The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans

“However, the molecular mechanism by which tissue-specific alternative splicing is regulated remains largely unknown, except for a few well-characterized examples (Smith and Valcarcel, 2000; Maniatis and Tasic, 2002; Black, 2003).”

“Disruption of the splicing machinery is known to cause various human diseases (Nissim-Rafinia and Kerem, 2002; Stoilov et al., 2002; Faustino and Cooper, 2003).”

“Actin depolymerizing factor (ADF)/cofilin proteins enhance actin filament dynamics by severing filaments and accelerating monomer dissociation from the pointed ends of the filaments (Bamburg, 1999; Bamburg et al., 1999; Maciver and Hussey, 2002; Ono, 2003).”

“The speckled localization of SUP-12 in the nucleus is similar to patterns that are commonly observed for other splicing factors (Misteli, 2000; Dundr and Misteli, 2001; Lamond and Spector, 2003).”

“Pre-messenger RNA splicing is mediated by spliceosomes that contain small ribonucleoprotein particles and splicing factors (Jurica and Moore, 2003; Nilsen, 2003).”

“However, the molecular mechanism by which tissue-specific alternative splicing is regulated remains largely unknown, except for a few well-characterized examples (Smith and Valcarcel, 2000; Maniatis and Tasic, 2002; Black, 2003).”

“However, the molecular mechanism by which tissue-specific alternative splicing is regulated remains largely unknown, except for a few well-characterized examples (Smith and Valcarcel, 2000; Maniatis and Tasic, 2002; Black, 2003).”

“In particular, myotonic dystrophy has been linked to perturbation of the mechanism of alternative splicing in muscle (Kanadia et al., 2003; Ho et al., 2004).”

“We further narrowed down sup-12 by the snipSNP mapping with polymorphisms in CB4856 (Wicks et al., 2001) to an interval between cosmid clones ZC64 and T06F4 that contained 20 genes.”

“In particular, myotonic dystrophy has been linked to perturbation of the mechanism of alternative splicing in muscle (Kanadia et al., 2003; Ho et al., 2004).”

“Essential functions of ADF/ cofilin in in vivo actin dynamics and cell viability have been demonstrated in several organisms (McKim et al., 1994; Gunsalus et al., 1995; Lappalainen and Drubin, 1997).”

“Thus, SUP-12 and TDP-43 may negatively regulate splicing in a similar manner by binding to UG repeats and directly competing with the U2 auxiliary factor, which is an essential splicing factor that binds to 3 -splice sites (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999).”

“The speckled localization of SUP-12 in the nucleus is similar to patterns that are commonly observed for other splicing factors (Misteli, 2000; Dundr and Misteli, 2001; Lamond and Spector, 2003).”

“Pre-messenger RNA splicing is mediated by spliceosomes that contain small ribonucleoprotein particles and splicing factors (Jurica and Moore, 2003; Nilsen, 2003).”

“Actin depolymerizing factor (ADF)/cofilin proteins enhance actin filament dynamics by severing filaments and accelerating monomer dissociation from the pointed ends of the filaments (Bamburg, 1999; Bamburg et al., 1999; Maciver and Hussey, 2002; Ono, 2003).”

“Actin depolymerizing factor (ADF)/cofilin proteins enhance actin filament dynamics by severing filaments and accelerating monomer dissociation from the pointed ends of the filaments (Bamburg, 1999; Bamburg et al., 1999; Maciver and Hussey, 2002; Ono, 2003).”

“Essential functions of ADF/ cofilin in in vivo actin dynamics and cell viability have been demonstrated in several organisms (McKim et al., 1994; Gunsalus et al., 1995; Lappalainen and Drubin, 1997).”

“…staining was performed on adult worms that were permeabilized with a freeze-crack method (Epstein et al., 1993) and fixed with methanol for 5 min at 20 C. Primary antibodies used were anti–UNC-60A (Ono et al., 1999) and anti-myoA (mAb 5.6, obtained from H.F. Epstein; Miller et al., 1983).”

“Thus, SUP-12 and TDP-43 may negatively regulate splicing in a similar manner by binding to UG repeats and directly competing with the U2 auxiliary factor, which is an essential splicing factor that binds to 3 -splice sites (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999).”

“Wild-type N2 and unc-60B(e677) (Waterston et al., 1980) were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN). sup-12(st89) and sup-12(st203) were obtained from G.R. Francis (Exelixis, Inc., South San Francisco, CA) and R. Waterston (University of Washington, Seattle, WA).…”

“Also, it is possible that SUP-12 affects RNA stability or interacts with transcription factors and regulates transcription and pre-mRNA processing because splicing factors and transcription factors functionally interact and regulate pre-mRNA splicing in many instances (Bentley, 2002).”

“Western blot was performed as described previously (Ono and Ono, 2002) using the following primary antibodies: anti–UNC-60A (Ono et al.”

“Thus, SUP-12 and TDP-43 may negatively regulate splicing in a similar manner by binding to UG repeats and directly competing with the U2 auxiliary factor, which is an essential splicing factor that binds to 3 -splice sites (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999).”

“The sup-12 single mutants had no detectable phenotype in the myofibril organization (Fig. 1 B; Francis and Waterston, 1985).”

“In mammals, three ADF/cofilin isoforms are encoded by separate genes and expressed in different patterns of tissue distribution (Matsuzaki et al., 1988; Moriyama et al., 1990; Ono et al., 1994; Gillett et al., 1996; Thirion et al., 2001; Vartiainen et al., 2002).”

“Disruption of the splicing machinery is known to cause various human diseases (Nissim-Rafinia and Kerem, 2002; Stoilov et al., 2002; Faustino and Cooper, 2003).”

“Our previous studies have indicated that the two ADF/cofilin isoforms have different activities: UNC-60A strongly depolymerizes filaments, whereas UNC-60B binds to filaments with only weak depolymerizing activity (Ono and Benian, 1998; Ono, 1999; Mohri and Ono, 2003).”

“The truncated region contains repeats of UG that have been reported to bind to several RNA-binding proteins (Mittag, 1996; Takahashi et al., 2000; Buratti et al., 2004).”

“645 brosis transmembrane conductance regulator pre-mRNA, the nuclear RRM protein TDP-43 binds to the UG repeats at the 3 end of intron 8 and causes exon skipping (Niksic et al., 1999; Pagani et al., 2000; Buratti et al., 2004).”

“Biotin-RNA pull-down assay ( Lee and Schedl, 2001 ) was performed with the following modifications.”

“645 brosis transmembrane conductance regulator pre-mRNA, the nuclear RRM protein TDP-43 binds to the UG repeats at the 3 end of intron 8 and causes exon skipping (Niksic et al., 1999; Pagani et al., 2000; Buratti et al., 2004).”

“The SUP-12 protein (248 aa) is a putative RNA-binding protein that has a single RNA recognition motif (RRM) domain (Shamoo et al., 1995) in its NH 2 terminus (residues 35–117) and alanine- and glutamine-rich (A/Qrich) sequence in its COOH terminus (26 A and 24 Q in residues 121–246; Fig.”

“(A) Worm motility was quantified as beating frequency (beats/min) as described previously (Epstein and Thomson, 1974).”

“Essential functions of ADF/ cofilin in in vivo actin dynamics and cell viability have been demonstrated in several organisms (McKim et al., 1994; Gunsalus et al., 1995; Lappalainen and Drubin, 1997).”

“, 1999), anti–UNC-60B (Ono et al., 1999), anti-actin monoclonal C4 (ICN Biomedicals), and anti– -tubulin (Amersham Biosciences).”

“unc-60B(su158) (Zengel and Epstein, 1980) was originally obtained from H.”

“3 B, lane 4). unc-60B(su158) has a 600-bp deletion in the unc-60B region without affecting unc-60A (Ono et al., 2003) and expressed a shorter unc-60B mRNA (Fig.”

“In mammals, three ADF/cofilin isoforms are encoded by separate genes and expressed in different patterns of tissue distribution (Matsuzaki et al., 1988; Moriyama et al., 1990; Ono et al., 1994; Gillett et al., 1996; Thirion et al., 2001; Vartiainen et al., 2002).”

“Actin filaments were visualized by staining adult worms with tetramethylrhodamine-phalloidin (Sigma-Aldrich) as described previously (Ono, 2001).”

“In mammals, three ADF/cofilin isoforms are encoded by separate genes and expressed in different patterns of tissue distribution (Matsuzaki et al., 1988; Moriyama et al., 1990; Ono et al., 1994; Gillett et al., 1996; Thirion et al., 2001; Vartiainen et al., 2002).”

“Although UNC-60A and UNC-60B have quantitatively different biochemical activities, both isoforms can enhance actin filament dynamics by depolymerizing actin filaments (Ono and Benian, 1998; Ono, 1999; Ono et al., 1999).”

“The truncated region contains repeats of UG that have been reported to bind to several RNA-binding proteins (Mittag, 1996; Takahashi et al., 2000; Buratti et al., 2004).”

“Disruption of the splicing machinery is known to cause various human diseases (Nissim-Rafinia and Kerem, 2002; Stoilov et al., 2002; Faustino and Cooper, 2003).”

“In mammals, three ADF/cofilin isoforms are encoded by separate genes and expressed in different patterns of tissue distribution (Matsuzaki et al., 1988; Moriyama et al., 1990; Ono et al., 1994; Gillett et al., 1996; Thirion et al., 2001; Vartiainen et al., 2002).”

“The truncated region contains repeats of UG that have been reported to bind to several RNA-binding proteins (Mittag, 1996; Takahashi et al., 2000; Buratti et al., 2004).”

“Our previous studies have indicated that the two ADF/cofilin isoforms have different activities: UNC-60A strongly depolymerizes filaments, whereas UNC-60B binds to filaments with only weak depolymerizing activity (Ono and Benian, 1998; Ono, 1999; Mohri and Ono, 2003).”

“Immunofluorescent staining was performed on adult worms that were permeabilized with a freeze-crack method (Epstein et al., 1993) and fixed with methanol for 5 min at 20 C.”

“Indeed, in case of polycistronic genes in C. elegans, an interaction between a 3 -end processing factor and a factor for trans-splicing is reported (Evans et al., 2001).”

“1 A), as well as other unc-60B loss of function alleles (McKim et al., 1988), and the motility of the unc-60B;sup-12 double mutants was restored nearly to the level of wild-type worms (Fig.”

“In mammals, three ADF/cofilin isoforms are encoded by separate genes and expressed in different patterns of tissue distribution (Matsuzaki et al., 1988; Moriyama et al., 1990; Ono et al., 1994; Gillett et al., 1996; Thirion et al., 2001; Vartiainen et al., 2002).”

“In Xenopus , two SEB-4 isoforms, XSEB-4 and XSEB-4R, are reported: XSEB-4 is expressed in muscle precursor cells (Fetka et al., 2000), whereas XSEB-4R is strongly expressed in neuronal cells and involved in neural differentiation (Boy et al.”

“, 2000), whereas XSEB-4R is strongly expressed in neuronal cells and involved in neural differentiation (Boy et al., 2004).”

“C. elegans has two ADF/cofilin isoforms, UNC-60A and UNC-60B, which are generated by the unc-60 gene by alternative splicing [McKim et al., 1994; Anyanful et al., 2004].”

“A previous study showed that the Caenorhabditis elegans homologue of these genes, sup12, specifically regulated expression of a muscle-specific gene during myogenic development (Anyanful et al. 2004).”

“elegans orthog SUP-12, providing further support for this motif [9,10].”

“Muscle-specific expression of UNC-60B is controlled by splicing factors, sup-12 (RBM24/SEB-4) and asd-2 (Quaking/QKI), which suppress expression of UNC-60A in muscle (Anyanful et al., 2004; Ohno et al., 2012).”

“Interestingly, previous genetic studies had identified alleles of sup-12 with no obvious visible phenotypes (Anyanful et al., 2004).”

“SEB-4 is likely involved in regulation of cytoskeletal events in muscle development, since it is a Xenopus homolog of the C. elegans protein SUP-12, which regulates splicing of unc-60 mRNA (Anyanful et al., 2004).”

“In C. elegans,the unc-60 gene encodes two ADF/cofilin isoforms, UNC-60A and UNC-60B, which are generated by alternative pre-mRNA splicing [McKim et al., 1994; Anyanful et al., 2004].”

“In C. elegans, two ADF/cofilin isoforms, UNC-60A and UNC-60B, are expressed from the unc-60 gene by alternative splicing (Anyanful et al., 2004; McKim et al., 1994).”

“Actin depolymerizing factor (ADF)/cofilin promotes actin filament turnover by severing and depolymerizing actin filaments (Bernstein and Bamburg, 2010).”

“Identi fi cation of SUP-12/RBM24 as a speci fi c regulator of touch neuron splicing is surprising, as previous work characterized SUP-12 as a muscle-speci fi c splicing factor in C. elegans 41,42 .”

“Other elements include the U-rich and UG repeat elements that are bound by embryonic lethal abnormal visual system protein (ELAV) and SUP-12, respectively, to repress splicing of the neuroglian 30 terminal exon and ADF/Cofilin exon 2A [37, 38], or UG motifs by TAR”

“Furthermore, the C Elegans ortholog of Rbm24 and Rbm38, SUP-12, has been implicated in muscle-specific splicing of ADF/cofilin and fibroblast growth factor receptor 2 (Fgfr2) [12, 13].”

“Splicing/alternative splicing regulated by RNA binding proteins has been reported to regulate tissue/organ specific expression in several animal (Anyanful et al. 2004; Underwood et al. 2005; Auweter et al. 2006; Kuroyanagi et al. 2006) as well as plant systems (Ito et al. 2002; Fang et al. 2004;…”

“…such as ASD-1, FOX-1, SUP-12, ASD-2, UNC-75 and PTB-1 have been characterized as the basis of tissue- and cell-type-specific alternative splicing (Anyanful et al., 2004; Kuroyanagi et al., 2006; Kuroyanagi et al., 2010; Kuroyanagi, Ohno, Mitani, & Hagiwara, 2007; Kuroyanagi, Watanabe, &…”

“The C. elegans homolog of vertebrate Rbm24 gene is sup-12 , which was cloned by screening mutations that suppress muscle defects resulted from decreased expression of muscle-specific actin depolymerizing factor (ADF)/cofilin (Anyanful et al. 2004).”

“An RNA segment centered on a GUGUG element was conserved in the regulatory intron region of both egl-15 and unc-60, and mutation of this motif prevented binding of SUP-12 (Anyanful et al. 2004; Kuroyanagi et al. 2007).”

“Previous studies suggested an essential role of Rbm38 in alternative splicing during late erythroid differentiation, neurogenesis, and myogenesis (Anyanful et al., 2004 ; Heinicke et al., 2013 ; Amrane et al., 2014 ; Ganaie et al., 2018 ; Li et al., 2020 ; Han et al., 2022 ).”

“From our muscle-expressed library, the 6-mer with the strongest ISE activity was GUGUGU (t-stat = 6.9), which closely resembles the consensus sequence recognized by SUP-12, a known muscle-expressed RBP splicing regulator [ 69 , 70 ] (Fig.”

“Nematode SUP-12 features an A/Q rich sequence in its C-terminus (Anyanful et al. 2004).”