Watabe, Eichi et al. (2019) International Worm Meeting "PES-4 regulates head-muscle-specific alternative splicing of the tropomyosin pre-mRNA."

KUROYANAGI, Hidehito, Watabe, Eichi

[International Worm Meeting, 2019]

In animals, many proteins involved in actin filament organization and functions have multiple isoforms owing to gene duplication and/or alternative pre-mRNA splicing. These isoforms are often expressed in cell-type-specific fashions. However, selection mechanisms and functional consequences of such protein isoforms in specific tissues largely remain to be elucidated. We have recently reported comprehensive analysis of splicing variants for the single tropomyosin gene lev-11 in C. elegans. We found that its novel alternative exon, 7a, is selected only in head muscles, whereas the other muscles and tissues select exon 7b (Mol Biol Cell, 2018; Cytoskeleton, 2018). Here we analyzed alternative splicing regulators of this head-muscle-specific splicing. We crossed dichromatic fluorescence lev-11 exon 7 splicing reporter worms with splicing factor mutants defective in muscle-specific alternative splicing and found that asd-2, a regulator for the let-2 gene and the unc-60 gene, are also involved at least in part in the head-muscle-specific splicing. We further searched recently published single-cell RNA-seq data for RNA-binding proteins enriched in the head muscles and identified pes-4, msi-1 and exc-7 as candidates. msi-1(os1) and exc-7(ok370) did not affect the lev-11 exon 7 splicing reporter. We then knocked out the pes-4 gene in the lev-11 exon 7 splicing reporter worms with CRISPR/Cas9 system and found that the pes-4 mutants completely lost the head-muscle-specific expression of exon 7a, although the homozygotes were arrested at the L1 stage. PES-4 protein has two KH-type RNA-binding domains, an alanine- and glutamine-rich (AQ-rich) region and a conserved C-terminal region. The C-terminal region matches the consensus of proline-tyrosine (PY)-type nuclear localization signal (PY-NLS) and were actually involved in the nuclear localization and function of PES-4. The AQ-rich region is not homologous to any other proteins in the primary sequence, but were essential for the splicing regulation. We are currently elucidating a model for exon 7a selection exclusively in the head muscles.

Kuroyanagi, Hidehito et al. (2021) International Worm Meeting "Alternative splicing through m6A modification at a 3' splice site for SAM synthetase homeostasis"

Togo-Ohno, Marina, Fukamizu, Akiyoshi, Watabe, Eichi, KUROYANAGI, Hidehito, Hirota, Keiko, Ishigami, Yuma, Suzuki, Tsutomu, Suzuki, Yutaka

[International Worm Meeting, 2021]

Alternative splicing of precursor messenger RNAs (pre-mRNAs) contributes not only to proteome diversity but also to regulation of gene expression levels by generating mRNA isoforms with a premature termination codon (PTC). Such unproductively spliced mRNAs are unstable and almost undetectable due to an mRNA surveillance system termed nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD), we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant, smg-2, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Gene ontology (GO) analysis revealed enrichment of genes related to metabolism in addition to those related to RNA translation and processing. Among them are S-adenosyl-L-methionine (SAM) synthetase (sams) genes. SAM synthetase activity negatively autoregulates sams gene expression through AS-NMD. METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation of the sams genes in vivo and specifically methylates in vitro the invariant AG dinucleotide at the distal 3' splice site (3'SS) used for the productive mRNAs. RNA immunoprecipitation with anti-m6A antibody and direct RNA sequencing with Nanopore technologies coupled with machine learning confirmed m6A modification of endogenous sams mRNAs. These results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6A modification at the 3'SS of the sams genes.