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一种改进的 MS2 系统，用于准确报告 mRNA 生命周期。

An improved MS2 system for accurate reporting of the mRNA life cycle.

机构信息

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA.

Department of Molecular Biology, Colorado College, Colorado Springs, Colorado, USA.

出版信息

Nat Methods. 2018 Jan;15(1):81-89. doi: 10.1038/nmeth.4502. Epub 2017 Nov 13.

DOI:10.1038/nmeth.4502
PMID:29131164
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5843578/
Abstract

The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.

摘要

MS2-MCP 系统使研究人员能够以高时间和空间分辨率对 mRNA 生命周期的多个步骤进行成像。然而，对于短寿命的 mRNAs 来说，MS2 外壳蛋白 (MCP) 与 MS2 结合位点 (MBS) 的紧密结合保护 RNA 免受有效降解，这使得 mRNA 调控的研究变得复杂。在这里，我们描述了一种具有降低的 MCP 亲和力的报告系统 (MBSV6)，它允许 mRNA 降解，同时保留通过单分子荧光原位杂交 (smFISH) 或活细胞成像确定的单分子检测。在酿酒酵母中，内源标记有 MBSV6 的组成型 mRNAs (MDN1 和 DOA1) 和高度调控的 mRNAs (GAL1 和 ASH1) 正常降解。结果，短寿命的 mRNAs 被完整地成像。MBSV6 报告显示，与之前的发现相反，在应激期间，mRNA 并未在 P 体等专门结构中协调募集，并且 mRNA 降解在细胞质中呈异质性分布。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/814239c083e9/nihms913718f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/1d13013a4cd9/nihms913718f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/2610fa7ecdf5/nihms913718f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/0d544c3d6414/nihms913718f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/b806cd7c2407/nihms913718f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/6d95142b164f/nihms913718f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/814239c083e9/nihms913718f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/1d13013a4cd9/nihms913718f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/2610fa7ecdf5/nihms913718f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/0d544c3d6414/nihms913718f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/b806cd7c2407/nihms913718f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/6d95142b164f/nihms913718f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2dfa/5843578/814239c083e9/nihms913718f6.jpg

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