449–21454, 1997.
- A. M. Chinnaiyan, D. Chaudhary, K. O'Rourke, E. V. Koonin, and V. M. Dixit, “Role of CED-4 in the activation of CED-3,” Nature, vol. 388, no. 6644, pp. 728–729, 1997.
- S. Seshagiri and L. K. Miller, “Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis,” Current Biology, vol. 7, no. 7, pp. 455–460, 1997.
- S. Shaham and H. R. Horvitz, “An alternatively spliced C. elegans ced-4 RNA encodes a novel cell death inhibitor,” Cell, vol. 86, no. 2, pp. 201–208, 1996.
- M. O. Hengartner, R. E. Ellis, and R. Horvitz, “Caenorhabditis elegans gene ced-9 protects cells from programmed cell death,” Nature, vol. 356, no. 6369, pp. 494–499, 1992.
- D. Chaudhary, K. O'Rourke, A. M. Chinnaiyan, and V. M. Dixit, “The death inhibitory molecules CED-9 and CED-4L use a common mechanism to inhibit the CED-3 death protease,” Journal of Biological Chemistry, vol. 273, no. 28, pp. 17708–17712, 1998.
- L. Wang, M. Miura, L. Bergeron, H. Zhu, and J. Yuan, “Ich-1, an ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death,” Cell, vol. 78, no. 5, pp. 739–750, 1994.
- Z.-H. Jiang, W.-J. Zhang, R. Yi, and J. Y. Wu, “Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 16, pp. 9155–9160, 1998.
- M. J. Moore, Q. Wang, C. J. Kennedy, and P. A. Silver, “An alternative splicing network links cell-cycle control to apoptosis,” Cell, vol. 142, no. 4, pp. 625–636, 2010.
- B. D. Galvin, D. P. Denning, and H. R. Horvitz, “SPK-1, an SR protein kinase, inhibits programmed cell death in Caenorhabditis elegans,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 5, pp. 1998–2003, 2011.
- H. Kuroyanagi, T. Kimura, K. Wada, N. Hisamoto, K. Matsumoto, and M. Hagiwara, “SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development,” Mechanisms of Development, vol. 99, no. 1-2, pp. 51–64, 2000.
- D. Longman, I. L. Johnstone, and J. F. Cáceres, “Functional characterization of SR and SR-related genes in Caenorhabditis elegans,” EMBO Journal, vol. 19, no. 7, pp. 1625–1637, 2000.