Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane.

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61ß (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61ß and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis.

The endoplasmic reticulum (ER) is a nexus for mRNA localization and translation, and recent studies have demonstrated that ER-bound ribosomes also play a transcriptome-wide role in regulating proteome composition. The Sec61 translocon (SEC61) serves as the receptor for ribosomes that translate secretory/integral membrane protein-encoding mRNAs, but whether SEC61 also serves as a translation site for cytosolic protein-encoding mRNAs remains unknown. Here, using a BioID proximity-labeling approach in HEK293T Flp-In cell lines, we examined interactions between ER-resident proteins and ribosomes in vivo Using in vitro analyses, we further focused on bona fide ribosome interactors (i.e. SEC61) and ER proteins (ribophorin I, leucine-rich repeat-containing 59 (LRRC59), and SEC62) previously implicated in associating with ribosomes. We observed labeling of ER-bound ribosomes with the SEC61ß and LRRC59 BioID reporters, comparatively modest labeling with the ribophorin I reporter, and no labeling with the SEC62 reporter. A biotin pulse-chase/subcellular fractionation approach to examine ribosome exchange at the SEC61ß and LRRC59 sites revealed that, at steady state, ribosomes at these sites comprise both rapid- and slow-exchanging pools. Global translational initiation arrest elicited by the inhibitor harringtonine accelerated SEC61ß reporter-labeled ribosome exchange. RNA-Seq analyses of the mRNAs associated with SEC61ß- and LRRC59-labeled ribosomes revealed both site-enriched and shared mRNAs and further established that the ER has a transcriptome-wide role in regulating proteome composition. These results provide evidence that ribosomes interact with the ER membrane via multiple modes and suggest regulatory mechanisms that control global proteome composition via ER membrane-bound ribosomes.

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/ß-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease.