Mayer S A, Dieckmann C L
Department of Biochemistry, University of Arizona, Tucson 85721.
Mol Cell Biol. 1991 Feb;11(2):813-21. doi: 10.1128/mcb.11.2.813-821.1991.
Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.
可变mRNA加工是一种从单个基因产生两种或更多种多肽的机制。虽然许多哺乳动物基因含有多个mRNA 3' 切割和聚腺苷酸化信号，这些信号在不同发育阶段或不同组织中使用时会改变成熟mRNA的编码序列，但只有一个酵母基因被鉴定具有这种能力。酿酒酵母核基因CPB1编码一种线粒体蛋白，该蛋白是细胞色素b mRNA稳定性所必需的。这种66 kDa的蛋白由从CPB1转录的2.2 kb mRNA编码。此前我们表明，第二个1.2 kb的转录本在CBP1启动子处起始，但3' 端位于编码序列中部附近。此外，研究表明，在诱导线粒体功能期间，2.2 kb CBP1 mRNA与1.2 kb mRNA的稳态水平之比降低了10倍，而两种mRNA的总水平保持不变。我们曾提出3' 端形成的调控决定了每个CBP1转录本的数量，现在我们表明，当将CBP1中部的一个146 bp片段插入酵母URA3基因时，它足以指导碳源调节的两种转录本的产生。通过对CBP1 cDNA克隆的序列分析绘制图谱，该片段包含野生型1.2 kb mRNA的七个聚腺苷酸化位点。聚腺苷酸化位点上游的缺失突变消除了1.2 kb转录本的形成，而删除其中三个位点仅导致1.2 kb mRNA丰度降低。我们的结果表明，两种CBP1转录本丰度的调控由该基因短片段中的元件控制，该元件指导1.2 kb转录本的3' 端形成，这在酵母细胞中是一个独特的例子。
