Transcriptome surveillance in S. cerevisiae by RNA synthesis and degradation coupling and selective termination of non-coding RNAs

Episode von Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06 von Ludwig-Maximilians-Universität München · 9. Oktober 2013

Eukaryotic gene transcription is highly complex and regulation occurs at multiple stages. RNA Polymerase II (Pol II) is recruited to promoter regions of the DNA to initiate transcription.

Shortly after initiation, Pol II exchanges initiation factors for elongation factors. After Pol II passes termination signals, the RNA is cleaved and Pol II eventually released from the DNA template. pre-mRNAs are polyadenylated and exported to the cytosol for translation and ultimately degradation. Mechanisms regulating transcription have been studied extensively, but mechanisms of mRNA degradation are less well understood. To monitor mRNA synthesis and degradation, we developed the comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae Sc cells with the use of Schizosaccharomyces pombe Sp cells as internal standard. We show that Sc mutants can buffer mRNA levels and that impaired transcription causes decreased mRNA synthesis rates compensated by decreased decay rates. Conversely, impairing mRNA degradation causes decreased decay rates, but also

decreased synthesis rates. Thus, although separated by the nuclear membrane, transcription

and mRNA degradation are coupled. In addition to regulated mRNA synthesis, pervasive transcription can be found throughout the genome, governed by an intrinsic affinity of Pol II for DNA. These divergent noncoding RNAs (ncRNAs) stem to a large extent from bidirectional promoters. However,

global mechanisms for the termination of ncRNA synthesis that could act as a transcriptome

surveillance mechanism are not known. It is also unclear if such a surveillance system protects the transcriptome from deregulation. Here we show that ncRNA transcription in Sc is globally restricted by early termination which relies on the essential RNA-binding factor Nrd1. Depletion from the nucleus results in Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) throughout the genome and can deregulate mRNA synthesis by antisense repression and transcription interference.

Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at essentially all promoters and antisense NUTs in most 3’-regions of genes. Nrd1 preferentially binds RNA motifs which are enriched in ncRNAs and depleted in mRNAs except in some mRNAs whose synthesis is controlled by transcription attenuation. These results describe a mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and prevent transcriptome deregulation

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