Gill Tina, Cai Ti, Aulds Jason, Wierzbicki Sara, Schmitt Mark E
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Mol Cell Biol. 2004 Feb;24(3):945-53. doi: 10.1128/MCB.24.3.945-953.2004.
RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.
核糖核酸酶线粒体RNA加工（RNase MRP）突变体已被证明存在有丝分裂退出缺陷，这是由CLB2 mRNA水平升高引起的，导致Clb2p（B-细胞周期蛋白）水平升高，进而导致后期延迟。在这里，我们描述了这种延迟背后的分子缺陷。CLB2 mRNA通常在细胞完成有丝分裂时迅速消失，但在RNase MRP突变体中其水平仍然很高。这与其他有丝分裂退出突变体形成直接对比，是CLB2 mRNA稳定性增加的结果。我们发现在体外试验中，高度纯化的RNase MRP在几个位置切割CLB2 mRNA的5'非翻译区（UTR）。在体内，我们在CLB2 mRNA上鉴定出与体外产物紧密匹配的依赖于RNase MRP的切割产物。这些产物的处理依赖于5'→3'外切核糖核酸酶Xrn1，而不是外泌体。我们的结果表明，核糖核酸内切酶RNase MRP特异性切割CLB2 mRNA的5'-UTR，以便通过Xrn1核酸酶进行快速的5'到3'降解。RNase MRP核酸内切酶对CLB2 mRNA的降解提供了一种调节细胞周期的新方法，它补充了蛋白质降解机制。此外，这些结果表明了一种在酿酒酵母中以前未见的mRNA降解新机制。
