Splicing factor SUP-12 and the molecular complexity of apparent cooperativity

Abstract

T he splicing factor SUP-12 from C. elegans, in combination with either ASD-1 or FOX-1 from the Fox-1 (RBFOX) family, is required for generating a muscle-specific isoform of the fibroblast growth factor receptor EGL-15. Biophysical techniques have revealed the sequence preference for the RNA Recognition Motif (RRM) domain from SUP-12 as well as the structural details of the RNA-bound complex. Detailed genetics have identified a requisite need for the presence of both SUP-12 and ASD-1/FOX-1 to regulate the alternative splicing event, prompting speculation of a cooperative mechanism between these proteins on binding RNA. In contrast, the interplay between SUP-12 and ASD-1 suggests that although the RRM domains from each protein are in direct contact on the egl-15 pre-mRNA, there is no simple contribution of binding cooperativity. Evidence for an independent binding mechanism by SUP-12 and ASD-1 will be discussed, including a model in which both positive and negative contributions are balanced during complex assembly. The ability to monitor tissue-specific alternative splicing in live nematodes will continue to provide a powerful method to test in vivo mechanistic models derived from atomic-level investigation.Interactions between cellular biomolecules such as proteins and nucleic acids are critical for life, and the precise regulation of these interactions in a time-and location-dependent manner allows for normal organism development.Alternative splicing can contribute to this process through the production of development-and tissuespecific protein isoforms. The spliceosome generally interacts with target premRNA with the help of constitutive protein trans factors that recognize RNA cis elements in order to define the intron boundaries and dictate the series of exons in the mature mRNA. To regulate this process, additional splicing factors also bind to the pre-mRNA in order to enhance or inhibit specific 5 0 and 3 0 intron splice sites. The mechanisms by which the final splice patterns are established are in general poorly understood, but predominantly involve cooperative or competitive interactions with the constitutive splicing machinery to alter spliceosome recruitment, premRNA motif availability or the catalytic efficiency.Several splicing factors have already been identified in C. elegans that display limited expression patterns and lead to cell type-specific changes in splicing regulation.1 In many cases, multiple factors are linked to a specific splicing event, including both positive and negative regulators that together dictate the final pattern of exon retention in the mature mRNA. 2Structures of RNA-binding domains from splicing proteins shed light on the basis for RNA motif preference and this information can explain the phenotypic effects of known genetic variations in the protein factors or RNA elements. The molecular details also help in the strategic design of mutants to selectively perturb the contribution of a splicing factor in a given splicing event. Less known are the atomic details that under...

Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

“…Transcripts of hundreds of structural genes are mis-spliced in Rbfox1 and Rbfox2 knockout mice, which display developmental defects in muscle structure and function and fail to maintain skeletal muscle mass as adults ( Pedrotti et al, 2015 ; Singh et al, 2018 ). Similarly, knockdown of Rbfox1 and Rbfox2 in zebrafish leads to defects in alternative splicing, myofiber morphology, and function of both heart and skeletal muscle ( Gallagher et al, 2011 ), and fox-1 mutants in C. elegans display aberrant myoblast migration and impaired egg-laying ( Kuroyanagi et al, 2006 ; Mackereth, 2014 ). We now show that knockdown of Rbfox1 in fly muscle causes behavioral deficits and impaired muscle function ( Figs 2 and 3 ).…”

Rbfox1 is required for myofibril development and maintaining fiber-type specific isoform expression inDrosophilamuscles

“…Knockdown of Rbfox1 and Rbfox2 in zebrafish leads to defects in alternative splicing, myofiber morphology, and function of both heart and skeletal muscle (Gallagher et al, 2011). Even mutants in the C. elegans homologue fox-1 lead to aberrant myoblast migration and impaired egg-laying (Kuroyanagi et al, 2006;Mackereth, 2014). We previously reported that muscle-specific knockdown of Rbfox1 in Drosophila results in short IFM sarcomeres (Nikonova et al, 2019).…”

“…In mouse, Rbfox1 and Rbfox2 regulate splicing of Mef2D exon α2 during myotube differentiation allowing Mef2D to escape inhibitory PKD signaling and activate the late-muscle gene expression program (Runfola et al, 2015). In C. elegans, FOX-1/ASD-1 and SUP-12 regulate a developmental switch in expression of the fibroblast growth factor receptor egl-15 that is necessary for myoblast migration and vulval muscle formation (Kuroyanagi et al, 2007;Mackereth, 2014). Rbfox1 is upregulated as cardiac cells differentiate and knockdown results in cardiac hypertrophy and splicing defects, consistent with the reduction in Rbfox1 expression in human patients with dilated cardiomyopathy and in hypertrophic heart from mouse and zebrafish (Gao et al, 2016).…”

Quantitative RNA-seq meta-analysis of alternative exon usage in C. elegans

“…The study of individual alternative splicing events can also be performed using fluorescent reporters in vivo (Kuroyanagi et al 2006(Kuroyanagi et al , 2007(Kuroyanagi et al , 2013Ohno et al 2008Ohno et al , 2012Tomioka et al 2016). To date, this is the most efficient technique to perform genetic screens in order to identify splicing factors that regulate those events and open the path to study the biochemical details of the regulatory interactions (Amrane et al 2014;Kuwasako et al 2014;Mackereth 2014).…”