Nicholas Proudfoot

We study molecular mechanisms that define the extent of transcription units in mammalian genomes by focusing on the molecular mechanism of RNA polymerase II (Pol II) termination.

Some genes possess G-rich transcriptional pause sites, closely following their polyA signals (PAS). These slow down elongating Pol II after the PAS, so that PAS cleavage followed by Xrn2 exonucleolytic degradation elicits termination (Pol II release from chromatin). G rich pause elements form RNA: DNA hybrid structures (R-loops) that require Senataxin resolution for Pol II termination. We now show that R-loops also induce localised antisense transcripts that recruit H3K9me2 heterochromatin marks via an RNAi-like mechanism to reinforce the termination process. Such genes tend to encode widely expressed housekeeping proteins.

Many genes do not terminate close to PAS but instead transcribe well into the 3' flanking region. Transcription of co-transcriptional cleavage (CoTC) elements results in transcript breakage and concomitant Pol II release. Cleavage at the upstream PAS subsequently occurs on released Pol II in the nucleoplasm. We have employed CLIP-seq to show that CoTC-mediated Pol II termination is a common occurrence and may have significant advantage in facilitating gene expression levels. These genes tend to encode regulated often tissue specific proteins.

We are carrying out detailed transcriptomic analysis focusing on nascent RNA either isolated from chromatin fractions or by using Pol II IP (NET-seq). These data will clarify the genomic distribution of pause type versus CoTC type termination. We also show that the microprocessor (Drosha-DGCR8) not only excises pre-microRNA from larger Pol II transcripts, but also promotes Pol II termination, especially of independently transcribed lncRNA genes.

Our studies on Pol II termination reveal a remarkably diverse range of mechanisms that may be tailored to specific genes classes.