Molecular recognition in post-transcriptional regulation

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

The functioning of the human body and of complex organisms in general requires different proteins to be produced in different types of cells. This cell-type-specific protein production is achieved by precisely regulating the translation of the genetic code into proteins. The two steps in this process of translation are, first, the translation of the DNA-encoded information into RNA and, then, the translation of the RNA code into a protein molecule. Both processes are regulated by biological machines, which are composed of proteins and, in some cases, RNA molecules. We focus on the regulation of the RNA-to-protein step (or post-transcriptional regulation) and investigate how the protein RNA machines assemble in a solution environment and regulate gene expression. Our structural studies complement other techniques such as X-ray crystallography, which can be used to study molecules in a static crystalline state. Analysing the structures of the molecules that govern regulation of protein synthesis has a direct medical relevance, as this process lies at the basis of common genetic diseases, cancer and viral infections. We work on an important regulatory mechanism, called ARE mediated mRNA decay (AMD), that increase the synthesis of specific proteins in inflammation and healing processes. This mechanism, if permanently switched on, can lead to inflammatory arthritis and cancer. We want to understand how the switch works at the molecular level and design specific therapies to switch in off when required.||Using a similar technical approach we are also investigating a key regulatory protein from herpes virus. This project wants to facilitate the design of anti-herpes drugs to treat people infected with this virus, which forms a major threat to immunodepressed patients, increases the risk of organ transplantation and chemotherapy and reduces the life expectancy of AIDS sufferers. Molecular insight into the interaction of ICP27 with its functional binding partners needs to be obtained if we are, for example, to design or optimise compounds to lock protein and RNA in a non-functional conformation or to (de)stabilise protein RNA complexes.

Technical Summary

Multifunctional eukaryotic regulatory proteins and their viral functional equivalents control gene expression by interacting with mRNAs in large macromolecular aggregates. Dissection of the molecular basis of post-transcriptional regulatory mechanisms has a direct medical relevance, as changes in the regulation of mRNA metabolism lie at the basis of common genetic diseases, cancer and viral infection. Current therapies for these diseases do not focus on the post-transcriptional steps of regulation but rather on the transcriptional ones, that are better understood. Our aim is to clarify the structure-function relation that is at the basis of mRNA recognition by post-transcriptional regulatory proteins and to suggest strategies to control this recognition. ||Adenine-uracil-rich element (ARE)-mediated mRNA decay (AMD) regulates the concentration of mRNAs that contain AREs within their 3 untranslated regions (3 UTRs) by promoting their degradation. Transient AMD shut-off up-regulates the stability of these mRNAs and is important for processes that require a fast response of the organism such as cellular growth, immune response, cardiovascular toning and external stress-mediated pathways. However, impaired AMD and the consequent pathological long-term increase in the stability of a subset of mRNAs have been related to several types of cancer (skin tumours, colorectal cancer, Hodgkins lymphoma, lung carcinoma and leukaemia) and auto-inflammatory diseases (Crohn-like inflammatory bowel disease and inflammatory arthritis). K-homology splicing regulator protein (KSRP) is an important player in AMD that interacts with several different AREs mediating the degradation of the corresponding mRNAs. Our work centers on the analysis of the structural and functional elements that contribute to KSRP-RNA interactions and to mRNA degradation. We are studying the structure and dynamics of the different domains of the protein and investigated their relationship to mRNA degradation. Using structural and functional information, we plan to dissect the details of the interaction with the RNA and help the design of a strategy for the tuning of KSRP activity.||Herpes viridae induced infections are a major threat to immunodepressed patients, increasing the risk of transplants and chemotherapy and reducing the life expectancy of AIDS sufferers. Current herpes virus therapies (e.g. Acyclovir) aim to block the synthesis of new DNA, but resistance to these treatments is increasing.||Successful viral replication is achieved through the tightly regulated expression of viral genes. A key component of the regulatory mechanism is ICP27, an essential HSV-1 RNA binding protein that regulates protein expression, both at the transcriptional and post-transcriptional level. We will study both ICP27 and the mechanism of post-transcriptional regulation acted upon by this protein, in particular 3 mRNA editing. Using the same strategy described above for the protein KSRP, we will obtain a better understanding of the regulatory cycle of the virus and define specific structural features amenable to structure-aided drug design

Publications

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Arkov AL (2010) Building RNA-protein granules: insight from the germline. in Trends in cell biology

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Arkov AL (2006) The role of Tudor domains in germline development and polar granule architecture. in Development (Cambridge, England)

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Beuth B (2007) Scaffold-independent analysis of RNA-protein interactions: the Nova-1 KH3-RNA complex. in Journal of the American Chemical Society

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Briata P (2011) KSRP, many functions for a single protein. in Frontiers in bioscience (Landmark edition)

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Briata P (2013) Functional and molecular insights into KSRP function in mRNA decay. in Biochimica et biophysica acta

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Castilla-Llorente V (2014) PolyQ-mediated regulation of mRNA granules assembly. in Biochemical Society transactions

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Castilla-Llorente V (2013) Terminal loop-mediated regulation of miRNA biogenesis: selectivity and mechanisms. in Biochemical Society transactions

 
Description FP7 Marie Curie
Amount € 200,371 (EUR)
Funding ID 301756 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 10/2012 
End 10/2014
 
Description Marie Curie FP7
Amount € 221,606 (EUR)
Funding ID 626920 
Organisation European Commission 
Department Research Executive Agency (REA)
Sector Public
Country European Union (EU)
Start 09/2014 
End 09/2016
 
Description Wellcome-Research Grant-2007
Amount £275,812 (GBP)
Funding ID WT022088MA 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2007 
End 09/2010
 
Title High-throughput analysis of protein-RNA interactions 
Description High-throughput analysis of protein-RNA interactions using Scaffold Independent Analysis and Principal Component Analysis 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This is a new tool. In the group has allowed to analyze a large number of protein-RNA binding domains 
 
Title Scaffold Independent Analysis 
Description Allows to define the sequence preference of a nucleic acid binding domain. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Allowed us to define the binding mode of KSRP protein on miRNA precursors. 
 
Description Development and application of NMR methods for the study of macromolecular complexes 
Organisation Medical Research Council (MRC)
Department MRC Biomedical NMR Centre
Country United Kingdom 
Sector Public 
PI Contribution Framework, samples, intellectual input, recording and processing of experiments, data analysis
Collaborator Contribution intellectual input, recording and processing of experiments, data analysis. Recent we started a collaboration on a related problem with the group of John Christodoulou in UCL, SMB where we provided data and intellectual input and the CVHristodoulou group provided the framework of analysis.
Impact PMID: 25586222; PMID: 23142982; PMID: 20711187; PMID: 20385598; PMID: 19198587; PMID: 17437720; PMID: 16407062; PMID: 15929005; PMID: 15665873; PMID: 27109776
 
Description Development and application of NMR methods for the study of macromolecular complexes 
Organisation University College London
Department Institute of Structural and Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Framework, samples, intellectual input, recording and processing of experiments, data analysis
Collaborator Contribution intellectual input, recording and processing of experiments, data analysis. Recent we started a collaboration on a related problem with the group of John Christodoulou in UCL, SMB where we provided data and intellectual input and the CVHristodoulou group provided the framework of analysis.
Impact PMID: 25586222; PMID: 23142982; PMID: 20711187; PMID: 20385598; PMID: 19198587; PMID: 17437720; PMID: 16407062; PMID: 15929005; PMID: 15665873; PMID: 27109776
 
Description KSRP 
Organisation University of Turin
Department Institute for Cancer Research and Treatment
Country Italy 
Sector Academic/University 
PI Contribution We cover the structural and biophysical aspects of the analysis of KSRP function and interactions with the RNA targets.
Collaborator Contribution They performed complementary functional studies
Impact We have dissected the function of KSRP in mRNA decay and miRNA biogenesis and the regulation by signalling pathways. PMID: 19198587 PMID: 19458619 PMID: 17437720 PMID: 22547390 PMID: 23142982 PMID: 25385579
Start Year 2006
 
Description gtetrads 
Organisation University of Naples
Country Italy 
Sector Academic/University 
PI Contribution I have collaborated to the NMR work on the structure of G-quartets involved in down regulation of telomerase
Collaborator Contribution It was a shared structural analysis
Impact We provide a structural analysis of G-quartets involved in down regulation of telomerase: PMID:18058552
Start Year 2006
 
Description rbm38 
Organisation Netherlands Cancer Institute (NKI)
Country Netherlands 
Sector Academic/University 
PI Contribution we have elucidated the bases of target selectivity and assessed interaction with functionally related protein factors
Collaborator Contribution we have completed and published a joint study on the role of RBM38 protein in mediating p53 action
Impact we published a joint paper in Nature Communications in 2011 - PMID: 22027593. There are unpublished results that we expect will result in a second paper.
Start Year 2010
 
Description tudor 
Organisation Skirball Institute of Biomolecular Medicine
Country United States 
Sector Academic/University 
PI Contribution Structural analysis of the domain of Tudor protein
Collaborator Contribution They performed functional studies on tudor protein
Impact We published a joint paper and a joint review: PMID: 16971472 and a joint review PMID: 20541937
Start Year 2006