1. Understanding the mechanisms of RNAPII transcription termination

RNAPII transcription termination is coupled to RNA 3′-end processing. In the yeast Saccharomyces cerevisiae, two alternative transcription termination mechanisms operate: the polyA-dependent and the NNS-dependent pathways. In the first pathway, termination is based on recognition and cleavage at the polyadenylation signal in the transcript, producing a stable mRNA on one side and an unprotected 5′ end on the RNA still attached to RNAPII. Degradation of this RNA by the Rat1 exonuclease triggers RNAPII dissociation by a yet unknown mechanism. In the NNS-dependent pathway, binding of Nrd1 and Nab3 to nascent RNA causes RNAPII termination. The NNS (Nrd1-Nab3-Sen1) complex recruits the exosome to the transcript, which is degraded in the case of non-coding RNAs or trimmed to the mature form in the case of snoRNAs. The signals present in the RNA, together with the phosphorylation state of the RNAPII CTD, determine the termination mechanism. We are interested in identifying new factors involved in RNAPII termination and, in particular, how chromatin structure affects the polyA-dependent pathway.

2. Determining the effect of cellular aging on transcription termination

Aging is a natural process for cells and organisms that increases the risk of cell malfunction and diseases such as Parkinson’s, Alzheimer’s and cancer. In addition, accelerated aging is the main syndrome of progeroid syndromes, a group of rare diseases that cause early death in patients. Therefore, a better understanding of the processes that are impaired during the functional decline of the cell during aging should help to improve strategies for extending cellular lifespan and could contribute to understanding the molecular basis of progeroid syndromes and reducing the incidence of age-related diseases. Chromatin is altered during cellular aging, with a reduction in the global level of histones leading to a decrease in nucleosome occupancy. As a result, cryptic transcription increases and aberrant transcripts accumulate in the cell. In this context, we aim to investigate whether the balance between transcription elongation and termination is affected by cell aging and whether this is mediated by changes in chromatin structure.

3. Causes and consequences of R-loop formation during transcription elongation.

The first step in genome expression is the synthesis of RNA, which transcribes the information contained in the template DNA molecule. Transcription can be conceptually divided into three phases: initiation, when RNA polymerase (RNAP) is recruited to the promoter; elongation, when RNAP actively synthesises the transcript; and termination, when RNAP dissociates from the template. During transcription, the nascent RNA is processed and assembled into an exportable ribonucleoparticle. Defects in mRNA processing increase the ability of the RNA to thread back into the template DNA, forming an RNA:DNA hybrid that leaves the non-template strand as ssDNA, a structure commonly referred to as an R-loop. R-loop formation is a major source of transcription-associated genome instability as it interferes with fundamental processes including transcription and replication. Although detrimental during elongation, R-loops are required for accurate termination at numerous human genes. We would like to understand how R-loops affect transcription at the elongation and termination stages.