Spatial Organization of Single mRNPs at Different Stages of the Gene Expression Pathway

mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single-molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compared to nuclear mRNPs and lncRNPs, association with ribosomes decompacts individual mRNAs, while pharmacologically dissociating ribosomes or sequestering them into stress granules leads to increased compaction. Moreover, translating mRNAs rarely show co-localized 5′ and 3′ ends, indicating either that mRNAs are not translated in a closed-loop configuration, or that mRNA circularization is transient, suggesting that a stable closed-loop conformation is not a universal state for all translating mRNAs. [Display omitted] •mRNA compaction determined by multi-color, single-molecule mRNA FISH•Nuclear mRNAs have extended morphology•Translating mRNAs rarely show interacting 5′ and 3′ ends•mRNAs in stress granules are more compacted than translating mRNAs Adivarahan et al. show that mRNA compaction varies depending on subcellular localization and mRNA translation state. Whereas translational inhibition and sequestration to stress granules leads to mRNA compaction, translation induces the separation of 5′ and 3′ ends, suggesting that mRNA translation does not occur in a stable circularized conformation.