Rasmussen T P, Culbertson M R
Laboratories of Genetics and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Mol Cell Biol. 1998 Dec;18(12):6885-96. doi: 10.1128/MCB.18.12.6885.
Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of approximately 1,400-nt 3'-extended forms (snR13R) and a 108-nt 5'-truncated form (snR13T) that is missing 16 nt at the 5' end. A subpopulation of snR13R contains the same 5' truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5' end and promotes maturation of the 3' end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5' end of snR13T and the 3' end of snR13F reside within C2U4 sequences that immediately flank the C and D boxes. A mutation in the 5' C2U4 repeat causes underaccumulation of snR13F, whereas mutations in the 3' C2U4 repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3' C2U4 repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3' C2U4 sequence act in a common pathway to facilitate 3' end formation. Based on these findings, we propose that Sen1p and the C2U4 repeats that flank the C and D boxes promote maturation of the 3' terminus and stability of the 5' terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.
来自酿酒酵母的Sen1p是一种核酸解旋酶，与DEAD盒RNA解旋酶和I型DNA解旋酶相关。位于解旋酶基序中的温度敏感型sen1-1突变改变了前体tRNA、前体rRNA和一些小核RNA的积累。在本报告中，我们表明携带sen1-1的细胞在温度转换后立即表现出几种小核仁RNA（snoRNA）积累的改变。使用Northern印迹、RNase H切割、引物延伸和碱基组成分析，我们在野生型细胞中检测到snoRNA snR13的三种形式：一种丰富的带TMG帽的124个核苷酸（nt）的成熟形式（snR13F）和另外两种含量较少的RNA，包括约1400 nt的3'延伸形式的异质群体（snR13R）和一种108 nt的5'截短形式（snR13T），其5'端缺失16 nt。snR13R的一个亚群包含相同的5'截短。在sen1-1细胞温度转换后，新合成的snR13R RNA随时间积累，以snR13F为代价，这表明可能存在前体-产物关系。在限制温度下，snR13R和snR13T的丰度均增加，表明Sen1p稳定5'端并促进3'端的成熟。snR13F包含许多snoRNA共有的典型C和D框。snR13T的5'端和snR13F的3'端位于紧邻C和D框的C2U4序列内。5' C2U4重复序列中的突变导致snR13F积累不足，而3' C2U4重复序列中的突变导致两种新RNA的积累，它们在500 nt范围内迁移。在限制温度下，携带sen1-1和3' C2U4重复序列突变的双突变体显示新RNA的积累减少，snR13R RNA的积累增加，这表明Sen1p和3' C2U4序列在促进3'端形成的共同途径中起作用。基于这些发现，我们提出Sen1p以及位于C和D框两侧的C2U4重复序列促进3'末端的成熟和5'末端的稳定性，并且是成熟snR13F最大合成速率和积累水平所必需的。
