Transcription is not limited to regions annotated for producing known functional RNAs, but occurs virtually everywhere in the genome, a phenomenon that is conserved from bacteria to human cells. The advent of techniques for analysing genome-wide the distribution of RNA polymerases with high-resolution and no preconception of pre-existing annotations has led to the explosion of the complexity of the transcriptome by revealing myriads of novel transcripts, mostly non-coding, and the consequential, unsuspected, existence of many novel transcription units. Because many of the non-coding RNAs produced are unstable and non-detected in normal cells, this phenomenon has been dubbed pervasive or hidden transcription. We are interested in how pervasive transcription is generated, how it is controlled and what is its functional importance. We use many techniques, from biochemistry to molecular biology and genome-wide analysis, in the yeast and human model. Our research interests can be divided into 4 global axes.
AXIS 1: Study of the mechanism of transcription termination for ncRNA and mRNA coding genes (PIs: D. Libri & O. Porrua)
Higher levels of transcription imply that DNA-associated events have to be coordinated to avoid potentially disruptive interferences. For instance the “cohabitation” of transcription complexes needs to be strictly regulated to prevent non-productive collisions or silencing of adjacent transcription units, both of which have been shown to occur to some extent in nature (see our review: Porrua and Libri, 2015). Transcription termination has important roles in this context, and we are interested in the underlying mechanisms. Transcription termination at the 3’end of protein-coding genes is mediated by a conserved multi-subunit complex called the CPF-CF complex. In budding yeast, the NNS complex, composed of the RNA-binding proteins Nrd1 and Nab3, and the helicase Sen1 terminates transcription of functional non-coding RNA genes but also of pervasive transcription events. Moreover, the NNS complex promotes the degradation of non-coding RNAs by the nuclear exosome and its cofactor the TRAMP complex. Because of its specific functions, the NNS complex has a major role in controlling pervasive transcription.
During the past years, we have extensively characterized the function of the NNS-complex. However, many aspects of the mechanisms of termination remain unclear, both for non-coding and mRNA coding genes. Using a technique that allows detecting in vivo the position of the RNAPII with single-nucleotide resolution (CRAC, Crosslinking Analysis of cDNAs), we have generated transcription maps in mutants of the NNS and the CPF-CF pathways. This allows comparative analysis with the same technique of the implication of many factors, and weighs the relative impact of each. We combine this high-resolution genomic technique with in vitro biochemical systems and structural approaches to gain a deep undertanding of the mechanisms of transcription termination and the interplay between the different termination pathways.
AXIS 2: Analysis of the impact of non-coding transcription in gene expression in different physiological conditions (PI: D. Libri & O. Porrua)
Non-coding transcription events can regulate gene expression by affecting the function of promoters of neighboring genes in the sense or antisense orientation. The regulatory potential of non-coding transcription has been generally neglected, mostly because many previous analyses relied on detection of the RNA as proxy for transcription and ncRNAs produced by potentially regulatory transcription events are often unstable and not easily detected in wild type cells. Mapping the transcribing RNAPII allows circumventing this problem. Because ncRNA transcription can alter gene expression, there is large potential for regulation mediated by its activation or by alterations in its termination efficiency. We are interested in further exploring pathways of regulation by non-coding transcription in different physiological and stress conditions. We have generated many high-resolution maps of transcribing RNAPII under conditions of stress, during different stages of the cell-cycle and in conditions in which the non-coding transcription termination machinery is subject to post-translational modifications. Interestingly we detected many novel ncRNA transcription events in these conditions, some of which derive from activated bidirectional promoters, some from “solo” non-coding transcription units and some from decreased transcription termination efficiency. Moreover, in some instances the occurrence of these novel non-coding transcription events correlate with the repression of neighboring protein-coding genes, suggesting a possible role in gene regulation. We are pursuing the study of the impact of non-coding transcription in gene expression using a variety of approaches and bioinformatic tools.
AXIS 3: Characterization of the mechanisms responsible for the resolution of transcription-replication conflicts (PI: D. Libri )
The existence of transcription events that transcend the limits of annotated canonical genes is a major challenge for the cohabitation of transcription and other DNA-associated events such as replication. We have been interested in the recent past the impact that pervasive transcription has on the function of yeast replication origins. We are now directing our interests to the conflicts between transcription and replication in relationship with the function of the helicase Sen1 that is required for transcription termination of ncRNA genes and that we have studied for many years. The mechanisms underlying the resolution of transcription-replication (TR) conflicts remain poorly understood and are the subject of intense investigation. Sen1 mutants have been shown to display defects related to an increase in TR conflicts, but to what extent these defects are also linked to defects in transcription termination remains unclear.
We are currently investigating the precise role of Sen1 in the resolution of TR conflicts as well as the interplay between Sen1 and other cellular machineries involved in this process. To this end, we use high-resolution techniques to map the presence of R-loops, a hallmark of TR conflicts, in various mutant and physiological contexts, as well as the presence of the factors involved in the resolution of these conflicts.
AXIS 4: Study of the molecular function of human senataxin and its involvement in neurodegeneration (PI: O. Porrua )
The human homologue of Sen1, senataxin (SETX), has attracted much attention because of its connection with two neurodegenerative diseases. Recessive loss-of-function SETX mutations have been associated with Ataxia with Oculomotor Apraxia type 2 (AOA2), whereas dominant gain-of-function mutations in SETX are linked to a juvenile form of Amyotrophic Lateral Sclerosis (ALS) dubbed ALS4. As Sen1, SETX has been assigned a role in transcription termination as well as in R-loop resolution, however the precise role of SETX in these processes remains poorly understood because of the absence of biochemical data on the properties and activities of SETX. In addition, a systematic identification of the protein interactors and the targets of SETX is missing. To fill these gaps, we will benefit from our expertise and the tools we have developed for the functional characterization of the yeast homologue of SETX to elucidate the molecular function of SETX.
In addition, we will address the molecular basis of SETX-associated ALS in collaboration with S. Nedelec (IFM, Paris). To this end, we will generate human motor neurons from induced pluripotent stem cells harbouring ALS4 mutations and we will employ a variety of approaches to investigate the impact of these mutations on motor neuron physiology. Finally, we will combine biochemical, proteomic and genomic approaches to unveil the deregulations responsible for motor neuron impairment in ALS4.