Roles of transcriptional events in determining RNA fate

Supervisors: David Tollervey, Guido Sanginetti

Project description:

Background:

RNA transcription, processing and assembly with protein complexes form the core of the gene expression system but many key features remain unclear. The Tollervey group is attempting to determine how the exosome complex can targeted to very large numbers of different targets [1]. In addition to mRNAs and stable, functional RNAs, all Eukaryotes examined to date synthesize a bewildering number of noncoding RNAs (ncRNAs). Both ncRNAs and mRNAs are transcribed by RNAPII and acquire 5’ caps. However, in contrast to mRNAs, ncRNAs are predominately nuclear-retained and rapidly degraded by the TRAMP and exosome nuclear surveillance complexes. The clearance of this massive transcriptional load is a major activity and important to maintain normal gene expression. In addition, the activity of the nuclear surveillance system and ncRNAs act together to help rapidly remodel gene expression under conditions of altered nutrition [2]. A recent collaboration between the Tollervey and Sanguinetti groups combined transcriptomics and modelling to show that mRNA and ncRNAs differ transcriptionally [3]. They have also recently jointly supervised an MSc student performing an initial analysis of ncRNA and mRNA promoter features.     

Proposed Work:

The aim of this project is to identify transcriptional features that distinguish the major RNA classes. In particular, we will test the hypothesis that the transcription complexes loaded onto mRNA and ncRNA genes are functionally distinct due to differences in the promoter regions, with consequences for the fate of the RNA transcripts. Bioinformatics and modelling will be used to define differences in the promoter regions of large groups of expression-matched ncRNAs and mRNAs. A synthetic biology approach, using the BBSRC-funded Edinburgh Genome Foundry, will generate large numbers of constructs based on mRNA or ncRNA promoters and flanking regions. These will be tested to determine the fate of the resulting transcripts. Most specific eukaryotic transcription initiates from promoter regions that are defined by nucleosome free regions (NFRs) flanked by precisely positioned nucleosomes. We will separate sequences from NFRs, +1 and -1 nucleosome locations, and the subsequent transcript regions of select mRNAs and unstable ncRNAs. Data from these experiments will be used to refine the models, which will guide subsequent gene construct design for experimental testing.

Research groups:

The groups involved have highly complementary skills. The Tollervey lab has extensive experience in the use of crosslinking and high-throughput sequencing to analyse RNA-protein interactions and RNA surveillance. The Sanguinetti group are leaders in the application of machine learning and modelling to understand biological problems.

Synpromics are developing and testing synthetic promoters for commercial use and insights derived from the proposed research are expected to be of direct relevance to future design.

Research training:

The student will acquire a range of advanced skills that will equip them to undertake many different projects in academia, medicine or industry. The project will involve: 1) Molecular genetic approaches in yeast, including transformation and the use of CRISPR/Cas9 techniques. 2) Biochemical purification of RNA and RNA-protein complexes. 3) High throughput sequencing and bioinformatics analyses of the resulting sequence data. 4) Modelling and machine learning techniques.

References:

1.         Delan-Forino, C., Schneider, C. and Tollervey, D. (2017) Transcriptome-wide analysis of alternative routes for RNA substrates into the exosome complex. PLOS Genetics, 13, e1006699.

2.         Bresson, S., Tuck, A., Staneva, D. and Tollervey, D. (2017) Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast. Mol. Cell, 65, 787-800.

3.         Milligan, L., Huynh-Thu, V.A., Delan-Forino, C., Tuck, A.C., Petfalski, E., Pascual, R.L., Sanguinetti, G., Kudla, G. and Tollervey, D. (2016) Strand-specific, high-resolution mapping of modified RNA polymerase II. Mol. Sys. Biol., 12, doi: 10.15252/msb.20166869.

Other: