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RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5′ terminal oligopyrimidine (5′ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Our results suggest that EVs and exosomes could play a role in the purging and intercellular transfer of excess free RNAs, including full-length tRNAs and other small ncRNAs.

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells. Human cells release molecules into their surroundings via membrane-bound packets called exosomes. These molecules can then circulate throughout the body and are protected from degradation. Among the cargos carried by exosomes are small molecules of RNA known as microRNAs, which are involved in regulating gene activity. Only a select subset of the hundreds of microRNAs in a human cell end up packaged into exosomes. This suggests that there might be a specific mechanism that sorts those microRNAs that are destined for export. However, few proteins or other factors that might be involved in this sorting process had been identified to date. Shurtleff et al. set out to identify these factors and started by purifying exosomes from human cells grown in the laboratory and looking for microRNAs that were more abundant in the exosomes than the cells. One exosome-specific microRNA, called miR-223, was further studied via a test-tube based system that uses extracts from cells rather than cells themselves. These experiments confirmed that miR-223 is selectively packed into exosomes that formed in the test tube. Using this system, Shurtleff et al. then isolated a protein called Y-box Protein I (or YBX1 for short) that binds to RNA molecules and found that it was required for this selective packaging. YBX1 is known to be a constituent of exosomes released from intact cells and may therefore be required to sort other RNA molecules into exosomes. Future studies will explore how YBX1 recognizes those RNA molecules to be exported from cells via exosomes. Also, because exosomes have been implicated in some diseases such as cancer, it will be important to explore what role exosome-specific microRNAs play in both health and disease.

Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.

Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential for accurate folding and post-translational stability rather than translation efficiency. Specifically, we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts an aberrant topology in the absence of the EMC leading to degradation. Together, our data highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing challenging transmembrane proteins during synthesis.

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes. Cells are surrounded and contained by a plasma membrane consisting of a double layer of fats and proteins. These proteins monitor and facilitate the movement of food, oxygen and messages in and out of the cell, and help neighboring cells communicate. Membrane proteins are manufactured in a cell compartment called the endoplasmic reticulum. Cellular machines called ribosomes visit this compartment’s membrane to manufacture proteins that need to be secreted or embedded into the cell’s membranes. As these proteins are made, they are pulled into the endoplasmic reticulum so they can be folded correctly and inserted in the membrane. A cellular machine in this compartment’s membrane that aids this process is the endoplasmic reticulum membrane protein complex (EMC). Many steps can go wrong during protein assembly, so to control protein quality, the EMC has to accommodate the variety of complex physical features that proteins can have. To explore the activity of the EMC, Miller-Vedam, Bräuning, Popova et al. studied the normal structure of the EMC in both yeast and human cells grown in the lab. These snapshots of the complex in different species had a lot in common, including how the complex was arranged within and around the membrane. Next, Miller-Vedam, Bräuning, Popova et al. generated 50 mutant versions of the EMC in human cells to determine how changing different parts of the complex affected the production of three proteins that rely on the EMC to fold correctly. These proteins were an enzyme called squalene synthase, a signaling protein called the beta adrenergic receptor and sigma intracellular receptor 2, a protein involved in the regulation of cholesterol levels. Mutations in the section of the EMC outside of the endoplasmic reticulum, within the main cellular compartment, negatively impacted the stability of squalene synthase. This section of the EMC provides a platform where proteins can associate before entering the membrane. The part of EMC that spans the membrane contains both a fat-filled cavity and a cavity with a ‘door’ that is either open or closed. Mutations in this section disrupted the insertion of both squalene synthase and the beta adrenergic receptor into the membrane, a role performed by the cavity with the door. The specific role of the fat-filled cavity is still not fully understood, but a mutation affecting this cavity disrupts the correct production of all three proteins studied. The largest section of the complex, which sits inside the endoplasmic reticulum, protected proteins as they folded, ensuring they were not destroyed for being folded incorrectly before they were fully formed. Mutations in this part of the EMC negatively impacted the stability of sigma intracellular receptor 2 without negatively affecting the other proteins. This molecular dissection of the activity of the EMC provides insights into how membrane proteins are manufactured, stabilized, coordinated, and monitored for quality. These findings could contribute towards the development of new treatments for certain congenital diseases. For example, cystic fibrosis, retinitis pigmentosa, and Charcot-Marie-Tooth disease are all thought to be caused by mutations within membrane proteins that require the EMC during their production.

Circulating proteins are common targets for the discovery of occupancy-based inhibitors including monoclonal antibodies. Effective inhibition of target pathogenicity with blocking approaches, however, is often challenged by target parameters that lead to insufficient occupancy and/or incomplete pharmacology limited by only single site binding. Extracellular targeted protein degradation approaches, such as lysosomal targeting chimeras (LYTACs), offer an opportunity to minimize these challenges by an event-driven mechanism that selectively, thoroughly and irreversibly eliminates drivers of disease. First generation LYTACs, designed to traffic to the lysosome, show limited durability since the therapeutic is degraded along with the target protein of interest. Here we describe cataLYTACs, which overcome this limitation by combining stabilized asialoglycoprotein (ASGPR) ligands, pH-sensitive target binding and recycling via the neonatal Fc receptor (FcRn). These cataLYTACs degraded superstoichiometric levels of a target protein, IgE, in vitro and demonstrated deep and sustained clearance of human IgE in mouse models. In non-human primates, cataLYTACs resulted in >98% clearance of circulating endogenous IgE for 2 weeks and outperformed the standard of care blocking antibody, omalizumab (Xolair), in both free IgE elimination and duration of action. CataLYTACs represent a new therapeutic modality for a wide range of disease states driven by circulating factors, with the potential for superior efficacy and duration of action compared to traditional inhibitors.Competing Interest StatementC.K., N.A.L., R.M.L., T.C., C.L.C., S.G., K.T.A., K.H.D., C.K.F., D.J.H., R.K., D.L., S.R., D.A.L., S.M.M., J.S.I., S.T.S., E.D.T. and M.J.S. are employees of Lycia Therapeutics Inc. R.M.L., I.E.J., K.T.A., K.H.D., D.L., T.T., R.Y., D.A.L., S.M.M., J.G.L. and S.T.S. are shareholders of Lycia Therapeutics Inc. R.W.H. and C.L. are employees of Thermo Fisher Scientific Inc. J.G.L. is a founder and employee of Chempiric Consulting, LLC. C.K., M.J.S., I.E.J., D.H.L., S.R., R.Y., D.A.L., J.S.I., J.G.L., S.T.S., E.D.T. and N.A.L. are inventors on provisional patent applications related to this work.

Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor derived EV subtypes and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two EV sub-populations from a metastatic cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV subtypes use different mechanisms of miRNA sorting, suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of some selectively packaged miRNAs.We found that two motifs embedded in miR-122 are responsible for high affinity binding to Lupus La and sorting into vesicles formed in vitro. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in cancer biology.

Abstract Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies revealed at least two separable EMC activities, as an insertase regulating tail-anchored protein levels and as a polytopic membrane protein holdase chaperone. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a novel, non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes. Competing Interest Statement The authors have declared no competing interest. Footnotes * Addition of Videos 1-3, addition of Figure 1 - figure supplement 8. Fixed typos in Main Figure 3 and 6.

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their co-isolation with contaminants, a lack of knowledge of the mechanisms of RNA sorting into EVs and limitations of conventional RNA-seq methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspannin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature tRNAs and other small non-coding RNAs encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long non-coding, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5 terminal oligopyrimidine (5 TOP) motif. Our results indicate that the RNA-binding protein YBX1, which we showed previously is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small non-coding RNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for post-transcriptional modification in the sorting of some RNA species into EVs. The identification of full-length small non-coding RNAs within EVs suggests a role for EVs in the export and possible intercellular functional transfer of abundant cellular transcripts.