RESUMO
Extracellular RNAs participate in intercellular communication, and are being studied as promising minimally invasive diagnostic markers. Several studies in recent years showed that tRNA halves and distinct Y RNA fragments are abundant in the extracellular space, including in biofluids. While their regulatory and diagnostic potential has gained a substantial amount of attention, the biogenesis of these extracellular RNA fragments remains largely unexplored. Here, we demonstrate that these fragments are produced by RNase 1, a highly active secreted nuclease. We use RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. We complement and extend our RNA sequencing results with northern blots, showing that tRNAs and Y RNAs in the non-vesicular extracellular compartment are released from cells as full-length precursors and are subsequently cleaved to distinct fragments. In support of these results, formation of tRNA halves is recapitulated by recombinant human RNase 1 in our in vitro assay. These findings assign a novel function for RNase 1, and position it as a strong candidate for generation of tRNA halves and Y RNA fragments in biofluids.
Assuntos
RNA de Transferência/metabolismo , RNA não Traduzido/metabolismo , Ribonucleases/metabolismo , Humanos , Células K562 , Mutação , Clivagem do RNA , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA não Traduzido/química , RNA-SeqRESUMO
Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion.
Assuntos
Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , RNA/metabolismo , Comunicação Celular/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Células K562RESUMO
The locus alx, which encodes a putative transporter, was discovered previously in a screen for genes induced under extreme alkaline conditions. Here we show that the RNA region preceding the alx ORF acts as a pH-responsive element, which, in response to high pH, leads to an increase in alx expression. Under normal growth conditions this RNA region forms a translationally inactive structure, but when exposed to high pH, a translationally active structure is formed to produce Alx. Formation of the active structure occurs while transcription is in progress under alkaline conditions and involves pausing of RNA polymerase at two distinct sites. Alkali increases the longevity of pausing at these sites and thereby interferes with formation of the inactive structure and promotes folding of the active one. The alx locus represents the first example of a pH-responsive riboregulator of gene expression, introducing a novel regulatory mechanism that involves RNA folding dynamics driven by pH.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Pareamento de Bases , Sequência Conservada , Proteínas de Escherichia coli/genética , Concentração de Íons de Hidrogênio , Mutação/genética , Conformação de Ácido Nucleico , Precursores de RNA/química , RNA Bacteriano/química , RNA Bacteriano/genéticaRESUMO
Previously, we described a novel pH-responsive RNA element in Escherichia coli that resides in the 5' untranslated region of the alx gene and controls its translation in a pH-dependent manner. Under normal growth conditions, this RNA region forms a translationally inactive structure, but when transcribed under alkaline conditions, it forms an active structure producing the Alx protein. We identified two distinct transcriptional pause sites and proposed that pausing at these sites interfered with the formation of the inactive structure while facilitating folding of the active one. Alkali increases the longevity of pausing at these sites, thereby promoting folding of the translationally active form of alx RNA. We show here that mutations that modify the extent and/or position of pausing, although silent with regard to structure stability per se, greatly influence the dynamics of folding and thereby translation. Our data illustrate the mechanistic design of alx regulation, relying on precise temporal and spatial characteristics. We propose that this unique design provides an opportunity for environmental signals such as pH to introduce structural changes in the RNA and thereby modulate expression.
Assuntos
Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/química , Sequências Reguladoras de Ácido Ribonucleico , Transcrição Gênica , Regiões 5' não Traduzidas , Sequência de Bases , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Dobramento de RNARESUMO
During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.
Assuntos
Análise da Expressão Gênica de Célula Única , Peixe-Zebra , Animais , Desenvolvimento Embrionário/genética , Transcrição Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the underlying regulation of mRNA transcription and degradation remains a challenge, especially within whole embryos with diverse cellular identities. Here, we collect temporal cellular transcriptomes of zebrafish embryos, and decompose them into their newly-transcribed (zygotic) and pre-existing (maternal) mRNA components by combining single-cell RNA-Seq and metabolic labeling. We introduce kinetic models capable of quantifying regulatory rates of mRNA transcription and degradation within individual cell types during their specification. These reveal different regulatory rates between thousands of genes, and sometimes between cell types, that shape spatio-temporal expression patterns. Transcription drives most cell-type restricted gene expression. However, selective retention of maternal transcripts helps to define the gene expression profiles of germ cells and enveloping layer cells, two of the earliest specified cell-types. Coordination between transcription and degradation restricts expression of maternal-zygotic genes to specific cell types or times, and allows the emergence of spatio-temporal patterns when overall mRNA levels are held relatively constant. Sequence-based analysis links differences in degradation to specific sequence motifs. Our study reveals mRNA transcription and degradation events that control embryonic gene expression, and provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.