RNA processing and eukaryotic gene expression regulation

Lead Research Organisation: University of Edinburgh

Abstract

DNA carries the genetic information of a cell and consists of thousands of genes that encode all the information required to make proteins. Genes (DNA) are copied into a molecule called RNA (pre-mRNA) in a process known as transcription. This pre-mRNA is then processed so that non-coding parts are removed and is then transported out of the nucleus to be ultimately translated into proteins in the cytoplasm. Our laboratory is interested in different aspects of how the pre-mRNA is accurately processed into mature mRNA, which then undergoes translation as part of the protein synthesis to produce proteins. Defects in RNA processing are linked to human disease. Our research program is at the basic end of the spectrum and we expect to contribute to the general knowledge in the areas of RNA processing and nuclear structure that could be applicable to human genetics.

Technical Summary

Gene expression is extensively regulated at the post-transcriptional level. The fundamental steps of eukaryotic RNA processing have been characterised in great detail, but knowledge of how the disruption of these processes contributes to human disease has only recently begun to emerge. The major aim of this programme is to study the mechanisms for the post-transcriptional regulation of gene expression. Our laboratory studies different aspects of RNA processing, including alternative splicing regulation, microRNA processing and nonsense-mediated decay.

The predominance of alternative splicing has become evident when completion of the human genome sequence revealed that a large proteomic complexity is achieved with a limited number of genes. We are particularly interested in the trans-acting factors that are involved in the regulation of alternative splicing, such as the SR proteins and hnRNP A/B type of proteins. These proteins have antagonistic activities and their molar ratio influence different modes of alternative splicing in vivo and may represent a mechanism for tissue-specific or developmental regulation of gene expression.

We also study the subcellular distribution of RNA processing factors and how this could be affected by extracellular signals.

The relevance of RNA processing, in particular pre-mRNA splicing, to human disease has emerged recently. Disturbances in pre-mRNA splicing have been found in a variety of disease processes. Splice site mutations are exemplified by some forms of thalassemia. However, in many cases, such as the dysregulated splicing of CD44 found in many human malignancies, mutations are not found in the target gene and it is presumed that the changes are due to changes in the milieu of splicing factors in the diseased cell. Therefore, cis-acting sequences and trans-acting factors that lead to aberrant RNA processing associated with human disease will be an important target of molecular therapies.

Interestingly, pre-mRNA splicing is not only important to form the mature mRNA, that is exported to the cytoplasm, but also influences processing events that act downstream of it. Therefore, we have expanded our interest to RNA processing events that act downstream of pre-mRNA splicing, including nonsense-mediated decay and translational regulation.

A combination of different methodologies is used in our research. We mostly rely on classical molecular biology and cellular biology techniques. Cell culture is widely used and we also are using two animal models, mice and the nematode C.elegans.
 
Description Wellcome Trust Senior Investigator Award
Amount £1,428,571 (GBP)
Funding ID 095518/Z/11/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 03/2017
 
Description Eyras-Computational methods -regulation of alternative pre-mRNA splicing 
Organisation Pompeu Fabra University
Country Spain 
Sector Academic/University 
PI Contribution Conception, design and interpretation of experiments
Collaborator Contribution Specialised bioinformatics analysis
Impact 1. Macias,S., Plass,M., Stajuda,A., Michlewski,G., Eyras,E. and Cáceres,J.F. (2012) DGCR8 HITS-CLIP reveals novel cellular functions for the microprocessor. Nat. Struct. Mol. Biol., 19, 760-766. 2. Heras,S.R.±, Macias,S.±, Plass,M., Fernandez,N., Cano,D., Eyras,E., Garcia-Perez,J.L.* and Cáceres,J.F.* (2013) The Microprocessor controls the activity of mammalian retrotransposons. Nat. Struct. Mol. Biol., 20, 1173-1181. ±Joint First authors *Co-corresponding authors 3. Gromak,N., Dienstbier,M., Macias,S., Plass,M., Eyras,E., Cáceres,J.F. and Proudfoot,N.J. (2013) Drosha regulates gene expression independently of RNA cleavage function. Cell Rep., 5, 1499-1510. 4. Maslon,M.M.*, Heras,S.R.*, Bellora,N., Eyras,E. and Cáceres,J.F. (2014) The translational landscape of the splicing factor SRSF1 and its role in mitosis. eLife, 3, e02028. *Joint First authors
Start Year 2011
 
Description Llorca-Structural studies-Electron Microscopy (EM)-Nonsense-mediated decay (NMD) 
Organisation Spanish National Cancer Research Center
Country Spain 
Sector Academic/University 
PI Contribution Experimental design, biochemical experiments
Collaborator Contribution Experimental design, structural analysis, electron microscopy
Impact 1. Melero,R.±, Hug,N.±, Lopez-Perrote,A., Yamashita,A., Cáceres,J.F.* and Llorca,O*. (2016) The RNA helicase DHX34 functions as a scaffold for SMG1-mediated UPF1 phosphorylation. Nat. commun., 7,10585 | doi: 10.1038/ncomms10585 ±Joint First authors/*Co-corresponding authors
Start Year 2013
 
Description Michael Sattler-Structural studies-Crystallography-NMR of factors involved in biogenesis of miRNAs 
Organisation Technical University of Munich
Country Germany 
Sector Academic/University 
PI Contribution Experimental design, biochemical experiments
Collaborator Contribution Experimental design, structural analysis, Crystallography, NMR
Impact Kooshapur,H., Choudhury,N.R., Simon,B., Mühlbauer,M., Jussupow,A., Fernandez,N., Dallmann,A., Gabel,F., Camilloni,C., Michlewski,G.*, Cáceres,J.F.* and Sattler,M.* (2018) Structural basis for terminal loop recognition and processing of pri-miRNA-18a by hnRNP A1. Nat commun., under revision *Co-corresponding authors
Start Year 2011
 
Description Professor Rory Duncan-Super Resolution Microscopy 
Organisation Heriot-Watt University
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell biological approaches to the study of Nonsense-mediated decay (NMD) factors
Collaborator Contribution Super resolution approaches, FRET-FLIM, Fluorescence correlation spectroscopy (FCS), RNA FISH coupled with high-resolution microscopy
Impact none, yet
Start Year 2016
 
Description Role of the Nonsense-mediated pathway in models of Neurodegeneration in nematodes 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have uncovered evidence that suggests the existence of a branch of the Nonsense-mediated decay pathway that is dedicated to Endoplasmic Reticulum (ER)-localized mRNAs. We are focusing on a novel NMD factor that we identified in our lab, which is termed NBAS, for Neuroblatoma-amplified gene. Given the established link between NMD and ER stress, we are investigating whether there is a protective role for the NMD pathway in pathological conditions where there is chronic activation of the Unfolded Protein Response (UPR), using established models for this process in neurodegenerative disease. In particular, in this collaboration we are focusing on models of Neurodegeneration in the nematode, C. elegans.
Collaborator Contribution The lab of Maria Doitsidou is providing her expertise in C.elegans and in models of Neurodegeneration in this organism
Impact This collaboration has just started
Start Year 2018