RESUMEN
Transcription elongation is a highly regulated process affected by many proteins, RNAs and the underlying DNA. Here we show that the nascent RNA can interfere with transcription in human cells, extending our previous findings from bacteria and yeast. We identified a variety of Pol II-binding aptamers (RAPs), prominent in repeat elements such as ACRO1 satellites, LINE1 retrotransposons and CA simple repeats, and also in several protein-coding genes. ACRO1 repeat, when translated in silico, exhibits ~50% identity with the Pol II CTD sequence. Taken together with a recent proposal that proteins in general tend to interact with RNAs similar to their cognate mRNAs, this suggests a mechanism for RAP binding. Using a reporter construct, we show that ACRO1 potently inhibits Pol II elongation in cis. We propose a novel mode of transcriptional regulation in humans, in which the nascent RNA binds Pol II to silence its own expression.
Asunto(s)
Aptámeros de Nucleótidos/genética , ARN Polimerasa II/genética , Transcripción Genética/genética , Aptámeros de Nucleótidos/metabolismo , Sitios de Unión/genética , Humanos , ARN Polimerasa II/metabolismoRESUMEN
The discovery of the catalytic properties of RNAs was a milestone for our view of how life emerged and forced us to reformulate many of our dogmas. The urge to grasp the whole spectrum of potential activities of RNA molecules stimulated two decades of fervent research resulting in a deep understanding of RNA-based phenomena. Most ribozymes were discovered by serendipity during the analysis of chemical processes, whereas RNA aptamers were identified through meticulous design and selection even before their discovery in nature. The desire to obtain aptamers led to the development of sophisticated technology and the design of efficient strategies. With the new notion that transcriptomes cover a major part of genomes and determine the identity of cells, it is reasonable to speculate that many more aptamers and ribozymes are awaiting their discovery in unexpected places. Now, in the genomic era with the development of powerful bioinformatics and sequencing methods, we are overwhelmed with tools for studying the genomes of all living and possibly even extinct organisms. Genomic SELEX (systematic evolution of ligands by exponential enrichment) coupled with deep sequencing and sophisticated computational analysis not only gives access to unexplored parts of sequenced genomes but also allows screening metagenomes in an unbiased manner.
Asunto(s)
Aptámeros de Nucleótidos/genética , ARN Catalítico/genética , Humanos , Riboswitch/genética , Técnica SELEX de Producción de AptámerosRESUMEN
The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca(2+), Mg(2+), Mn(2+), and Sr(2+) have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.