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.
Highlights
Alternative pre-mRNA processing is a major way to produce a number of different mRNAs and proteins from one gene [1,2]
We reveal regulation mechanisms of pre–mRNA processing of the unc-60 gene locus, encoding two tissue-specific isoforms of actin depolymerising factor (ADF)/cofilin in C. elegans
Biochemical, and immunohistochemical evidence that muscle-specific RNA–binding proteins ASD-2 and SUP-12 cooperatively bind to specific motifs in intron 1A to retain intron 1A, which leads to skipping of exon 2A through 5A and splicing between exon 1 and 2B
Summary
Alternative pre-mRNA processing is a major way to produce a number of different mRNAs and proteins from one gene [1,2]. Recent transcriptome analyses by deep sequencing estimated that more than 90% of human multi-exon genes undergo alternative processing and most alternative processing events are regulated in tissue-specific manners [3,4]. These alternative pre-mRNA processing events are classified into seven elementary events: cassette exons, mutually exclusive exons, alternative 59 splice sites, alternative 39 splice sites, intron retention, alternative first exons and alternative polyadenylation sites [5,6]. Pre-mRNA processing in multicellular organisms is often complex due to various combinations of the elementary events and the molecular mechanisms by which tissue-specific factors regulate such complex alternative processing of the entire gene in vivo remain to be elucidated. Global analyses of splicing patterns during development of heart and skeletal muscle revealed that splicing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
