Imaging of single mRNA translation repression reveals diverse interactions with mRNP granules

Abstract

During cellular stress mRNAs exit translation and accumulate in stress granules and Pbodies, although the dynamics of these interactions remain unclear. We imaged in real-time single mRNAs, their translational output, and mRNA-granule interactions during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionality between them. While translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable associations that rigidly immobilize the mRNA within the granule. Imaging thousands of individual mRNA-granule interactions showed the probability of stable association increases with both mRNA length and granule size.Therefore, the recruitment of mRNAs to RNP granules involves both highly dynamic and stable interactions, influenced by several parameters, demonstrating a new layer of complexity in mRNA regulation during stress.One Sentence Summary: mRNAs interact with stress granules and P-bodies in stable and dynamic manners influenced by ribosome association, mRNA length, and granule size.3 Main text:To survive environmental stress, cells reduce bulk protein production to promote translation of stress responsive proteins. During this response, mRNAs accumulate into cytoplasmic granules of non-translating mRNAs and proteins (RNPs), referred to as stress granules (SGs) and P-bodies (PBs) (1). While it is known that mRNAs within these mRNP granules are translationally repressed, is it not known when or where translation is repressed, how dynamic mRNA-granule interactions are, and the order of mRNA association with PB and SG. Resolving these issues are important because mRNA-granule interactions are implicated in multiple processes beyond the modulation of the stress response (2, 3), including maternal mRNA storage in early development (4), synaptic plasticity (5), tumor progression (6, 7), and neurodegeneration (8-10).To quantify the relationship between translation of single mRNAs and their interactions with mRNP granules in living cells, we used Nascent Chain Tracking (11). The translation of individual mRNAs labeled by fluorescent MS2 coat protein (MCP-Halo with JF646 dye (12)) was monitored via the binding of fluorescent antibody fragments (Fab conjugated with the Cy3 dye) to epitopes at the N-terminus of the nascent peptide ( Fig. 1A). Thus, translating mRNAs were labeled by both MCP and Fab, while non-translating mRNAs were only labeled by MCP.By imaging these constructs in U-2 OS cells stably expressing the SG marker GFP-G3BP1 (13), we examined the translation status of single mRNAs during arsenite stress in relation to their interactions with SGs.We first imaged cells at lower temporal resolution (1 volume every 3 minutes for 1 hour) to determine the timing of translation repression during stress. The translation of single KDM5B reporter mRNAs declined upon stress, with SGs forming after 10 minutes of stress ( Fig. 1B, 4 Movie S1). The interaction of KDM5B mRNAs with SGs slightly lagged behind SG assembly, reaching a maxim...

mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

“…In other work, we had observed that during stress the 5′ and 3′ ends of the AHNAK mRNA were often close together (Moon et al, 2018 Preprint ). Similar compaction of three other mRNAs under a variety of stress conditions has been observed (Adivarahan et al, 2018).…”

“…This argues that mRNAs must fully disengage from elongating ribosomes before stable accumulation in SGs. Additional evidence in support of this model comes from single-molecule experiments showing that mRNAs engaged with ribosomes can form only a transient association with SG and do not enter a stable association, which can be seen with nontranslating mRNAs (Moon et al, 2018 Preprint ).…”

“…Specific smFISH probes were created using the Stellaris Probe Designer software designed by Biosearch Technologies. smFISH probes that bind to DYNC1H1, NORAD, PEG3, ZNF704, CDK6, and the 5′ and 3′ ends of AHNAK mRNAs were designed previously (Khong et al, 2017; Moon et al, 2018 Preprint ). Newly designed smFISH probes include probes that bind to the middle of AHNAK mRNAs, 5′ end, middle, 3′ end, and SunTag DYNC1H1 mRNAs.…”

mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

“…SGs is due to elongating ribosomes. First, treatment of U-2 OS cells with arsenite and 138 puromycin, which releases all elongating ribosomes from mRNAs, causes the AHNAK and 139 DYNC1H1 mRNAs to be recruited to SGs at earlier times ( Figure 1A In other work, we had observed that during stress the 5' and 3' ends of the AHNAK 146 mRNA were often close together (Moon et al, 2018). Similar compaction of three other mRNAs 147 under a variety of stress conditions have been observed (Srivathsan et al, 2018).…”

Germ Cell Responses to Stress: The Role of RNP Granules

“…Stress induces disassembly of the polysome, and the released mRNPs are sequestered in stress granules to protect mRNA from degradation and prevent translation until the stress subsides (Kedersha and Anderson, 2002). A recent study examining single mRNAs during arsenite stress shows that mRNAs stably associated with stress granules are non-translating (Moon et al, 2018). Stress granules have also been implicated in protein quality control mechanisms, with deregulation contributing to neurodegenerative diseases, among other disorders (Alberti et al, 2017).…”