Multicolour single-molecule tracking of mRNA interactions with RNP granules

Ribonucleoprotein (RNP) granules are non-membrane-bound organelles that have critical roles in the stress response^1,2, maternal messenger RNA storage³, synaptic plasticity⁴, tumour progression^5,6 and neurodegeneration^7-9. However, the dynamics of their mRNA components within and near the granule surface remain poorly characterized, particularly in the context and timing of mRNAs exiting translation. Herein, we used multicolour single-molecule tracking to quantify the precise timing and kinetics of single mRNAs as they exit translation and enter RNP granules during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionally between them. Although translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable, and sometimes rigid, associations with RNP granules with stability increasing with both mRNA length and granule size. Live and fixed cell imaging demonstrated that mRNAs can extend beyond the protein surface of a stress granule, which may facilitate interactions between RNP granules. Thus, the recruitment of mRNPs to RNP granules involves dynamic, stable and extended interactions affected by translation status, mRNA length and granule size that collectively regulate RNP granule dynamics.