Recruitment of endoplasmic reticulum-targeted and cytosolic mRNAs into membrane-associated stress granules

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.