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

The MS2-MCP system allows imaging 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, confounding the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, allowing mRNA degradation while preserving single molecule detection determined by smFISH or live imaging. Constitutive mRNAs (MDN1 and DOA1) or highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in S. cerevisiae degrade normally. As a result, rapidly turning over mRNAs were imaged throughout their complete life cycle. MBSV6 provided single molecule detection in live mammalian cells. The MBSV6 reporter revealed that coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur and that degradation was heterogeneously distributed in the cytoplasm.