PMC:4502370 / 15764-19741 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"Synthetic genetics of Snp1 truncation mutants\nSnp1, a 300-aa polypeptide, is the yeast homolog of human U1-70K (437-aa). Alignment of their primary structures highlights 98 positions of side-chain identity/similarity over the N-terminal 207-aa segment of Snp1 (Figure 1A). In the 3.3 Å crystal structure of the core human U1 snRNP (Kondo et al. 2015), the N-terminal 60-aa segment of U1-70K is a highly extended polypeptide that drapes across the surface of the U1 particle, making contacts to U1-C/Yhc1 near the U1 snRNA 5′ terminus, to each of the Sm ring subunits, and to the U1 snRNA 3′ of the Sm site. The segment of U1-70K from aa 61-202 (underlined in Figure 1A), comprising a long α helix and an RRM domain, binds to the conserved stem-loop 1 (SL1) of the U1 snRNA (Kondo et al. 2015). The C-terminal domains of Snp1 and U1-70K differ in length and amino acid sequence and are expected to be poorly structured based on their amino acid composition. The conserved N-terminal 21-aa peptide of Snp1 that interacts with U1-C/Yhc1 and SmD3 could be deleted without effect on yeast vegetative growth at any temperature (Schwer and Shuman 2015). A SNP1-(22-300) tgs1∆ double-mutant displayed the same cs growth defect as tgs1∆ (Schwer and Shuman 2015).\nFigure 1 Snp1 C-terminal truncations suppress tgs1∆ cold sensitivity. (A) The amino acid sequence of the 300-aa S. cerevisiae Snp1 protein is aligned to the homologous segment of the 437-aa human U1-70K polypeptide. Positions of side chain identity/similarity are indicated by • above the alignment. Arrowheads indicate the boundaries of the C-terminal truncations of Snp1. (B) The wild-type and truncated SNP1 alleles were tested for activity by plasmid shuffle in snp1∆, snp1∆ mud2∆, snp1∆ nam8∆, snp1∆ mud1∆, and snp1∆ tgs1∆ strains. Viable FOA-resistant snp1∆ strains bearing the indicated SNP1 allele on a CEN HIS3 plasmid in an otherwise wild-type (top panel), mud1∆, or nam8∆ background as indicated were spot-tested for growth on YPD agar at the temperatures specified. Synthetic growth defects are denoted by •. snp1∆ tgs1∆ strains bearing the indicated SNP1 allele on a CEN HIS3 plasmid were spot-tested for growth on YPD agar at the temperatures specified (bottom panel). Suppressors of the tgs1∆ cs phenotype are denoted by arrowheads. Here, we constructed three C-terminal truncation mutants of Snp1 with distal margins indicated by the reverse arrowheads in Figure 1A. The wild-type and truncated alleles were placed on CEN HIS3 plasmids under the control of the native SNP1 promoter and tested by plasmid shuffle for complementation of a snp1∆ p[CEN URA3SNP1] strain. The resulting SNP1-(1-223), SNP1-(1-208), and SNP1-(1-193) strains were viable after FOA selection and grew as well as wild-type YHC1 cells on YPD agar (Figure 1B).\nWe surveyed genetic interactions of the benign Snp1 C-terminal truncations with mud2∆, nam8∆, and mud1∆. The results (Figure 1B) disclosed an informative hierarchy of synthetic mutational effects. SNP1-(1-223), SNP1-(1-208), and SNP1-(1-193) were lethal at all temperatures in the absence of Mud2, indicating that the essential contributions of the Snp1 segment downstream of the RRM module to early spliceosome assembly/stability are buffered by the cross-intron bridging interactions of Mud2 (engaged with Msl5 at the branchpoint) with U1 snRNP at the 5′ splice site.\nSNP1-(1-223) and SNP1-(1-208) were barely viable in the nam8∆ genetic background and SNP1-(1-193) was synthetically lethal with nam8∆. By contrast, SNP1-(1-223) and SNP1-(1-208) supported normal growth of mud1∆ cells at 20°–34° and slightly slowed growth at 37° (Figure 1B). The salient finding was that the more truncated SNP1-(1-193) allele was synthetically lethal in the mud1∆ strain, signifying that the Snp1 peptide 194FKPRRLGGGLGGRGY208 is critical for U1 snRNP function in vivo in the absence of Mud1. The corresponding peptide in U1-70K makes direct contacts to the SL1 loop (Kondo et al. 2015).","divisions":[{"label":"title","span":{"begin":0,"end":45}},{"label":"p","span":{"begin":46,"end":1253}},{"label":"figure","span":{"begin":1254,"end":2302}},{"label":"label","span":{"begin":1254,"end":1262}},{"label":"caption","span":{"begin":1264,"end":2302}},{"label":"p","span":{"begin":1264,"end":2302}},{"label":"p","span":{"begin":2303,"end":2802}},{"label":"p","span":{"begin":2803,"end":3372}}],"tracks":[]}