SignificanceCells release vesicles containing selectively packaged cargo, including RNA, into the extracellular environment. Prior studies have identified RNA inside extracellular vesicles (EVs), but due to limitations of conventional sequencing methods, highly structured and posttranscriptionally modified RNA species were not effectively captured. Using an alternative sequencing approach (thermostable group II intron reverse transcriptase sequencing, TGIRT-seq), we found that EVs contain abundant small noncoding RNA species, including full-length transfer RNAs and Y RNAs. Using a knockout cell line, we obtained evidence that the RNA-binding protein YBX1 plays a role in sorting small noncoding RNAs into a subpopulation of EVs termed exosomes. These experiments expand our understanding of EV–RNA composition and provide insights into how RNA is sorted into EVs for cellular export.

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling.

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.

Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN. INTRODUCTIONMannose 6-phosphate receptors (MPRs) bind newly synthesized lysosomal hydrolases in the Golgi complex and deliver them to prelysosomes (Ghosh et al., 2003). There, the enzymes are released from MPRs, which are then recycled to the trans-Golgi network (TGN) for another round of enzyme delivery. There are two MPRs in most cell types: the cationindependent (CI)-MPR of ϳ300 kDa and the cation-dependent (CD)-MPR, a dimer of ϳ45 kDa. Both MPRs seem to require similar cellular components for their transport between compartments within mammalian cells (Ghosh et al., 2003).This laboratory has studied the process by which MPRs are transported from late endosomes to the Golgi. We have shown that MPR recycling requires Rab9 GTPase and its effector p40 (Lombardi et al., 1993;Riederer et al., 1994;Diaz et al., 1997), a cargo adaptor named TIP47 (for tail-interacting protein of 47 kDa; Diaz and Pfeffer, 1998;Burguete et al., 2005) and several other general transport factors (Itin et al., 1997). Using live cell video microscopy, we were able to track yellow fluorescent protein (YFP)-Rab9 -containing transport vesicles and watch them fuse with a cyan fluorescent protein-labeled trans-Golgi compartment (Barbero et al., 2002). That study showed that Rab9 is present on transport vesicles en route to the Golgi complex, but it did not provide additional clues to the mechanisms by which such vesicles are targeted.In an effort to understand the full scope of Rab9 function, we sought to identify additional partners for the active form of this GTPase. We report here the discovery that a Golgilocalized, putative tether of the Golgin protein family binds specifically to Rab9. Golgins contain long, predicted coiledcoil motifs and are important for Golgi organization, vesicle tethering, and secretory protein transport (for reviews, see Gillingham and Munro, 2003;Gleeson et al., 2004;Short et al., 2005). Several ...

Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel templateswitching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5′ to 3 ′ gene coverage and identify more splice junctions, particularly near the 5 ′ ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq.