RNAs are involved in many key cellular processes, where minor alterations in mRNA levels are often detrimental and can lead to diseases in humans. Levels of individual mRNAs are regulated in response to environmental cues, during cell differentiation and development; however the mechanisms underpinning these processes remains poorly understood.

Our research aims to understand how RNA polymerase II (Pol II) transcription and the RNA exosome complex, a major cellular RNA decay machinery, regulate levels of individual mRNAs. To achieve this, our laboratory is using multidisciplinary approaches, combining biochemistry, structural analyses, genetics and genomics

RESEARCH

RNAs are involved in many key cellular processes, where minor alterations in mRNA levels are often detrimental and can lead to diseases in humans. Levels of individual mRNAs are regulated in response to environmental cues, during cell differentiation and development; however the mechanisms underpinning these processes remains poorly understood. Our research aims to understand how RNA polymerase II (Pol II) transcription and the RNA exosome complex, a major cellular RNA decay machinery, regulate levels of individual mRNAs. To achieve this, our laboratory is using multidisciplinary approaches, combining biochemistry, structural analyses, genetics and genomics.

We are focused on three central questions:

1. How is mRNA processing coupled to RNA polymerase II transcription?

To form functional mRNA molecules that can be translated in the cytoplasm to proteins, mRNAs are subjected to extensive processing such as capping, splicing and 3’end cleavage and polyadenylation. Accuracy and efficiency of mRNA processing is regulated by Pol II transcription. Phosphorylation of the C-terminal domain (CTD) of the largest subunit of Pol II plays a key role in mRNA processing, however the mechanisms underpinning CTD function are not well understood. Recently, we have discovered that conserved proteins that form multivalent interactions with CTD, Pol II core, nascent RNA and mRNA 3’end processing machinery are key in mediating CTD function in mRNA 3’end processing at the end of transcription cycle (Figure 1) (Wittmann et al., 2017 Nature Comm; Kecman et al., 2018 Cell Reports). We have solved the structures of the CTD-Interacting Domain (CID) and RNA binding module (RRM), providing insights into how these proteins interact with Pol II CTD and nascent pre-mRNA (Figure 2). Building on these exciting findings, we now aim to uncover the molecular mechanisms involved in coupling transcription and mRNA 3’end processing to make functional mRNA.

Figure 1. Conserved CID-RRM proteins in RNA polymerase II transcription.

CID- CTD–Interacting Domain,

RRM- RNA Recognition Motif.

Figure 2. Novel organization of the RNA-binding module consisting of the canonical RRM and RRM-like domain (green) (1.02Å) PDB 5MDU (Wittmann et al., 2017 Nature Comm).

2. How is Pol II dislodged from DNA at the end of transcription?

All three stages of the transcription cycle of mRNAs (initiation, elongation and termination) are tightly regulated. Pol II interacts with a different set of factors to regulate the transition between these stages. The factors modulate how Pol II behaves, changing its ability to interact with DNA, incorporate nucleotides or pause. At the transition between initiation and elongation, initiation factors bound to the core are replaced by elongation factors, causing Pol II to pause. Termination is essential for 3' end formation of functional mRNA, mRNA release, and Pol II recycling, but the changes that take place as Pol II moves from elongation to termination are not fully understood.

We have discovered that factor exchange takes place during the transition from elongation to termination as well as from initiation to elongation (Kecman et al., 2018 Cell Reports). The exchange of factors is regulated by phosphorylation of the elongation factor Spt5 and Pol II CTD. Our future research will provide insights into molecular events leading to Pol II and mRNA release.

3. How are individual transcripts targeted for degradation?

The RNA exosome complex, a major RNA degradation machinery, plays a key role in nearly every step of RNA metabolism, including mRNA maturation, degradation and surveillance (Figure 3). The RNA exosome complex consists of 10-11 subunits that possesses 3’-5’ exo- and endonucleolytic activity is central to RNA regulation. The exosome controls expression levels of specific mRNAs in response to environmental cues, during cell differentiation and development. Deregulation of the exosome function leads to severe neurological diseases such as spinal muscular atrophy, pontocerebellar hypoplasia and infantile leukodistrophy. The mechanisms by which RNA is targeted to (or escapes from) the exosome are not known, preventing us from understanding how these defects arise when exosome function is perturbed. In the past, we have identified RNA-binding proteins that regulate targeting of the exosome complex to specific RNAs (Exosome-Specificity-Factors, ESFs). One of our current goals is to understand how ESFs regulate targeting of the exosome to RNAs.

Figure 2. RNA exosome complex. Model of the EXO11 Dis3+Rrp6 complex. Surface structure of EXO11 Dis3+Rrp6 was generated by superimposing the structures of EXO10 Dis3+Rrp6 C-term (Rrp6 in red and the cap complex in green (Csl4, Rrp4, and Rrp40; PDB 4IFD) and EXO10 Rrp6 (PH ring complex in blue (Rrp41, Rrp45, Rrp42, Mtr3, Rrp43, and Rrp46), Dis3 in purple and RNA in black; PDB 4OO1).

(Right) Schematics of an RNA molecule threading 3' to 5' through the central channel of the cap and PH ring to the Dis3 exonucleolytic centre, where it is degraded (Kilchert et al., 2016 Nature Rev Mol Cell Biol).

In the news:

Our recent paper on the mechanism regulating Pol II transition from elongation to termination stage of transcription:

https://www.bioch.ox.ac.uk/article/new-paper-shows-how-transcription-terminates-with-the-help-of-a-phosphatase

Our paper discovering a key role for conserved factors in terminating RNA

polymerase II transcription:

http://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=1401

Contact: lidia.vasilieva@bioch.ox.ac.uk

Graduate and postdoctoral positions: enquiries with CV welcome