Umen J G, Guthrie C
Department of Biochemistry and Biophysics, University of California at San Francisco 94143, USA.
Genes Dev. 1995 Apr 1;9(7):855-68. doi: 10.1101/gad.9.7.855.
The choice of a 3' splice site in Saccharomyces cerevisiae introns involves recognition of a uridine-rich tract upstream of the AG dinucleotide splice junction. By isolating mutants that eliminate the normal preference for uridine-containing 3' splice sites in a cis-competition, we identified a mutation that is an allele of PRP8, prp8-101. This was unexpected because previous analysis has demonstrated that the U5 snRNP protein encoded by PRP8 is required for spliceosome assembly prior to the first catalytic step of splicing. In contrast, the uridine recognition defect caused by the prp8-101 mutation selectively inhibits the second catalytic step of splicing. This defect is seen not only in 3' splice site cis-competitions but also in the splicing of an unusual intron in the TUB3 gene and in the ACT1 intron when utilization of its 3' splice site is rate limiting for splicing. Consistent with a direct role in 3' splice site selection, Prp8 can be cross-linked to the 3' splice site during the splicing reaction. These data demonstrate a novel function for Prp8 in 3' splice site recognition and utilization.
酿酒酵母内含子中3'剪接位点的选择涉及对AG二核苷酸剪接接头上游富含尿苷序列的识别。通过在顺式竞争中分离消除对含尿苷3'剪接位点正常偏好的突变体，我们鉴定出一个突变，它是PRP8的一个等位基因，即prp8 - 101。这是出乎意料的，因为先前的分析表明，PRP8编码的U5 snRNP蛋白在剪接的第一个催化步骤之前的剪接体组装中是必需的。相比之下，prp8 - 101突变引起的尿苷识别缺陷选择性地抑制剪接的第二个催化步骤。这种缺陷不仅在3'剪接位点顺式竞争中可见，在TUB3基因中一个不寻常内含子的剪接以及ACT1内含子的剪接中（当其3'剪接位点的利用对剪接起限速作用时）也可见。与在3'剪接位点选择中起直接作用一致，Prp8在剪接反应过程中可与3'剪接位点交联。这些数据证明了Prp8在3'剪接位点识别和利用中的新功能。
