Peg MacMorris et al. (2007) International Worm Meeting "A novel family of C. elegans snRNPs contains proteins associated with trans-splicing."

Madhur Kumar, Erika Lasda, Alison Larsen, Tom Blumenthal, Brian Kraemer, Peg MacMorris

[International Worm Meeting, 2007]

In approximately 70% of C. elegans pre-mRNAs, the RNA sequence between the 5 cap and the first 3 splice site is replaced by trans-splicing a short spliced leader (SL) from the Sm snRNP, SL1. C. elegans also utilizes a similar Sm snRNP, SL2, to trans-splice at sites between genes in polycistronic pre-mRNAs from operons. How do SL1 and SL2 snRNPs function in different contexts? Here we show that the SL1 snRNP contains a complex of SL75p and SL21p, homologs of novel proteins previously reported in the Ascaris SL snRNP. Interestingly, the SL2 snRNP does not contain either of these proteins. However, SL75p and SL26p, a paralog of SL21p, are components of another Sm snRNP that contains a novel snRNA species, Sm Y. Knockdown of SL75p is lethal. However, knockdown of either SL21p or SL26p alone leads to cold-sensitive sterility, whereas knockdown of both SL21p and SL26p is lethal. This suggests that these two proteins have overlapping functions even though they are associated with different classes of snRNP. These phenotypic relationships, along with the association of SL26p with SL75p imply that, like the SL1 RNA/Sm/SL75p/SL21p complex, the Sm Y/Sm/SL75p/SL26p complex is associated with trans-splicing. We hypothesize that the Sm Y snRNP is somehow associated with SL2-specific trans-splicing. This idea is supported by the fact that the sequences of SL2 RNA and Sm Y allow base pairing of the two snRNPs through their second stem/loops. We are currently testing for this base pairing interaction using psoralen/UV crosslinking.

Scott A Barbee et al. (2001) International C. elegans Meeting "The splicesomal Sm proteins play a role in P granule localization during early embryogenesis."

Thomas C Evans, Scott A Barbee, Alex L Lublin

[International C. elegans Meeting, 2001]

In early C. elegans embryos, development is initiated by asymmetric cell division, which leads to the precise localization cell fate regulators to specific cells. One feature of early asymmetry is the localization of cytoplasmic RNP particles, the P granules, to the germ cell precursors at each of several polarized cell divisions. The composition and function of P-granules is not well understood. Further, the mechanisms that control the localization of P-granules and other factors in the embryo are poorly understood. We used RNA interference (RNAi) to screen a cDNA library for new genes involved in early polarity. Surprisingly, one of the genes identified is the ortholog of human SmE. SmE is one of several Sm proteins that form a multisubunit complex required for snRNP assembly, nuclear import, and mRNA splicing. RNAi of Sm subunits in C. elegans caused mislocalization of P granules to somatic sisters of germ cells after the 4-8 cell stages in the early embryo. In addition, Sm RNAi disrupted the subcellular distribution of PGL-1 (a P-granule component) in both mature germ cells and in germ cell precursors after the 8-cell stage. P-granules in wild type germ cells are primarily attached to the nucleus, but in Sm (RNAi) germ cells and embryos, the nuclear attachment of many P-granules is lost. At lower penetrance, GLP-1 protein was inappropriately expressed in posterior cells following Sm RNAi. By contrast, RNAi of the core splicing factors U1 70K and U2AF65, or of RNA polymerase II, had no effect on P granule segregation, subcellular distribution, or GLP-1 asymmetry, although all caused severe embryonic defects. These data suggest that the P-granule and GLP-1 asymmetry phenotypes from Sm RNAi are not likely to result from a general defect in splicing. Therefore, Sm proteins may have a role in P-granule localization that is independent of splicing. Interestingly, antibodies against the Sm complex stain P granules at all stages of development. Sm RNAi attenuates this staining. Therefore, Sm proteins may be P granule components that affect the localization of P granules by controlling their integrity or nuclear attachment in germ cells and their precursors. The Sm proteins are also found in the nucleus of most cells. However, nuclear localization of the Sm’s is dynamically regulated during oogenesis and germ cell precursor formation, suggesting that regulation of the Sm complex or snRNPs may be important for germ cell development.

Scott A Barbee et al. (2003) International Worm Meeting "The spliceosomal Sm proteins regulate germ cell fate during early C. elegans embryogenesis."

Thomas C Evans, Scott A Barbee

[International Worm Meeting, 2003]

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. However, we have previously shown that the Sm proteins, core components of the spliceosome, also regulate germ granule localization during early C. elegans embryogenesis independently of mRNA splicing. Here we provide evidence that the Sm proteins are involved in the specification of germ cells by regulating transcriptional quiescence and protein expression in germ cell precursors of the embryo. In early development of several metazoans, germ cell precursors are transcriptionally inactive. One indication of this general transcriptional repression is the absence of phosphorylation of the C-terminal domain of RNA polymerase II. When Sm protein activity was disrupted by RNA interference (RNAi), CTD phosphorylation occurred inappropriately in germ cell precursors. In addition, at least one germ cell promoter is inappropriately activated in germ cell precursors in SmE(RNAi) embryos. These results suggest that the Sm complex is necessary for transcriptional silencing in these cells. In early C. elegans development, inhibition of transcription in germ cell precursors requires PIE-1, a CCCH-type zinc-finger protein that control germ cell fate. Disruption of Sm protein activity causes reduction and possibly mislocalization of PIE-1 in early embryos. Loss of PIE-1 in SmE(RNAi) embryos occurs after the 1-cell stage, suggesting that translation or stability of maternal PIE-1 is regulated by the Sms in the embryo. Additionally, expression of NOS-2, a germ cell-specific factor whose translation requires PIE-1, is attenuated following Sm (RNAi). Taken together, this suggests that the Sm proteins may control germ cell fate by regulating PIE-1 expression. In contrast, loss of other splicing factors had little or no impact on each of the germ precursor markers. These observations suggest that the Sm proteins regulate germ cell fate or function in the early embryo by a mechanism distinct from their role in pre-mRNA splicing. Sm and Sm-related proteins bind small non-coding RNAs and possibly mRNAs. Therefore, the Sm complex may control germ cell fate and other processes as a novel broad-based chaperone for RNP assembly and/or RNA:RNA interactions.

Kamal, M. et al. (2017) International Worm Meeting "Sphingomyelin abundance is likely a key determinant in regulating drug accumulation in C. elegans."

Moshiri, H., Magomedova, L., Kamal, M., Szlapa, K., Bagg, R., Hall, D., Roy, P., Yeo, M., Fraser, A., Zheng, H., Cummins, C.

[International Worm Meeting, 2017]

One major barrier to a drug's access to target is the endothelium's plasma membrane (PM). In metazoans, the four major classes of phospholipids include phosphatidylcholine (PC) and sphingomyelin (SM), which are enriched in the outer leaflet of the PM, and phosphatidylserine and phosphatidylethanolamine, which are enriched in the inner leaflet. How different phospholipids impact the PM's permeability to drugs is not fully understood. We previously identified 275 synthetic small molecules that kill C. elegans (PMID 26108372). We discovered that a subset of these, including a molecule called wact-190, accumulate as crystals within the marginal cells of the anterior pharynx and perforate the luminal PM. Dose-response analyses indicate a tight correlation (r=0.99) between crystal formation and death. Towards understanding crystal formation, we screened for mutants that resist wact-190's lethality and identified 46 resistant mutants. We found that reduction-of-function of a presumptive sphingomyelin (SM) synthesis pathway and a p-glycoprotein pump called PGP-14 are each sufficient to confer resistance to both the lethality and crystal formation that results from wact-190 and other crystal-forming compounds. Mutants in the SM pathway and pgp-14 not only resist the lethality induced by crystal-forming compounds, but are hypersensitive to relatively smaller molecules. Mass-spectrometry revealed that 'resistant' molecules accumulate less in the mutants, while 'hypersensitive' molecules accumulate more in the mutants relative to wild type. We found that the SM synthase, which is the terminal enzyme in the SM synthesis pathway, is expressed exclusively in the pharynx and the spermatheca, while pgp-14 is expressed exclusively in the marginal cells of the anterior pharynx. Both proteins function cell-autonomously in the marginal cells with respect to wact-190 resistance. A human homolog of PGP-14 is ABCB4, which translocates PCs from the inner to the outer leaflet of the PM. SM synthases use the choline head from PCs to make SM. Hence our current model is that PGP-14 provides SM synthase with PCs to generate SM in the PM. Indeed, preliminary analyses indicate that both pgp-14 mutants and an SM synthase mutant are deficient in SM in anterior pharynx. Together, our work suggests that SM abundance in the PM is a key determinant of small molecule accumulation within animal cells. Further implications will be discussed.

Keiko Gengyo-Ando et al. (2005) International Worm Meeting "Functional analysis of the C. elegans SM gene vps-45 and its cognate syntaxins"

Tetsuo Kobayashi, Shohei MItani, Keiko Gengyo-Ando, Yoichi Seyama

[International Worm Meeting, 2005]

Sec1/Munc-18 (SM) protein family is involved in various vesicular trafficking from yeast to mammals, but its function is less well understood. To investigate systematically molecular structure and function of SM family in multicellular organism, we isolated deletion mutants of SM genes and found that four of six SM gene mutations lead to a lethal phenotype. Hence, SM genes appear to be also indispensable factors for intracellular trafficking in C. elegans. The putative null mutation of vps-45 (tm246), whose orthologous protein in yeast plays an important role in membrane trafficking from the Golgi to vacuole, showed a ts lethality and defects of endocytosis. Maternal rescued homozygotes (m+ z-) exhibit a Cup (coelomocyte uptake defective) and Rme (receptor mediated endocytosis defective) phenotypes in restrictive temperature, suggesting that vps-45 may be required for a common pathway between Rme and non-Rme endocytosis. Most SM proteins interact with syntaxin family, a subclass of Q-SNAREs, and may regulate SNARE complex formation. To identify the cognate syntaxin for C. elegans vps-45, we performed co-immunoprecipitation and yeast two hybrid studies between vps-45 and eight syntaxin genes identified on the C. elegans genome (Jantsch-Plunger and Glotzer, 1999). We found that vps-45 exhibits specific interactions with tlg-2 and pep-12. To elucidate whether vps-45 functions with the cognate syntaxins in vivo, we isolated deletion mutations of these syntaxins. tlg-2 (tm1560), pep-12 (tm1198, tm1764), tlg-2; pep-12 were viable, and endocytic trafficking in coelomocytes of these mutant animals were not severely blocked. These results suggest that specific binding of SM proteins to syntaxins has probably important aspects, but SM proteins might also have syntaxin-independent functions in endocytic trafficking. Biosynthetic pathways from Golgi to lysosome in coelomocytes of the isolated mutants are now analyzed using a visible marker of cathepsin D/ fluorescent fusion protein.

Adikes, R.C. et al. (2019) International Worm Meeting "Cell cycle and cytoskeletal dynamics during C. elegans muscle progenitor migration."

Kim, N., Goldstein, B., Ahmed, O.B., Pani, A.M., Adikes, R.C., Matus, D.Q.

[International Worm Meeting, 2019]

Defects in cell cycle regulation or morphogenetic movements are associated with many pathogenic processes and are regarded as hallmarks of cancer metastasis. Here we explore the migration and differentiation of mesodermal precursor cells in the nematode C. elegans. The C. elegans sex myoblasts (SMs) are derived from progenitors that migrate during larval development and then proliferate and differentiate into the adult vulval muscles. The two SM founder cells travel anteriorly during the second larval stage until they flank the gonad and begin to divide. Several signaling pathways and guidance cues important for SM specification, migration, and differentiation have been identified by classical methods, but the cell biological mechanisms underlying SM migration remain largely unknown. To explore cellular mechanisms, we used high-resolution, time-lapse microscopy to analyze the migratory behaviors of SM cells in vivo. We show that these cells generate numerous filopodia and exhibit complex cytoskeletal dynamics. Furthermore, using a newly developed cell cycle state sensor we establish that these cells are in G0 quiescence when they migrate. To further investigate the roles that cell cycle state and cytoskeletal dynamics play during SM migration and pre-differentiation we are performing an RNAi screen of candidate genes that we hypothesize may regulate SM behaviors. These findings lay the groundwork for establishing C. elegans SMs as a cell-biological model for cell migration in vivo and for studying the relationship between migration, cell-cycle state and cytoskeletal regulation.

MacMorris, Peg et al. (2009) International Worm Meeting "Novel proteins bound to SL1 and Sm Y RNAs: possible roles in recycling Sm proteins."

Blumenthal, Tom, Saldi, Tassa, MacMorris, Peg

[International Worm Meeting, 2009]

C. elegans codes for two classes of SL RNA, SL1 RNA and SL2 RNA, that donate their 5'' 22 nt to mRNAs in trans-splicing. A two-polypeptide complex, SNA-2/SNA-1, unique to nematodes, is bound to the SL1 RNA but not to SL2 RNA. Surprisingly however, a different class of snRNA, whose function is unknown, also unique to nematodes, Sm Y RNA, contains a SNA-2/SUT-1 complex, where SUT-1 is a paralog of SNA-1. Deletions of sna-1 or sut-1 are viable, but 15 deg sterile. They can also phenocopy a variety of morphological abnormalities at 15 deg . Both the sna-2 deletion and the sna-1/sut-1 double deletion are lethal. Thus SNA-1 and SUT-1 are functionally redundant, even though they are bound to different classes of snRNP. Why does SL2 RNA have neither of these proteins? How can these results be rationalized? The answer may lie in the issue of how the Sm ring proteins are recycled after trans-splicing. SL1, SL2 and Sm Y snRNPs all contain the Sm proteins, which are normally components of stable spliceosomal snRNPs, U1, U2 etc. We think the SNA and SUT-1 proteins may collaborate with Sm Y RNA to recycle the Sm proteins after splicing. The Evans lab showed that Sm proteins are present in P granules and are required for fertility. We hypothesize that at 15 deg , when either SUT-1 or SNA-1 is missing, there is a failure to recycle some of the Sm protein used in trans-splicing, which could result in splicing problems, producing morphological defects, or failure to make active P granules, producing sterile worms. In support of this idea, we have found a strong genetic interaction between prmt-5, whose protein product modifies Sm proteins, and both sut-1 and sna-1: whereas the prmt-5 deletion alone is without detectable phenotype, this mutation suppresses the sut-1 and enhances the sna-1 deletion phenotypes. Interestingly, these genes also interact with mutations in genes encoding P granule proteins, pgl-1 and glh-1.

Sundaram MV et al. (1996) West Coast Worm Meeting "let-60 RAS IS REQUIRED IN THE SEX MYOBLASTS TO CONTROL PRECISE POSITIONING"

Han M, Sundaram MV, Yochem JJ

[West Coast Worm Meeting, 1996]

The two sex myoblasts (SMs) in the hermaphrodite are born during the L1 larval stage in the posterior region of the animal, one on the left side and one on the right. The SMs then migrate anteriorly during the L2 stage to occupy very precise final positions flanking the gonad in the mid-body region, near the site of the future vulva. Two separable components of SM guidance have been described: a gonad-independent mechanism sufficient for the initial anterior migration to the mid-body region, and a gonad-dependent mechanism required for precise final positioning (Thomas et al., 1990). We have demonstrated a role for a Ras-mediated signal transduction pathway in controlling SM migration. Loss-of-function mutations in let-60 ras, ksr-1, lin-45 raf, let-537/mek-2 or sur-1/mpk-1 cause the SMs to adopt a broadened range of final positions that resembles what is seen in gonad-ablated wild-type animals, while constitutively active let-60 ras(G13E) transgenes allow fairly precise SM positioning to occur in the absence of the gonad. A recent mosaic analysis has demonstrated that let-60 ras is required within the SMs to control proper positioning. Based on these results, we propose a model in which gonadal signals normally stimulate let-60 ras activity in the SMs, thereby making the SMs competent to sense or respond to positional cues in the mid-body environment. Several observations suggest that let-60 ras could have additional roles in SM guidance as well: 1) Several unusual but apparently reduction-of-function let-60 alleles [WBG 13(1), 13(3)] cause a striking posterior displacement of the SMs similar to that caused by mutations in egl-15 FGFR (DeVore et al., 1995); 2) The SMs sometimes adopt abnormal dorsal postions in let-60 ras and other Ras pathway mutants; and 3) Mosaic animals lacking let-60 ras in the SMs appear to have a more posterior SM distribution than mosaic animals lacking let-60 ras in both the SMs and gonad. We are currently pursuing these observations. DeVore, D. L., Horvitz, H. R. and Stern, M. J. (1995). Cell 83, 611-620. Thomas, J. H., Stern, M. J. and Horvitz, H. R. (1990). Cell 62, 1041-1052.

Sundaram MV et al. (1995) International C. elegans Meeting "SEX MYOBLAST MIGRATION, SUR-3 AND THE RAS PATHWAY"

Sundaram MV, Han M

[International C. elegans Meeting, 1995]

During the L2 stage in the hermaphrodite, the two sex myoblasts (SMs) migrate from the posterior to positions precisely flanking the center of the somatic gonad. Thomas, Stern and Horvitz (1990) have previously shown that while the initial anterior migration of the SMs is gonad-independent, the precise final positioning of the SMs requires gonadal cues: if the gonad is ablated prior to the time of SM migration, the SMs still migrate anteriorly but adopt final positions spanning a broader anterior-posterior range than normal. We have found that mutations reducing Ras pathway activity (i.e. those in let-60, lin-45, mek-2, sur-1/mpk-1, and sur-3) affect SM migration in a similar manner to gonad ablations, suggesting that a Ras- mediated signaling pathway is required for the SMs to respond to gonadal cues. Surprisingly, a gain of function let-60 ras allele, let-60(n1046), also causes a weak SM migration defect and strongly enhances the defects caused by other Ras pathway mutations, including other let-60 mutations. Thorough analysis of the role of Ras pathway genes in controlling SM migration is complicated by the fact that null alleles in these genes are lethal. For now, we have most closely analyzed the effects of sur-3 mutations (see other abstract by Sundaram and Han). We find that sur-3 mutations mimic gonad ablation in WT, egl-15, egl-17 and dig-1 backgrounds, but not in a let-60(n1046) background. We have also made transgenic lines bearing integrated arrays of either let-60(+) or let-60(n1046), and have used these lines to explore the relationship between let-60 and other mutations affecting SM migration. We find that either type of let-60 array can partially suppress mutations in egl-15 and egl-17. Our results suggest that the Ras pathway and egl-15/egl-17 may have both overlapping and distinct roles in regulating migration.

Lublin AL et al. (2000) West Coast Worm Meeting "The splicing Sm proteins colocalize with P granules in germ cells and participate in P granule localization in the early embryo"

Barbee SA, Lublin AL, Evans TC

[West Coast Worm Meeting, 2000]

Early embryonic polarity in C. elegans is initiated by asymmetric cell division which leads to the localization of maternally contributed proteins and mRNAs. In turn, these factors control the temporal and spatial expression of gene products that determine the developmental fate of cells. Conventional genetic screening has identified more than 20 genes involved in this process. However, these screens may not uncover pleiotropic genes that have a necessary function at other stages. With this in mind, we have used RNA interference (RNAi) to screen a cDNA library for new genes involved in generating asymmetries in the early embryo. One gene identified in the RNAi screen is a homolog to human SNRPE (SmE). SmE is one of several Sm proteins which form a complex required for mRNA splicing and import of snRNAs (U1, U2, U4, and U5) into the nucleus. RNAi of SmE and other Sm proteins, which together form a subcomplex, resulted in disruption of P granule segregation in the embryo. P granules are cytoplasmic ribonucleoprotein particles that segregate to the posterior germline precursor cells. RNAi of several subunits of the Sm complex caused mislocalization of P granules at various stages. Other aspects of early polarity may also be disrupted. However, RNAi of other distinct Sm proteins had no effect. RNAi of the core splicing factors U170K and U2AF65, or of RNA polymerase II (ama-1), also had no effect on P granule localization. These data suggest that the P granule phenotype is not the result of a general splicing defect. Therefore, Sm proteins may have a role in generating early embryonic polarity that is independent of snRNA import and splicing. Interestingly, using a monoclonal antibody against mammalian Sm proteins, we found colocalization of the Sm's with PGL-1 in P granules at all stages of development. Sm proteins, therefore, may be P granule components that participate in P-granule localization and/or other polarity functions.