SMU-2 and SMU-1, Caenorhabditis elegans Homologs of Mammalian Spliceosome-Associated Proteins RED and fSAP57, Work Together To Affect Splice Site Choice

Abstract

Mutations in the Caenorhabditis elegans gene smu-2 suppress mec-8 and unc-52 mutations. It has been proposed that MEC-8 regulates the alternative splicing of unc-52 transcripts, which encode the core protein of perlecan, a basement membrane proteoglycan. We show that mutation in smu-2 leads to enhanced accumulation of transcripts that skip exon 17, but not exon 18, of unc-52, which explains our finding that smu-2 mutations suppress the uncoordination conferred by nonsense mutations in exon 17, but not in exon 18, of unc-52. We conclude that smu-2 encodes a ubiquitously expressed nuclear protein that is 40% identical to the human RED protein, a component of purified spliceosomes. The effects of smu-2 mutation on both unc-52 premRNA splicing and the suppression of mec-8 and unc-52 mutant phenotypes are indistinguishable from the effects of mutation in smu-1, a gene that encodes a protein that is 62% identical to human spliceosomeassociated protein fSAP57. We provide evidence that SMU-2 protects SMU-1 from degradation in vivo. In vitro and in vivo coimmunoprecipitation experiments indicate that SMU-2 and SMU-1 bind to each other. We propose that SMU-2 and SMU-1 function together to regulate splice site choice in the pre-mRNAs of unc-52 and other genes.The excision of introns from pre-mRNA is carried out by the spliceosome, a complex machine composed of five snRNAs and many-approximately 145 in humans (52)-different proteins. Understanding how the spliceosome regulates splicing and splice site choice is complicated by the phenomenon of alternative RNA splicing, in which a given transcript can be spliced in alternative ways to generate different mRNA isoforms, which may be translated into different protein products with different functions (2). In a few cases, alternative splicing factors that regulate the alternative splicing of specific premRNA targets in a development-or tissue-specific manner are known. Well-studied examples include the Drosophila melanogaster Sxl, Tra, and Tra2 proteins, which regulate alternative splicing of genes involved in fly sex determination.The Caenorhabditis elegans protein MEC-8 also behaves as a specific regulator of alternative splicing. It contains two RNA recognition motifs and promotes the accumulation of two alternatively spliced isoforms of unc-52 pre-mRNAs (29). mec-8 null mutants are viable and fertile but exhibit defects in mechanosensation, chemosensation, and embryogenesis (7, 28), which have been attributed to defects in the processing of premRNA targets of MEC-8 action other than unc-52 (28, 43). The specific phenotypes conferred by mec-8 mutation along with the tissue-specific pattern of mec-8 expression (42) suggest that MEC-8 is a specific rather than general regulator of splicing, and no apparent orthologs of MEC-8 have been found in purified human spliceosomes.MEC-8's action on unc-52 transcripts accounts for a synthetic lethal interaction exhibited by mec-8 mutations when they are combined with those for any of several otherwiseviable alleles of unc-52. The unc-52 g...

Mast Cells Produce Novel Shorter Forms of Perlecan That Contain Functional Endorepellin

“…A human homologue of Smu-1 has been described and termed fZAP57, which is ϳ62% identical in nucleotide sequence. It has been localized to the nucleus and has RNA binding motifs suggesting that it might be part of the spliceosome (56). However, it awaits further investigation to determine if it performs this function in human cells and whether it is up-regulated in mast cells.…”

A Genetic Screen for Suppressors of a Mutated 5′ Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly

“…To do this, we took advantage of unc-73(e936), in which modest increases in the use of the wt splice site lead to dramatic increases in coordination, as a sensitive screen for changes in cryptic splice site choice. In this article we report that the proteins SMU-1 and SMU-2, which are nonessential factors previously shown to have a role in alternative splicing (Spartz et al 2004), have a role in selection of cryptic 59 splice sites. We also report the identification of a new dominant suppressor of cryptic splicing, snrp-27, which encodes a C. elegans homolog of the human tri-snRNP 27K protein.…”

The Maize Zmsmu2 Gene Encodes a Putative RNA-Splicing Factor That Affects Protein Synthesis and RNA Processing during Endosperm Development

“…The human RED protein is a component of spliceosomes (Neubauer et al, 1998;Makarov et al, 2002;Rappsilber et al, 2002;Zhou et al, 2002). In C. elegans, SMU-2 and a related protein, SMU-1, were shown to play an important role in splice site selection during alternative RNA splicing (Spartz et al, 2004). Consequently, we designated the maize mutant gene Zmsmu2.…”

“…The nematode homolog, smu-2, was isolated from a screen for genetic suppressors of the mec-8; unc-52 double mutant (Lundquist and Herman, 1994). SMU-2 plays a role in pre-mRNA splicing, because the smu-2 mutant accumulates unc-52 transcripts that lack exon 17, which contains a premature stop codon, thereby preventing the mec-8; unc-52 double mutant from being lethal (Spartz et al, 2004). The gene product of another suppressor, smu-1, physically interacts with SMU-2, and a human protein homologous to SMU-1 is also found in spliceosomes (Jurica and Moore, 2003).…”

“…Characterization of the gene associated with this fragment showed that it encodes a homolog of mammalian RED (for Arg-, Glu-, and Asp-rich repeats) proteins, which are known components of spliceosomes (Neubauer et al, 1998;Makarov et al, 2002;Rappsilber et al, 2002;Zhou et al, 2002). The comparable mutation in Caenorhabditis elegans, smu-2 (Lundquist and Herman, 1994), was shown to play a role in pre-mRNA splicing (Spartz et al, 2004). Hence, we designated the mutant maize gene zmsmu2 (Zea mays homolog of nematode smu-2).…”