Structural studies on macromolecular complexes in translational control and ribosome biogenesis

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

The ribosome is a large molecular machine central to the survival of all living cells. Genetic information is brought to the ribosome in the form of messenger RNA (mRNA) which is a mobile, molecular copy of a given gene sequence. The ribosome reads the information stored in the mRNA and translates it by synthesising a protein with a specific structure and function. By understanding the structure of a particular protein, we can often gain insights into its function (and malfunction in disease states) in the cell. Producing ribosomes requires a large proportion of a growing cell‘s resources and several genetic diseases are associated with inefficient ribosome production. Ribosome synthesis goes through a number of ordered steps, involving many proteins that ensure synthesis proceeds correctly. My research will examine two related aspects of protein production, firstly how a particular protein recognises certain mRNAs and prevents them from being translated. Interestingly, this protein is also hijacked by hepatitis C virus, which disguises its genes by mimicking cellular mRNA. The virus might use the normal function of this protein to aid its reproduction. Secondly, I will look at proteins involved in producing ribosomes and how they control the final stages of ribosome maturation.

Technical Summary

Translation of messenger RNA (mRNA) into protein on the ribosome is a fundamental process at the core of cellular function. Ribosomes constitute a large protein-RNA assembly that, in eukaryotes, requires ~170 soluble factors during ribosome biogenesis. In eukaryotes, the export of pre-ribosomal particles across the nuclear envelope requires adaptor proteins to recruit components of the nuclear export machinery. Pre-ribosomal particles are only exported once they have reached a particular maturation state and translation does not occur until ribosome maturation is completed in the cytoplasm. The rate of translation of a given mRNA is dependent not only on its availability but also on protein factors bound to the mRNA that can either promote or inhibit translation, sequester mRNAs into stress granules or processing bodies, or otherwise alter the location of mRNAs within the cell. Many transiently expressed mRNAs contain cis-acting sequences in their 3‘ untranslated regions that mark them as unstable and recruit protein factors that activate mRNA turnover. Signalling events can induce binding of other protein factors that protect these mRNAs from degradation. The work proposed here will use a combination of biochemistry and X-ray crystallography to gain insights into the functions of macromolecular complexes involved in translational control of mRNAs and in ribosome biogenesis. Understanding the structures of proteins in different states and/or complexes often provides key insights into their function, and allows hypotheses about the molecular mechanisms of cellular processes to be developed. These hypotheses can then be tested by creating site-directed mutants that are examined in biochemical and cell-based assays. The first project examines how structured cis-acting sequences in certain mRNAs recruit a protein complex known as NF90/NF45. This complex promotes the stability of bound mRNAs and inhibits translation. This complex is also a cellular host factor recruiteby hepatitis C virus to its RNA genome and co-localises at viral replication foci in infected hepatocytes. This project aims to understand the molecular basis for RNA recognition by NF90/NF45 in these different systems. The second project will address the molecular basis for the final maturation stages in eukaryotic ribosome biogenesis, based initially on studies in budding yeast. Removal and recycling of ribosomal export factors in the cytoplasm is often coupled to the insertion of ribosomal proteins. I will examine complexes of ribosomal processing factors with ribosomal proteins to understand the mechanism behind this functional coupling.
 
Title Fused glass cells 
Description I collaboration with Sarah Keer-Keer (Outreach Manager, Wellcome Trust Centre for Cell Biology), I have created some fused glass items that can be used to explain the process of crystallisation. 
Type Of Art Artefact (including digital) 
Year Produced 2013 
Impact Fused glass objects are very useful in public engagement activities. Unlike 2D images they are tactile and can have both colour and sculptural qualities. These items help to attract members of the public and serve as a talking point to discuss aspects of your work. 
URL http://sbsweb2.bio.ed.ac.uk/cook/node/9
 
Title Structural Biology Picture Wall 
Description A dynamic picture wall was created for to give visitors to our department a snapshot of some of the work ongoing in the Edinburgh Structural Biology groups. The pictures of protein structures are framed and attached with velcro strips so that pictures can easily be replaced and moved around to make an ever changing composition. 
Type Of Art Artwork 
Year Produced 2016 
Impact This artwork is primarily seen by departmental visitors such as academics from external institutions and visitors from grant funding bodies. 
URL http://cook.bio.ed.ac.uk/node/9
 
Description Nanolitre pipetting robot for crystallisation of macromolecular complexes
Amount £61,100 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 01/2013 
End 06/2013
 
Description Two cold light source microscopes for macromolecular crystal viewing and manipulation
Amount £36,900 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 01/2014 
End 06/2014
 
Description Wellcome Trust Biomedical Vacation Scholarship
Amount £1,440 (GBP)
Funding ID 099618/Z/12/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Vacation Scholarship
Sector Charity/Non Profit
Country United Kingdom
Start 07/2012 
End 08/2012
 
Description Wellcome Trust Biomedical Vacation Scholarship
Amount £1,500 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Vacation Scholarship
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 08/2015
 
Description Wellcome Trust Biomedical Vacation Scholarship
Amount £1,500 (GBP)
Funding ID 202605/Z/16/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Vacation Scholarship
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 08/2016
 
Description Wellcome Trust Senior Research Fellowship
Amount £1,568,534 (GBP)
Funding ID 200898/Z/16/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Senior Research Fellowship
Sector Private
Country United Kingdom
Start 10/2016 
End 09/2021
 
Description A histone-like code for RNA binding proteins 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Niki Gray's laboratory was recently awarded a BBSRC grant to study the functional importance of histone-like modifications on RNA binding proteins involved in transcriptional control. Prior to this award, I co-supervised a PhD student with Niki Gray and provided the student with training in biochemical methods. Part of the student's work formed the basis of this grant. I currently provide support for the PDRA named on this grant for structural biology studies, that form one of the key aims.
Collaborator Contribution The Gray laboratory have shared research data and expertise with my group.
Impact This collaboration has resulted in a collaborative grant from BBSRC with the Gray laboratory: "Can histone code-like 'switches' govern the multi-functionality of RNA-binding proteins?" ref: BB/P022065/1 award value is ~£735K on which Atlanta Cook is named as a co-investigator.
Start Year 2013
 
Description Adrian Bird/MeCP2/TBLR1 complex 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution I have supervised a PhD student on a collaborative project. The primary aim of this work is to understand the structural basis for the recruitment of nuclear co-repressor complexes to DNA by a methylated DNA binding protein.
Collaborator Contribution Adrian Bird provides co-supervision and a PhD stipend for this project.
Impact We have solved a co-crystal structure of a fragment of a DNA binding protein with a domain from a co-repressor complex. A publication describing this work is currently under review.
Start Year 2013
 
Description David Tollervey/Ribosome Biogenesis 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Transfer of information and reagents
Collaborator Contribution Transfer of information and reagents
Impact Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases. McCaughan UM, Jayachandran U, Shchepachev V, Chen ZA, Rappsilber J, Tollervey D, Cook AG. (2016) Nat Commun. 7:11789 doi: 10.1038/ncomms11789. PMID: 27250689
Start Year 2011
 
Description Sander Granneman 
Organisation Universities UK
Country United Kingdom 
Sector Academic/University 
PI Contribution I contributed data analysis and figures to a publication from Sander Granneman's laboratory.
Collaborator Contribution Our partner laboratory has provided training and support in data analysis of NGS data.
Impact Nucleic Acids Res. 2014 Oct 29;42(19):12138-54. doi: 10.1093/nar/gku815. Epub 2014 Sep 8. Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution. Hector RD, Burlacu E, Aitken S, Le Bihan T, Tuijtel M, Zaplatina A, Cook AG, Granneman S.
Start Year 2012
 
Description Ulrike Kutay, Mammalian-specific ribosome biogenesis factors 
Organisation ETH Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution I assisted the group of Ulrike Kutay on a study of the role of nuclear factors 90 and 45 in ribosome biogenesis in mammalian cells. I provided vector expression constructs and advice on the use of site-specific mutants.
Collaborator Contribution Ulrike Kutay's laboratory shared information prior to publication.
Impact Mol Cell Biol. 2015 Oct;35(20):3491-503. doi: 10.1128/MCB.00306-15. Epub 2015 Aug 3. The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology. Wandrey F, Montellese C, Koos K, Badertscher L, Bammert L, Cook AG, Zemp I, Horvath P, Kutay U.
Start Year 2013
 
Description modified RNAs 
Organisation Isis Pharmaceuticals
Country United States 
Sector Private 
PI Contribution We have used materials from this company for crystallisation experiments
Collaborator Contribution The company provided us with modified oligonucleotides for crystallisation experiments
Impact We have solved a structure of an RNA/protein complex using materials from this company
Start Year 2012
 
Description "Life through a Lens" workshop for children at the Scottish Storytelling Centre 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact In 2012, along with our public engagement manager, I helped to develop a workshop based on the early history of microscopy and cell biology, known as "Life through a Lens". This workshop was reformatted for a story-telling and activity workshop based at the Scottish Storytelling centre in Edinburgh. I helped with the delivery of this workshop and helped children and parents examine and understand specimens viewed by early pioneers in light microscopy.
Year(s) Of Engagement Activity 2017
 
Description Diamond light source MX User working group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Feedback to DLS management committee about new MX developments

This is an ongoing working group
Year(s) Of Engagement Activity 2013,2014,2015
 
Description Kickstart 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Secondary School children attend workshops and talks around the university campus to help them decide on which subjects they wish to study for their degree. This workshop involves dissection of fruitfly testes to examine the process of meiosis.
Year(s) Of Engagement Activity 2011,2012,2013,2014
URL http://www.ed.ac.uk/student-recruitment/widening-participation/projects/partnership-projects/kicksta...
 
Description Life Through a Lens 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public outreach event highlighting research that goes on in the Wellcome Trust Centre for Cell Biology, where I work, with an opportunity to present my work to the public.

Based on feedback from participants, this outreach event is always very well received.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2016
URL http://outreachwcb.bio.ed.ac.uk/wcb/Life_Through_a_Lens.html
 
Description Zorbing the Cell 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We set up a zorb ball in the Botanic gardens in Edinburgh to represent the cell and its nucleus. Members of the public were invited to climb into the ball where they found sets of homologous chromosomes. In addition to the zorb ball, we had information about our research activities (fliers) and other glass and sculptural objects to explain our work on cell biology.
Year(s) Of Engagement Activity 2014
URL http://sbsweb2.bio.ed.ac.uk/cook/node/9