Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans

• Type of Material: Article
• Authors: Genta Ohno, Tokyo Medical and Dental University; Kanako Ono, Emory University; Marina Togo, Tokyo Medical and Dental University; Yohei Watanabe, Tokyo Medical and Dental University; Shoichiro Ono, Emory University; Masatoshi Hagiwara, Tokyo Medical and Dental University; Hidehito Kuroyanagi, Tokyo Medical and Dental University
• Language: English
• Date: 2012-10-11
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2012 Ohno et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pgen.1002991
• Title of Journal or Parent Work: PLoS Genetics
• ISSN: 1553-7390
• Volume: 8
• Issue: 10
• Start Page: e1002991
• End Page: e1002991
• Grant/Funding Information: GO was supported by Japan Society for the Promotion of Science (JSPS), including JSPS Research Fellowships for Young Scientists.; We acknowledge support from grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and from Japan Science and Technology Agency (JST) to HK and MH, and a grant from the National Institute of Health (R01 AR48615) to SO.
• Keywords: C-ELEGANS; IN-VIVO; UNC-60 GENE; ACTIN-FILAMENTS; ENCODES; DEPOLYMERIZING FACTOR/COFILIN PROTEINS; SERINE/THREONINE KINASE; BINDING PROTEIN; Science & Technology; GENETICS & HEREDITY; Life Sciences & Biomedicine; Genetics & Heredity; MYOGENIC DIFFERENTIATION; CHOLINERGIC LOCUS
• Research Categories: Health Sciences, Pathology

Abstract

Pre-mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre-mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre-mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre-mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre-mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA-binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT-PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.