Kuroyanagi, Hidehito et al. (2009) International Worm Meeting "Auto-regulation of asd-1, a Fox-1 family alternative splicing regulator."

Kikuchi, Yuriko, KUROYANAGI, Hidehito, Hagiwara, Masatoshi

[International Worm Meeting, 2009]

Alternative splicing of pre-mRNAs is a major source of proteome diversity in metazoan. Recent studies on alternative splicing regulators in cultured vertebrate cells suggest a complex regulatory network of alternative splicing regulators. However, the splicing regulatory network in vivo still remains to be elucidated. We have recently developed a transgenic reporter system that enables visualization of alternative splicing patterns in living organisms by utilizing multiple fluorescent proteins and demonstrated that Fox-1 family proteins, Alternative Splicing Defective-1 (ASD-1) and FOX-1, and muscle-specific regulator SUP-12 coordinately regulate muscle-specific selection of egl-15 exon 5A. The Fox-1 family proteins are evolutionarily conserved alternative splicing regulators that specifically bind to UGCAUG stretch in target pre-mRNAs. Here we report that expression of functional asd-1 mRNA is negatively auto-regulated. We identified an alternative asd-1 mRNA isoform, E2D, in which skipping of exon 2 caused a frame-shift near N-terminus. E2D mRNA was accumulated in smg-2 mutant, indicating that it is a target of nonsense-mediated mRNA decay (NMD) and therefore is non-functional. The amount of E2D mRNA was remarkably reduced in asd-1; fox-1 double mutant in the smg-2 background, indicating that skipping of asd-1 exon 2 depends on the Fox-1 family. In order to analyze the asd-1 splicing regulation in vivo, we constructed a pair of asd-1 alternative splicing reporter mini-genes that visualize inclusion and skipping of exon 2 by expression of RFP and GFP, respectively. When expressed under asd-1 promoter, RFP expression was observed in a wide variety of tissues that express asd-1, while expression of GFP was restricted to muscles and some neurons but not in intestine. Consistent with the requirement of the Fox-1 family for exon 2 skipping, GFP expression was remarkably reduced in asd-1; fox-1 double mutant, indicating that reporter expression from the mini-genes reflects regulation of endogenous exon 2. We also found that only one out of four UGCAUG stretches in the upstream and downstream introns from exon 2 is required for efficient GFP expression. These results indicate that C. elegans Fox-1 family RNA binding proteins ASD-1 and FOX-1 negatively regulate asd-1 expression by repressing inclusion of exon 2 via a UGCAUG stretch in intron 2 in a tissue-specific manner.

Sugimoto, Asako et al. (2017) International Worm Meeting "Establishing genetic techniques to study Caenorhabditis sp. 34, a sister species of C. elegans."

Kanzaki, Natsumi, Hoshi, Yuki, Kumagai, Ryohei, Sugimoto, Asako, Kikuchi, Taisei, Namai, Satoshi, Tsuyama, Kenji

[International Worm Meeting, 2017]

Caenorhabditis sp. 34 is a sister species of C. elegans recently isolated from the syconia of the fig Ficus septica on Ishigaki Island, Japan (see abstract by T. Kikuchi, et al.). C. sp. 34 is gonochoric and shares typological key characters with other Elegans supergroup species, but strikingly, adults are nearly twice as long as C. elegans. The optimal culture temperature for C. sp. 34 is significantly higher (27 deg C) than that of C. elegans (20 deg C). Young adult males and females tend to form clumps, and Dauer larvae are rarely observed in laboratory culture conditions. Recently the C. sp. 34 genome assembly was produced into six chromosomes (see abstract by T. Kikuchi, et al.). The marked differences from C. elegans in morphology, behaviors and ecology, and the availability of the complete genome sequence make C. sp. 34 highly attractive for comparative and evolutionary studies. To make C. sp. 34 genetically tractable, we have been developing genetic and molecular techniques and tools. Stable transgenic lines of C. sp.34 could be obtained by microinjecting marker plasmids commonly used in C. elegans, although the efficiency was lower than that in C. elegans. Both soaking and feeding RNAi was as effective as in C. elegans. A panel of antibodies against C. elegans proteins successfully recognized expected structures in C. sp. 34 by immunofluorescence. Thus, many of the rich genetic and molecular resources for C. elegans can be directly used for C. sp. 34 studies. We well present some of the comparative analyses of gene functions regarding the body size, germ cell formation and sex determination.

Stevens, Lewis et al. (2019) International Worm Meeting "Nothing in Caenorhabditis biology makes sense except in the light of evolution: The Caenorhabditis Genomes Project."

Blaxter, Mark, Stevens, Lewis

[International Worm Meeting, 2019]

Caenorhabditis elegans is one of the preeminent model organisms in modern biology, but only recently have we started to understand the details of its natural ecology and evolutionary history. Studying C. elegans alongside its closest relatives provides an important evolutionary context for the origins and constraints that have resulted in the particular instance analyzed in the laboratory. A focussed search for new Caenorhabditis species over the last decade has led to the discovery of over 50 species of Caenorhabditis. As part of an international collaboration, the Caenorhabditis Genomes Project (CGP), we have sequenced the genomes of all Caenorhabditis species currently in culture. We exploit these new genome sequences to perform the most comprehensive reconstruction of the Caenorhabditis phylogeny to date, providing an essential evolutionary framework for downstream analyses. We reveal extensive variation in genome size in the genus, ranging from the 48 Mb genome of C. drosophilae to the 160 Mb genome of C. vivipara. Investigating the origins of this variation, we find that protein-coding gene number is highly correlated with genome size, with C. drosophilae possessing just 13,000 genes due to extensive gene loss. Other genomic features, such as repeat (mobile element) proliferation also contribute to genome size changes. Among the non-coding portions of these genomes, we identified rapid and extensive intron loss across the genus, including in the group that includes C. elegans. We demonstrate the utility of these genome sequences for C. elegans research by analyzing the evolutionary history of key signaling pathways, revealing unexpected complexity. We have made these genomic resources, including genus-wide orthology sets, publicly available via the CGP website (http://www.caenorhabditis.org) which includes a dedicated genome browser and a BLAST server. These new species and their associated genomic resources add to the arsenal of tools available to the C. elegans researcher to interrogate biology of this important nematode. The CGP is a collaboration between the following labs: Blaxter (Edinburgh), Felix (Paris), Ailion (Washington), Andersen (Northwestern), Braendle (Nice), Cutter (Toronto), Fitch (NYU), Fierst (Alabama), Kikuchi (Miyazaki), Rockman (NYU), Schwarz (Cornell), Wang (Academia Sinica).