Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

Zachary B Katz,Timothée Lionnet,Nilah Monnier,Ben Ovryn,Robert H Singer,Young J Yoon,Mark Bathe,Brian P English

doi:10.7554/elife.10415

Abstract

Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

Highlights

MRNA compartmentalization is a conserved mechanism cells use to spatially control protein translation through zipcode sequences often in the transcript’s 3’UTR (Kislauskis and Singer, 1992; Holt and Bullock, 2009; Pratt and Mowry, 2012)
Previous studies identified that mRNA: (1) exhibited multiple movement profiles, (2) zipcode sequences facilitated directed transport of b-actin mRNA along microtubules, and (3) zipcode binding protein 1 (ZBP1) delivered b-actin mRNA to leading edge adhesions where it could dwell for more than a minute
To investigate mRNA movement based on cytoplasmic location we first established a method to track and map all endogenous b-actin mRNA in live fibroblasts

Summary

Introduction

MRNA compartmentalization is a conserved mechanism cells use to spatially control protein translation through zipcode sequences often in the transcript’s 3’UTR (Kislauskis and Singer, 1992; Holt and Bullock, 2009; Pratt and Mowry, 2012). Previous work has shown that MS2 binding sites (MBS) inserted adjacent to the zipcode sequence in the 3’UTR of b-actin mRNA can be used to label and track the movement of single mRNA molecules (Fusco et al, 2003; Yamagishi et al, 2009; Lionnet et al, 2011; Katz et al, 2012). Previous studies identified that mRNA: (1) exhibited multiple movement profiles (directed, diffusive, and corralled), (2) zipcode sequences facilitated directed transport of b-actin mRNA along microtubules, and (3) zipcode binding protein 1 (ZBP1) delivered b-actin mRNA to leading edge adhesions where it could dwell for more than a minute. The actin cytoskeleton was previously shown to be necessary for mRNA localization and maintenance at the leading edge of migrating fibroblasts (Sundell and Singer, 1991). A recent report argued that the RNP size is the main

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Conclusion