Regulation of mammalian transcription by noncoding RNA

Transcription is a fundamental cellular process that allows the expression of the genetic information stored in genes; its misregulation can lead to developmental arrest or disease. The products of transcription are ribonucleic acids (RNAs) of two types: messenger RNAs, which encode proteins, and noncoding RNAs, which have a large variety of functions. Messenger RNAs are transcribed by a large protein enzyme, RNA polymerase II, through a highly regulated process. The first step of transcription is initiation, which requires RNA polymerase II and many other proteins to come together at a promoter to form a preinitiation complex. Whereas the functions of many protein regulators of transcription have been clarified, RNA-based mechanisms of regulation are less well understood. In response to cellular stress, the levels of certain noncoding RNAs increase via transcription of repetitive genomic elements. Such RNAs have been shown to bind to the promoters of a subset of genes after heat shock, to repress transcription, and to associate directly with RNA polymerase II. How these RNA sequences physically interact with RNA polymerase II and its preinitiation complexes to exert their biological effect is poorly understood. In this project, we plan to investigate the interactions of RNA polymerase II and a minimal transcription preinitation complex with regulatory noncoding RNAs. Through the use of state-of-the art cryo-electron microscopy techniques, we will determine the three-dimensional structures of these complexes. Biochemical experiments will be performed to test the insights gained from the structures, and to further address how these complexes would be destabilized in the cell. This work will provide a new understanding of the mechanisms by which noncoding RNA regulates transcription.

Regulation of mammalian transcription by noncoding RNA Transcription is a fundamental cellular process that allows the expression of the genetic information stored in genes; its misregulation can lead to developmental arrest or disease. The products of transcription are ribonucleic acids (RNAs) of two types: messenger RNAs, which encode proteins, and noncoding RNAs, which have a large variety of functions. Messenger RNAs are transcribed by a large protein enzyme, RNA polymerase II, through a highly regulated process. The first step of transcription is initiation, which requires RNA polymerase II and many other proteins to come together at a promoter to form a preinitiation complex. Whereas the functions of many protein regulators of transcription have been clarified, RNA-based mechanisms of regulation are less well understood. In response to cellular stress, the levels of certain noncoding RNAs increase via transcription of repetitive genomic elements. Such RNAs have been shown to bind to the promoters of a subset of genes after heat shock, to repress transcription, and to associate directly with RNA polymerase II. How these RNA sequences physically interact with RNA polymerase II to exert their biological effect had been poorly understood. In this project, we applied state-of-the art cryo-electron microscopy techniques to determine the three-dimensional structures of Pol II-noncoding RNA complexes. These high-resolution structures revealed that the noncoding RNA "looks" like DNA and RNA that binds to RNA polymerase II as it transcribes messenger RNAs, and in that way blocks RNA polymerase II from accessing genomic DNA until stress is over and levels of the noncoding RNA decrease. Biochemical experiments revealed that domains of the general transcription factor TFIIF affect complex dynamics and control repressive activity. Through this work, we have revealed how a noncoding RNA can regulate mammalian gene expression.