Broad role for YBX1 in defining the small noncoding RNA composition of exosomes.

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

A novel retinoblastoma therapy from genomic and epigenetic analyses.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss

DESIGNING A MICROBIAL PROLYL PEPTIDASE DELIVERY SYSTEM FOR THE TREATMENT OF CELIAC DISEASE

Celiac disease is an autoimmune enteropathy resulting from the ingestion of gluten and gluten-like proteins from wheat, barley and rye in afflicted individuals. Indigestible gluten-derived peptides rich in proline residues are known to be responsible for eliciting the inappropriate immune response characteristic of the disease. In this investigation, surface level expression of prolyl peptidase activity by genetically engineered probiotic lactobacilli was postulated to be a possible treatment for this disease. Plasmid-based reporter vectors were constructed utilizing a novel, homology-based cloning technique to assess the expression and localization signals from the S-layer protein gene of Lactobacillus acidophilus. These plasmids were mutated during construction due to toxicity associated with the cloned cassettes. The toxicity of the Slayer secretion and/or anchoring domains in E. coli was confirmed by cloning the fused components into an inducible expression system. When the prolyl peptidase, Xaa-Pro, from L. reuteri was incorporated into the S-layer expression cassette, the full-length protein was efficiently expressed but was not active, likely due to protein aggregation and inclusion body formation. Future research directions are discussed and a modified experimental design strategy is presented. This work provides a foundation for continued investigation into the feasibility of utilizing genetically engineered lactobacilli as a potential treatment strategy for celiac disease

Buoyant Density Fractionation of Small Extracellular Vesicle Sub-populations Derived from Mammalian Cells.

Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography-SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight differences in their buoyant density. Moreover, this technique also allows sEVs purification from vesicle-free RNA-protein complexes co-isolating in the high-speed pellet or by the use of crowding agents. This protocol describes cultivation of mammalian cells for sEV collection, sEV sedimentation, buoyant density fractionation of sEV sub-populations and immunoblots for sEV markers. This protocol can be used to fractionate distinct sEV sub-populations produced by a variety of mammalian cells

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

Abstract 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

Data from: Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

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

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction.

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