Heinrich Stephanie, Sidler Corinne L, Azzalin Claus M, Weis Karsten
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
RNA. 2017 Feb;23(2):134-141. doi: 10.1261/rna.057786.116. Epub 2016 Nov 10.
The binding of sequence-specific RNA-interacting proteins, such as the bacteriophage MS2 or PP7 coat proteins, to their corresponding target sequences has been extremely useful and widely used to visualize single mRNAs in vivo. However, introduction of MS2 stem-loops into yeast mRNAs has recently been shown to lead to the accumulation of RNA fragments, suggesting that the loops impair mRNA decay. This result was questioned, because fragment occurrence was mainly assessed using ensemble methods, and their cellular localization and its implications had not been addressed on a single transcript level. Here, we demonstrate that the introduction of either MS2 stem-loops (MS2SL) or PP7 stem-loops (PP7SL) can affect the processing and subcellular localization of mRNA. We use single-molecule fluorescence in situ hybridization (smFISH) to determine the localization of three independent mRNAs tagged with the stem-loop labeling systems in glucose-rich and glucose starvation conditions. Transcripts containing MS2SL or PP7SL display aberrant localization in both the nucleus and the cytoplasm. These defects are most prominent in glucose starvation conditions, with nuclear mRNA processing being altered and stem-loop fragments abnormally enriching in processing bodies (PBs). The mislocalization of SL-containing RNAs is independent of the presence of the MS2 or PP7 coat protein (MCP or PCP).
序列特异性RNA相互作用蛋白，如噬菌体MS2或PP7外壳蛋白，与它们相应的靶序列的结合极其有用，并被广泛用于在体内可视化单个mRNA。然而，最近有研究表明，将MS2茎环引入酵母mRNA会导致RNA片段的积累，这表明这些环会损害mRNA的降解。这一结果受到质疑，因为片段的出现主要是使用整体方法评估的，而且它们在细胞中的定位及其影响在单个转录本水平上尚未得到解决。在这里，我们证明引入MS2茎环（MS2SL）或PP7茎环（PP7SL）都会影响mRNA的加工和亚细胞定位。我们使用单分子荧光原位杂交（smFISH）来确定在富含葡萄糖和葡萄糖饥饿条件下，用茎环标记系统标记的三个独立mRNA的定位。含有MS2SL或PP7SL的转录本在细胞核和细胞质中均显示出异常定位。这些缺陷在葡萄糖饥饿条件下最为明显，核mRNA加工发生改变，茎环片段在加工体（PBs）中异常富集。含茎环RNA的错误定位与MS2或PP7外壳蛋白（MCP或PCP）的存在无关。
