Rutz B, Séraphin B
EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
EMBO J. 2000 Apr 17;19(8):1873-86. doi: 10.1093/emboj/19.8.1873.
The MSL5 gene, which codes for the splicing factor BBP/ScSF1, is essential in Saccharomyces cerevisiae, yet previous analyses failed to reveal a defect in assembly of (pre)-spliceosomes or in vitro splicing associated with its depletion. We generated 11 temperature-sensitive (ts) mutants and one cold-sensitive (cs) mutant in the corresponding gene and analyzed their phenotypes. While all mutants were blocked in the formation of commitment complex 2 (CC2) at non-permissive and permissive temperature, the ts mutants showed no defect in spliceosome formation and splicing in vitro. The cs mutant was defective in (pre)-spliceosome formation, but residual splicing activity could be detected. In vivo splicing of reporters carrying introns weakened by mutations in the 5' splice site and/or in the branchpoint region was affected in all mutants. Pre-mRNA leakage to the cytoplasm was strongly increased (up to 40-fold) in the mutants. A combination of ts mutants with a disruption of upf1, a gene involved in nonsense-mediated decay, resulted in a specific synthetic growth phenotype, suggesting that the essential function of SF1 in yeast could be related to the retention of pre-mRNA in the nucleus.
编码剪接因子BBP/ScSF1的MSL5基因在酿酒酵母中至关重要，但先前的分析未能揭示（前）剪接体组装或与其缺失相关的体外剪接存在缺陷。我们在相应基因中产生了11个温度敏感（ts）突变体和1个冷敏感（cs）突变体，并分析了它们的表型。虽然所有突变体在非允许温度和允许温度下在承诺复合体2（CC2）形成过程中均被阻断，但ts突变体在体外剪接体形成和剪接方面没有缺陷。cs突变体在（前）剪接体形成方面存在缺陷，但可以检测到残余的剪接活性。携带因5'剪接位点和/或分支点区域突变而弱化的内含子的报告基因的体内剪接在所有突变体中均受到影响。突变体中前体mRNA泄漏到细胞质的情况大幅增加（高达40倍）。ts突变体与参与无义介导衰变的upf1基因破坏的组合导致了一种特定的合成生长表型，表明SF1在酵母中的基本功能可能与前体mRNA保留在细胞核中有关。
