RESUMO
Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Transcrição Gênica/genética , Fator de Transcrição YY1/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Genoma Humano/genética , Células Hep G2 , Humanos , Células K562 , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , RNA-Seq , Transcriptoma , Fator de Transcrição YY1/genéticaRESUMO
mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.
Assuntos
Códon , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Fúngico/química , RNA Mensageiro/químicaRESUMO
The Drosophila Dscam1 gene encodes a vast number of cell recognition molecules through alternative splicing. These exhibit isoform-specific homophilic binding and regulate self-avoidance, the tendency of neurites from the same cell to repel one another. Genetic experiments indicate that different cells must express different isoforms. How this is achieved is unknown, as expression of alternative exons in vivo has not been shown. Here, we modified the endogenous Dscam1 locus to generate splicing reporters for all variants of exon 4. We demonstrate that splicing does not occur in a cell-type-specific fashion, that cells sharing the same anatomical location in different individuals express different exon 4 variants, and that the splicing pattern in a given neuron can change over time. We conclude that splicing is probabilistic. This is compatible with a widespread role in neural circuit assembly through self-avoidance and is incompatible with models in which specific isoforms of Dscam1 mediate homophilic recognition between processes of different cells.
Assuntos
Processamento Alternativo , Moléculas de Adesão Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Neurônios/metabolismo , Isoformas de Proteínas/genética , Animais , Drosophila melanogaster/metabolismo , Éxons , Neurônios/classificação , ProbabilidadeRESUMO
Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport, and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive crosstalk between gene regulatory layers takes advantage of dynamic spatial, physical, and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control.
Assuntos
Redes Reguladoras de Genes , Splicing de RNA , Processamento Alternativo , Animais , Núcleo Celular/genética , Cromatina/metabolismo , Humanos , Sequências Reguladoras de Ácido Ribonucleico , Transcrição GênicaRESUMO
The RNA isoform repertoire is regulated by splicing factor (SF) expression, and alterations in SF levels are associated with disease. SFs contain ultraconserved poison exon (PE) sequences that exhibit greater identity across species than nearby coding exons, but their physiological role and molecular regulation is incompletely understood. We show that PEs in serine-arginine-rich (SR) proteins, a family of 14 essential SFs, are differentially spliced during induced pluripotent stem cell (iPSC) differentiation and in tumors versus normal tissues. We uncover an extensive cross-regulatory network of SR proteins controlling their expression via alternative splicing coupled to nonsense-mediated decay. We define sequences that regulate PE inclusion and protein expression of the oncogenic SF TRA2ß using an RNA-targeting CRISPR screen. We demonstrate location dependency of RS domain activity on regulation of TRA2ß-PE using CRISPR artificial SFs. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2ß-PE detected in breast tumors, altering breast cancer cell viability, proliferation, and migration.
Assuntos
Neoplasias da Mama/patologia , Diferenciação Celular , Éxons , Síndromes Mielodisplásicas/patologia , Proteínas do Tecido Nervoso/metabolismo , Splicing de RNA , Fatores de Processamento de Serina-Arginina/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Humanos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas , Fatores de Processamento de Serina-Arginina/genética , Células Tumorais CultivadasRESUMO
In this issue of Cell, Gabut and colleagues (2011) identify a new splice variant of FOXP1 that directly regulates the expression of pluripotency genes. It endows human embryonic stem cells with their pluripotent nature and is required for the reprogramming of somatic cells to induced pluripotent stem cells.
RESUMO
The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.
Assuntos
Bases de Dados Genéticas , Genoma/genética , Genômica , Anotação de Sequência Molecular , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Bases de Dados Genéticas/normas , Bases de Dados Genéticas/tendências , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Genômica/normas , Genômica/tendências , Histonas/metabolismo , Humanos , Camundongos , Anotação de Sequência Molecular/normas , Controle de Qualidade , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismoRESUMO
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética , Processamento Alternativo/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/genética , Masculino , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade por SubstratoRESUMO
To achieve adaptive and heritable immunity against viruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide in vivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.
Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , DNA Intergênico/genética , Edição de Genes , Motivos de Nucleotídeos , Pyrococcus furiosus/genética , Imunidade Adaptativa , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/química , DNA Bacteriano/imunologia , DNA Bacteriano/metabolismo , DNA Intergênico/química , DNA Intergênico/metabolismo , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/imunologiaRESUMO
Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.
Assuntos
Exorribonucleases/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Citoplasma/metabolismo , Endonucleases/metabolismo , Exorribonucleases/genética , Poli A/metabolismo , Poliadenilação , Estabilidade de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif.
Assuntos
Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , RNA/química , RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/genética , Relação Estrutura-AtividadeRESUMO
The prevalence of obesity is increasing in older adults and contributes to age-related decline. Caloric restriction (CR) alleviates obesity phenotypes and delays the onset of age-related changes. However, how late in life organisms benefit from switching from a high-(H) to a low-calorie (L) diet is unclear. We transferred male flies from a H to a L (HL) diet or vice versa (LH) at different times during life. Both shifts immediately change fly rate of aging even when applied late in life. HL shift rapidly reduces fly mortality rate to briefly lower rate than in flies on a constant L diet, and extends lifespan. Transcriptomic analysis uncovers that flies aged on H diet have acquired increased stress response, which may have temporal advantage over flies aged on L diet and leads to rapid decrease in mortality rate after HL switch. Conversely, a LH shift increases mortality rate, which is temporarily higher than in flies aged on a H diet, and shortens lifespan. Unexpectedly, more abundant transcriptomic changes accompanied LH shift, including increase in ribosome biogenesis, stress response and growth. These changes reflect protection from sudden release of ROS, energy storage, and use of energy to growth, which all likely contribute to higher mortality rate. As the beneficial effects of CR on physiology and lifespan are conserved across many organisms, our study provides framework to study underlying mechanisms of CR interventions that counteract the detrimental effects of H diets and reduce rate of aging even when initiated later in life.
Assuntos
Ingestão de Energia , Longevidade , Animais , Masculino , Longevidade/fisiologia , Envelhecimento/fisiologia , Restrição Calórica , Drosophila melanogaster/fisiologia , ObesidadeRESUMO
RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with â¼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.
Assuntos
Bases de Dados Factuais , Imagem Óptica , Proteínas de Ligação a RNA , Humanos , Anticorpos/metabolismo , Células HeLa , RNA/química , Proteínas de Ligação a RNA/metabolismo , Células Hep G2RESUMO
CRISPR-Cas systems are functionally diverse prokaryotic antiviral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis, and S. thermophilus) were each expressed on a single plasmid in E. coli, and the efficiency and specificity of gene knockdown was assessed by northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and noncoding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.
Assuntos
Sistemas CRISPR-Cas , Escherichia coli , Escherichia coli/genética , Técnicas de Silenciamento de Genes , RNA/genética , Staphylococcus epidermidis , TecnologiaRESUMO
Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.
Assuntos
Proteínas Arqueais/imunologia , Pyrococcus furiosus/imunologia , Interferência de RNA , RNA Arqueal/imunologia , Proteínas Arqueais/metabolismo , Sequência de Bases , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Pyrococcus furiosus/virologia , RNA Arqueal/química , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA Viral/imunologia , Pequeno RNA não TraduzidoRESUMO
RNA structure is intimately related to function, yet methods to identify base-paired RNA strands in a transcriptome-wide manner in cells have remained elusive. One recent paper in Cell and two in Molecular Cell describe related methods to identify RNA sequences that interact in living cells, setting the stage for breakthroughs in our understanding of RNA structure and function.
Assuntos
RNA/química , Animais , Pareamento de Bases , Humanos , Conformação de Ácido Nucleico , RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Neuritos/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Diferenciação Celular/genética , Reprogramação Celular/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Isoformas de Proteínas , Estrutura Terciária de Proteína , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Transcriptoma/genéticaRESUMO
Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell lines, tissues, and developmental stages is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology, and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/).
Assuntos
Bases de Dados Genéticas , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Transcriptoma/genética , Sítios de Ligação , Humanos , Ligação Proteica , RNA/genética , RNA Interferente Pequeno/classificação , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Type III CRISPR-Cas systems have a unique mode of interference, involving crRNA-guided recognition of nascent RNA and leading to DNA and RNA degradation. How type III systems acquire new CRISPR spacers is currently not well understood. Here, we characterize CRISPR spacer uptake by a type III-A system within its native host, Streptococcus thermophilus. Adaptation by the type II-A system in the same host provided a basis for comparison. Cas1 and Cas2 proteins were critical for type III adaptation but deletion of genes responsible for crRNA biogenesis or interference did not detectably change spacer uptake patterns, except those related to host counter-selection. Unlike the type II-A system, type III spacers are acquired in a PAM- and orientation-independent manner. Interestingly, certain regions of plasmids and the host genome were particularly well-sampled during type III-A, but not type II-A, spacer uptake. These regions included the single-stranded origins of rolling-circle replicating plasmids, rRNA and tRNA encoding gene clusters, promoter regions of expressed genes and 5' UTR regions involved in transcription attenuation. These features share the potential to form DNA secondary structures, suggesting a preferred substrate for type III adaptation. Lastly, the type III-A system adapted to and protected host cells from lytic phage infection.
Assuntos
Sistemas CRISPR-Cas , Streptococcus thermophilus/genética , Streptococcus thermophilus/virologia , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Plasmídeos , Streptococcus thermophilus/metabolismoRESUMO
Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.