Liang S, Briggs M W, Butler J S
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14618, USA.
RNA. 1997 Jun;3(6):648-59.
A 26-nt sequence from the 3' UTR of the yeast GAL7 mRNA directs accurate and efficient cleavage and polyadenylation to form the 3' end of the GAL7 mRNA in vivo and in vitro. Here we asked whether this polyadenylation signal can function within the context of a tRNA. Insertion of the GAL7 signal into the intron of the dominant SUP4 nonsense suppressor allowed us to judge the effect of the insert on SUP4 function by observation of nonsense suppression efficiency in vivo. The GAL7 signal impairs the function of SUP4 in an orientation-dependent manner in vivo, consistent with its ability to specify cleavage and polyadenylation in this context in vitro. Mutation of a UA repeat within the GAL7 signal restores SUP4 function partially, consistent with the role of this repeat as an efficiency element in polyadenylation. Mutations that impair the mRNA 3' end-processing factors Rna14p and Rna15p restore suppressor function partially. Northern blot analysis, PCR amplification, and DNA sequence analysis show that the GAL7 signal directs polyadenylation within the body of pre-SUP4 and within the terminator, suggesting that polyadenylation inhibits 5' and 3' end processing, as well as removal of the pre-tRNA intron. These findings indicate that the GAL7 polyadenylation signal is capable of targeting a pre-tRNA to the mRNA processing pathway.
酵母GAL7 mRNA 3'UTR的一段26个核苷酸的序列在体内和体外均可引导准确且高效的切割和聚腺苷酸化反应，从而形成GAL7 mRNA的3'末端。在此，我们探究了该聚腺苷酸化信号能否在tRNA的背景下发挥作用。将GAL7信号插入显性SUP4无义抑制基因的内含子中，使我们能够通过观察体内无义抑制效率来判断插入片段对SUP4功能的影响。在体内，GAL7信号以方向依赖的方式损害SUP4的功能，这与其在体外此背景下指定切割和聚腺苷酸化的能力一致。GAL7信号内UA重复序列的突变部分恢复了SUP4的功能，这与该重复序列作为聚腺苷酸化效率元件的作用相符。损害mRNA 3'末端加工因子Rna14p和Rna15p的突变部分恢复了抑制基因的功能。Northern印迹分析、PCR扩增和DNA序列分析表明，GAL7信号在pre-SUP4内部和终止子内引导聚腺苷酸化，这表明聚腺苷酸化抑制了5'和3'末端加工以及前体tRNA内含子的去除。这些发现表明，GAL7聚腺苷酸化信号能够将前体tRNA靶向至mRNA加工途径。
