RESUMO
Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3' ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.
Assuntos
RNA não Traduzido/genética , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos , Moldes GenéticosRESUMO
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.
Assuntos
Exossomos/fisiologia , Pequeno RNA não Traduzido/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , DNA/química , DNA/metabolismo , Exossomos/química , Vesículas Extracelulares , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Biotecnologia , RNA Guia de Sistemas CRISPR-Cas , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.
Assuntos
Escherichia coli , Edição de Genes , Animais , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/genética , RNA , Sistemas CRISPR-Cas/genética , Mamíferos/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Autoantígenos/metabolismo , Transporte Biológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
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.