An improved MS2 system for accurate reporting of the mRNA life cycle.
Nat Methods
; 15(1): 81-89, 2018 01.
Article
em En
| MEDLINE
| ID: mdl-29131164
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
/
RNA Fúngico
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RNA Mensageiro
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Estabilidade de RNA
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Proteínas de Saccharomyces cerevisiae
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Proteínas do Capsídeo
Limite:
Humans
Idioma:
En
Revista:
Nat Methods
Assunto da revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos