mRNA Partitioning to the Endoplasmic Reticulum

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2021

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Abstract

The signal recognition particle (SRP) pathway has long been regarded as the primary mechanism of transcriptome and translatome partitioning to the endoplasmic reticulum (ER). This co-translational targeting mechanism is conserved in all living organisms studied to date. By the SRP pathway, ribosomes translating secretory and membrane protein-encoding mRNAs in the cytosol are selected for recruitment to the ER following presentation of a peptide signal sequence early in translation. SRP recognizes and binds the peptide signal and targets the mRNA-ribosome-nascent chain complex to the ER membrane via interaction with the ER-resident SRP receptor (SR). The membrane-targeted signal peptide is then passed to the translocon and the secretory/membrane protein is co-translationally translocated into the ER lumen or inserted into the ER membrane. Via this positive selection strategy, mRNAs encoding secretory and membrane proteins are translated by ER-associated ribosomes, while non-signal encoding mRNAs are translated by free ribosomes in the cytosol. Yeast and bacterial species possess post-translational protein translocation mechanisms which allow SRP-independent translocation. Mammalian cells, by contrast, rely on the SRP pathway for protein targeting and translocation. The precise role of the SRP pathway in subcellular mRNA localization, however, remains elusive. In fact, studies which investigate RNA localization to the ER suggest many diverse strategies could anchor a broad representation of all cellular mRNAs to the ER membrane and regulate their translation. Here we evaluate the extent to which the SRP pathway contributes to transcriptome partitioning in mammalian cells via CRISPR/Cas9- and RNAi-mediated depletion of SR, followed by sequential detergent fractionation into cytosol and ER subcellular fractions and deep sequencing of the compartmentalized mRNAs. We found that disruption of the SRP pathway does not impact steady-state mRNA localization to the ER, though minor defects in protein expression were observed in a quantitative proteomic study, thereby decoupling SRP pathway function in protein biogenesis from general mRNA partitioning. Assessment of de novo subcellular localization patterns of newly synthesized mRNAs, via deep sequencing of 4-thiouridine metabolically labeled mRNAs in the cytosol and ER fractions of parental and SR-deficient cells, revealed that mRNAs are predominately localized to the ER membrane upon nuclear export, independently of a functional SRP pathway or encoded signal sequence. We further found that translation inhibition, through physiological stress or chemical inhibitors, enhanced the ER localization of mRNAs, especially non-signal encoding mRNAs. This suggests that translation-coupled events release this mRNA cohort into the cytosol to establish steady-state subcellular distributions. Additional investigation into RNA localization patterns by single molecule RNA imaging under conditions of stress-induced translation inhibition, which promotes the formation of ribonucleoprotein stress granules (SG), revealed that newly synthesized mRNAs serve as primary substrates for SG biogenesis, as transcription inhibition prevented mRNA recruitment into SGs. Furthermore, SG formation was found to occur in association with the ER membrane for both signal- and non-signal-encoding mRNAs. Collectively these data support a novel mRNA trafficking model by which newly synthesized mRNAs are exported from the nucleus and localized directly to the ER membrane independently of the SRP pathway, likely via interaction with ER-resident ribosome and/or RNA binding proteins, implicating the ER as a regulatory center for initiation of subcellar transcriptome partitioning.

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Child, Jessica Rae (2021). mRNA Partitioning to the Endoplasmic Reticulum. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/23816.

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