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

Shurtleff, Matthew J.; Yao, Jun; Qin, Yidan; Nottingham, Ryan M.; Temoche-Diaz, Morayma M.; Schekman, Randy; Lambowitz, Alan M.

doi:10.1073/pnas.1712108114

Cited by 264 publications

(318 citation statements)

References 58 publications

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“…Cytoplasmic proteins including members of the endosomal sorting complex required for transport (ESCRT) machinery, syntenin, and certain chaperones are also enriched in EVs [32,34]. In addition, a variety of RNAs are present and thought to carry signals involved in cell-cell communication [35][36][37]. Several packaged miRNAs have been shown to exert specific biological effects supporting the notion that EV cargo is actively selected.…”

Section: Introductionmentioning

confidence: 93%

A cell-based assay for CD63-containing extracellular vesicles

Cashikar

Hanson

2019

PLoS ONE

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Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.

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Section: Introductionmentioning

confidence: 93%

A cell-based assay for CD63-containing extracellular vesicles

Cashikar

Hanson

2019

PLoS ONE

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“…GsI-IIC RT (TGIRT-III) has been used for a variety of applications, including comprehensive profiling of whole-cell, exosomal and plasma RNAs Qin et al 2016;Shurtleff et al 2017;Boivin et al 2018); quantitative tRNA-seq based on the ability of the TGIRT enzyme to obtain full-length end-to-end reads of tRNAs with or without demethylase treatment (Shen et al 2015;Zheng et al 2015;Qin et al 2016); determination of tRNA aminoacylation levels (Evans et al 2017); high-throughput mapping of posttranscriptional modifications by distinctive patterns of misincorporation (Katibah et al 2014;Zheng et al 2015;Shen et al 2015;Shurtleff et al 2017;Li et al 2017;Safra et al 2017); identification of protein-bound RNAs by RIP-Seq or CLIP (Katibah et al 2014;Zarnegar et al 2016); and RNA-structure mapping by DMS-MaPseq (Zubradt et al 2017;Wang et al 2018) or SHAPE (Mohr et al 2018). A study comparing TGIRT-seq to benchmark TruSeq v3 datasets of rRNA depleted (ribodepleted) fragmented Universal Human Reference (UHR) RNA with External RNA Control Consortium (ERCC) spike-ins showed that TGIRT-seq: (i) better recapitulates the relative abundance of mRNAs and ERCC spike-ins; (ii) is more strand-specific;…”

Section: Introductionmentioning

confidence: 99%

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Yao

et al. 2018

Preprint

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Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNAseq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5'and 3'-end biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5'-and 3'-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.

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“…In particular, tRNA halves can be generated in response to diverse stimuli including viral infection and sex hormone [42,53,54,63] and have been proposed to be new signaling molecules in immune biology [46]. Moreover, tRNA halves were found abundantly in body fluids, including blood/serum, sperm, cerebrospinal fluid and more [63][64][65][66][67][68][69][70]. tRNA halves in mouse sperm were shown to be regulated by parental diet and reported to modulate offspring development [19,41,45].…”

Section: Discussionmentioning

confidence: 99%

tRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model

Frost

Lammert

et al. 2019

Preprint

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tRNA-derived small fragments (tRFs) and tRNA halves have emerging functions in different biological pathways, such as regulating gene expression, protein translation, retrotransposon activity, transgenerational epigenetic changes and response to environmental stress. However, small RNAs like tRFs and microRNAs in the maternal-fetal interface during gestation have not been studied extensively. Here we investigated the small RNA composition of mouse placenta/decidua, which represents the interface where the mother communicates with the fetus, to determine whether there are specific differences in tRFs and microRNAs during fetal development and in response to maternal immune activation (MIA). Global tRF expression pattern, just like microRNAs, can distinguish tissue types among placenta/decidua, fetal brain and fetal liver. In particular, 5' tRNA halves from tRNA Gly , tRNA Glu , tRNA Val and tRNA Lys are abundantly expressed in the normal mouse placenta/decidua. Moreover, tRF and microRNA levels in the maternal-fetal-interface change dynamically over the course of embryonic development. To see if stress alters non-coding RNA expression at the maternal-fetal interface, we treated pregnant mice with a viral infection mimetic, which has been shown to promote autism-related phenotypes in the offspring. Acute changes in the levels of specific tRFs and microRNAs were observed 3-6 hours after MIA and are suppressed thereafter. A group of 5' tRNA halves is down-regulated by MIA, whereas a group of 18-nucleotide tRF-3a is up-regulated. In conclusion, tRFs show tissue-specificity, developmental changes and acute response to environmental stress, opening the possibility of them having a role in the fetal response to MIA.

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

Cited by 264 publications

References 58 publications

A cell-based assay for CD63-containing extracellular vesicles

A cell-based assay for CD63-containing extracellular vesicles

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

tRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model

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