The putative nucleic acid helicase sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae
The putative nucleic acid helicase sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae
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DOI：
10.1128/mcb.18.12.6885
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发表时间：
1998-12-01
影响因子：
5.3
通讯作者：
Culbertson, MR
中科院分区：
文献类型：
--
作者：
Rasmussen, TP;Culbertson, MR
来自酵母的Sen1p是一种与DEAD盒RNA解旋酶和I型DNA解旋酶相关的核酸解旋酶，位于解旋酶基序的温度敏感突变sen1-1改变了前trnas，前RNAs和一些小核RNA的积累。在本报告中，我们发现携带sen1-1的细胞在温度变化时立即表现出几种小核核RNA （snoRNAs）的积累改变。通过Northern blotting、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三种形式的snoRNA snR13：一种是丰富的ttg覆盖的124核苷酸（nt）成熟形式（snR13F）和两种较少丰富的rna，包括类似于1400 nt的3‘延伸形式（snR13R）和108 nt 5’截断形式（snR13T）的异质群体，在5'端缺失16 nt。snR13R的一个亚群包含相同的5'截断。sen1-1细胞温度变化后，新合成的snR13R RNA以snR13F为代价随时间积累，提示可能存在前体-产物关系。snR13R和snR13T在限制性温度下丰度均增加，说明Sen1p稳定了5′端，促进了3′端成熟。snR13F包含许多snorna常见的规范C和D盒，snR13T的5‘端和snR13F的3’端位于C2U4序列中，直接连接C和D盒。5' C2U4重复序列的突变导致snR13F的积累不足，而3' C2U4重复序列的突变导致两种新rna的积累，这些rna在500-nt范围内迁移。在限制性温度下，携带sen1-1和3' C2U4重复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序列在一个共同的途径上促进3’末端的形成。我们认为，位于C和D框两侧的Sen1p和C2U4重复序列促进了3‘末端的成熟和5’末端的稳定性，并且是最大合成速率和成熟snR13F积累水平所必需的。
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 similar to 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 Bank 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.