1

Contents

Welcome 2

Acknowledgements 3

Parti cipant Informati on 3

Meals & Refreshments 4

Social Programme 4

Venue Faciliti es 5

Locati on Map 5

Transport 6

Accommodati on 8

LA - moti f containing proteins 9

Agenda 2nd September 10

Agenda 3rd September 12

Speakers’ Abstracts 14

Poster Abstracts 27

Delegate List 31

Full Author Index 31

Floor Plans 32

2 3

The organisers would like to thank the sponsors and supporters of the 1st Annual Meeti ng of the LARP Society, without whose support this meeti ng would not have been possible.

• Nick Jenkins Charitable Foundati on

• Imperial College

• University of Perpignan

We would also like to thank Uta Boeger-Brown and the staff of BioMedEx for their help with the organisati on of this conference. www.biomedex.co.uk

Parti cipant Informati on

VenueLectures and registrati on will take place at:

W12 Conferences Hammersmith Campus, Imperial College, 150 Du Cane Road, London, W12 0HS.

Tel: +44 (0) 20 3313 1606Fax: +44 (0) 20 3313 1610

Please refer to page 32 for the venue layout

Registrati onThe registrati on desk is located in The Lounge area of the conference centre and will be open at the following ti mes:

Thursday 2nd September 09:00 - 18:30

Friday 3rd September 08:30 - 15:30

Upon registrati on, all parti cipants will receive the fi nal programme book, further conference informati on and a name badge. Badges must be worn for the durati on of the conference for security purposes and for entry to the lectures and social events.

AcknowledgementsWelcome

Welcome Notes on behalf of the LARP SocietyIt is with great pleasure that we welcome you to the fi rst of our annual meeti ngs of the Larp Society (LarpSoc). The formati on of LarpSoc and this meeti ng was inspired by the discovery that there are a number of groups around the world with a shared interest in La-containing proteins. The varied geographical and disciplinary distributi on of these groups means we would be unlikely to meet in an ordinary conference setti ng. So we have designed this conference with the specifi c aim of bringing everyone together for the fi rst ti me.

We have been delighted by the number of presti gious scienti sts who have agreed to come and speak or join in the discussion about this fascinati ng area of research. The aim of this year’s programme is to give an overview of Larp proteins in general and an update on what is known about the individual Larp family members. This will cover their structural biology, evoluti onary history, role in mRNA translati on and the proteins with which they interact.

We hope this will be a sti mulati ng meeti ng and that you may also contribute to our discussion about the future directi on of LarpSoc. We thank you again for coming and hope that this will be the beginning of a long and fruitf ul relati onship!

Cécile Bousquet-Antonelli & Sarah Blagden

Cover Picture

Crystal structure of the N-terminal domain of the human La protein bound to an AUUUU RNA oligonucleoti de (shown in sti cks). The La Moti f (yellow) and RRM1 (light brown) clamp together to bind the 3´-end of the RNA. (Koti k-Kogan et al., (2008) Structure, 16: 852-862).

Kindly contributed by Stephen Curry and Sasi Conte

4 5

Medical ServicesPlease contact the registrati on desk in the case of a minor incident. If it is an emergency, please call 999 directly and inform BioMedEx staff at the registrati on desk.

Internet faciliti esFree Wifi is available throughout the conference centre. No password is required for this service simply connect to ‘Imperial Guest’ for access.

Shopping and banking faciliti esThere is a convenience store and a NatWest cash machine at the main recepti on of the Hammersmith Hospital and the Conference Centre is a 20 minute walk from Westf ield Shopping Centre, Europe’s largest inner city shopping mall.

LiabilityNeither the Organisers of the 1st Annual Meeti ng of the Larp Society, nor BioMedEx will assume any responsibility whatsoever for damage or injury to persons or property during the conference. Parti cipants are advised to arrange their own personal travel and health insurance.

Locati on Map

Venue Faciliti es

Bus Stop

Meals and Refreshments

Lunches and refreshments will be served in The Lounge, at the following ti mes:

Thursday 2nd SeptemberRefreshments 09:00 – 10:00

Refreshments 11:25 – 11:50

Lunch 13:00 – 14:30

Refreshments 15:40 – 16:30

Friday 3rd SeptemberRefreshments 11:00 – 11:30

Lunch 12:05 – 14:00

Social Programme

Wednesday 1st September 19:30-22:30 Pre-conference Dinner at The Grove Bar & Restaurant 83 Hammersmith Grove, London W6 0NQ www.groverestaurants.co.uk

Thursday 2nd September 19:30 – 22:30 Conference Dinner at Los Molinos Restaurant 127 Shepherds Bush Road, Hammersmith, London W6 7LP www.losmolinosuk.com

6 7

Lost Property Enquiries about lost property should be made to the Excess Baggage Company on: +44 (0)20 8745 7727. If property has been left on an aircraft , please contact the relevant airline, tour operator or handling agent.

Taxis from Heathrow AirportLondon black taxis stop at the taxi ranks outside all Heathrow terminals. The journey to central London costs between £50 and £55 and usually takes between 30 minutes and one hour. Journeys which are both inside London and within 20 miles of the airport are priced using the taxi meter or a fi xed price can be agreed.

Addison Lee is London’s largest licenced minicab service, off ering a fi xed-price service to and from any desti nati on. All private hire minicabs must be pre-booked online or by telephone. Drivers will meet you from your fl ight in the arrivals area.

Taxi Services Addison Lee 0844 55 60 747 (UK) or +44 203 214 5149 (From outside the UK)

Public Transport from Heathrow AirportFollow signs to Underground and take the Piccadilly line to ’Hammersmith’, exit the Piccadilly line and follow signs for Hammersmith Bus Stati on. The 72 Bus stops outside the Main Entrance to Hammersmith Hospital.

By Public Transport:Please visit www.tf l.gov.uk for best directi ons from your locati on to W12 Conference Centre.

Bus: The 72 and 272 bus routes stop directly outside the main entrance to Hammersmith Hospital. Both buses leave from Hammersmith Broadway bus stati on (next to the Holiday Inn Hotel) approximately every 8 minutes and the journey ti me takes approximately 25 minutes. On arrival, turn left and walk along Du Cane Road, passed Queen Charlott e and Chelsea Maternity Hospital (QCCH) and at the South West Gate Entrance turn right into Arti llery Lane, turn fi rst right again, then fi rst left turning into the court yard in front of the W12 Conferences Building.

Underground: Alternati vely, the centre is situated on the central line underground; the nearest tube stati ons are White City and East Acton both are within a 10 minute walk.

ParkingBecause of shortage of space, delegates are not able to park in the hospital grounds. You may park in the Wormwood Scrubs car park at the rear of the hospital, next to the Linford Christi e Stadium via Arti llery Lane. Arti llery Lane is situated on the right-hand side past the hospital if approaching from White City and situated on the left -hand side past Wormwood Scrubs if approaching from East Acton.

The car park is operated by the local council and uses a pay and display system, currently being charged at £1.80 per hour.

By AirThe closest airport to the Centre is Heathrow Airport, which is situated approx 14 miles away. You can travel between the airport and the Centre by the underground, bus or taxi.

Heathrow Airport Limited The Compass Centre, Nelson Road, Hounslow, Middlesex, TW6 2GW

T: +44 (0)844 335 1801www.heathrowairport.com

Transport

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A special rate for delegates of the 1st Annual Meeti ng of the LARP Society has been arranged at the Holiday Inn Express London Hammersmith.

Holiday Inn Express London Hammersmith124 King Street, Hammersmith, London W6 0QU, UKReservati ons: T - +44 (0) 208 746 5100 E - reservati ons@expresshammersmith.co.uk www.expresshammersmith.co.uk

Locati on:www.expresshammersmith.co.uk/Holiday-Inn-Express-Hammersmith-Our-Locati on/our-locati on.html

Holiday Inn Express London Hammersmith is located just off King’s Street in the bustling Hammersmith district in West London (entrance opposite Hammersmith and Fulham Council Offi ces and beside The Plough and Harrow Pub). The hotel is within Zone 2 of the Underground network, with the Tube stati on just a 3 minute walk from the hotel, allowing easy access to both central London and Heathrow Airport. The Holiday Inn Express is also within two miles of the Westf ield Shopping centre & exhibiti on halls of Earl’s Court and Olympia and within walking distance to the Hammersmith Apollo theatre. The hotel is fully air conditi oned and off ers modern accommodati on. Rates also include complimentary hot buff et and conti nental breakfast.

Internet Access: The hotel off ers 24 hour internet access in each hotel room at a charge of £12. Alternati vely free WiFi internet access (for up to 6 hours) is available in the restaurant area on the ground fl oor. Internet vouchers, password and login details can be obtained from the recepti on desk.

The Holiday Inn Express London Hammersmith is located 3 miles away from the W12 Conference Centre at the Hammersmith campus of Imperial College. The hotel is within easy commuti ng distance to the conference centre via

• Number 72 bus from the Hammersmith Broadway Bus stati on (buses leave approximately every 8 minutes, journey ti me 25 minutes)

• The Hammersmith and City / Circle and Central underground lines (Hammersmith Stati on, to White City Stati on - approximately 12-15 minutes commute)

Please refer to www.tf l.gov.uk/getti ngaround/default.aspx

Phylogeneti c relati onship and structural organizati ons among LA-Moti f containing proteins(adapted from Bousquet-Antonelli and Deragon, (2009) RNA; Bayfi eld et al. (2010) BBA)

La-moti f proteins fall into 5 evoluti onary conserved families. Members of the same family share conserved domain organizati on. As for the genuine LA proteins, the LAM-RRM organizati on is conserved in every LA-Related Protein families. With the excepti on of the LARP7 family which has a canonical RRM1, other LARP families display a variant RRM named: RRM-L3a/3b; RRM-L4 and RRM-L5. In each family, the most commonly represented domain organizati on is highlighted (black arrow).

Accommodati on LA - moti f containing proteins

ProtistaFungiPlantsAnimals

ProtistaAnimals

ProtistaPlantsAnimals

ProtistaAnimals

ProtistaFungiPlantsAnimals

0.98

0.86

0.98 LARP1

0.99 LARP4

0.88LARP6

0.90 LARP7

0.97 Genuine LA

Subfamily Membersin

Structural organizations

LAM RRM-L4

PAM2/PAM2v

LAM RRM-L5

LAM RRM-L5 DM15

LAM DM15

65%

LAM RRM-L3a

LSA

LAM RRM-L3b

80%

LAM RRM1 RRM2

LAM RRM1

80%

LAM RRM1 RRM2

LAM RRM1

80%

10 11

9:00 – 10:00 Registrati on and Refreshments (The Lounge)

10:00 – 10:15 Welcome (Maple & Ash Room)

Session 1: An overview of the LARP family of proteins

10:15 – 10:50 The La Module: variati ons on the theme (S01) Dr Sasi Conte King’s College London, UK

10:50 – 11:25 The evoluti onary history of LAM-containing proteins (S02) Professor Jean-Marc Deragon Université de Perpignan, France

11:25 – 11:50 Refreshments (The Lounge)

11:50 – 12:25 Looking backwards and forwards at the structural biology of the La Protein (S03)Professor Stephen Curry Imperial College, London, UK

12:25 – 13:00 A functi onal overview of the LARP family of proteins (S04) Dr Richard Maraia NIH, Bethesda, USA

13:00 – 14:30 Lunch and Poster Session (The Lounge)

Plenary 1

14:30 – 15:05 7SK RNA, a non-coding RNA that controls elongati on of transcripti on elongati on (S05) Professor Olivier Bensaude IBENS, France

Session 2: The functi on of individual LARPs

The LARP7-p65 family

15:05 – 15:40 The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and aff ects transcripti on of cellular and viral polymerase II genes (S06)Professor Dr Utz Fischer Unviersity of Würzburg, Germay

15:40 – 16:30 Refreshments and Poster Session (The Lounge)

16:30 – 18:30 Discussion Session: Remit of the LARP Society

19:30 Dinner at “Los Molinos”

Programme: Day 1 Thursday 2nd September 2010

12 13

Plenary 2

09:00 – 09:35 Regulati on of translati on under conditi ons of cell stress (S07) Professor Anne Willis Leicester University, UK

Session 2: The functi on of individual LARPs (conti nued)

The LARP1 family

09:35 – 10:10 Larp1: a role in EMT and cancer progression? (S08) Dr Sarah Blagden Imperial College, London, UK

10:10 – 10:45 The La-Moti f containing proteins of the model plant Arabidopsis thaliana: functi onal study of the AtLARP1a protein (S09) Dr Cécile Bousquet-Antonelli CNRS / Université de Perpignan, France

10:45 – 11:00 Loss of LARP1 results in multi ple phenotypes of spermatogenesisin Drosophila (P01) Dr Yoshihiro Inoue,Kyoto Insti tute of Technology, Japan

11:00 – 11:30 Refreshments (The Lounge)

The LARP6 family

11:30-12:05 Binding of LARP6 to the 5’ stem-loop of collagen mRNAs and regulati on of translati on (S10) Dr Branko Stefanovic College of Medicine, Florida State University, USA

12:05 – 14:00 Lunch and Poster Session (The Lounge)

13:30 – 14:00 LARP Society – Offi cers’ Meeti ng (Maple & Ash Room)

The LARP4 family

14:00 – 14:35 A sti mulatory role for the la-related protein 4B in translati on (S11) Professor Dr Utz Fischer University of Würzburg, Germany

14:35 – 15:10 La-related protein-4 (LARP4) binds poly (A) and interacts with PABP on polysomes to promote gene expression by stabilizing mRNA (S12)Dr Richard Maraia NIH, Bethesda, USA

15:10 Close of meeti ng

15:30 Departure

Programme: Day 2 Friday 3rd September 2010

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S01The La Module: variati ons on the themeDr Sasi (Maria R) Conte¹, Dr Luigi Marti no¹, Dr Simon Pennell², Dr Olga Koti k-Kogan³, Professor Stephen Curry³, Dr Geoff Kelly²

¹ King’s College London, ² Nati onal Insti tute for Medical Research, London, ³ Imperial College, London

The specifi c interacti on between the genuine La protein and 3’ polyU RNA is achieved via the extremely well conserved La moti f (LM), in synergy with the immediately following RNA recogniti on moti f (RRM1), with a mechanism characterised in our lab by NMR and X-ray crystallography. This RNA binding unit, named the ‘La module’, is also found in LARPs, although for these proteins the determinants for RNA binding affi nity and specifi city have just started to be investi gated. Preliminary data on the structure and RNA recogniti on properti es of the La module of LARPs will be presented and discussed. Furthermore, the ability of the La module to work together with other domain/sequences within the proteins will also be described.

S02The evoluti onary history of LAM-containing proteinsProfessor Jean-Marc Deragon, Dr Cecile Bousquet-AntonelliUniversité de Perpignan / CNRS, France

The extremely well conserved La-Moti f (LAM), in synergy with the immediately following RNA Recogniti on Moti f (RRM), allows direct binding of the (genuine) La autoanti gen to RNA polymerase III primary transcripts. This moti f is not only found on La homologues, but also on La-Reglated Proteins (LARPs) of unrelated functi ons. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA binding proteins fulfi lling crucial cellular functi ons. We recently published a comprehensive evoluti onary analysis of the LAM-containing protein superfamily that allowed the defi niti on of fi ve families: a “genuine” La family and four LARP families (LARP1, LARP4, LARP6 and LARP7). Recently, we performed a more detailed analysis of the evoluti onary history of LARP4, LARP6 and LARP7 families throughout eukaryotes. I will summarize in my presentati on, the data obtained from this new analysis and how this informati on can be used to design structure / functi on experiments.

Speakers’ Abstracts

16 17

S03Looking backwards and forwards at the structural biology of the La ProteinProfessor Stephen Curry¹, Dr Olga Koti k-Kogan¹, Dr Sasi (Maria R) Conte²

¹Imperial College, London, ² King’s College, London

Originally identi fi ed as the Lupus Autoanti gen in the 1980s, the eukaryoti c La protein has emerged as an important player in the maturati on pathway of non-coding RNAs. Through its N-terminal domains, La specifi cally recognizes the poly(U) sequences found at the 3´-end of nascent RNA transcripts. However, at least in the case of pre-tRNAs, the protein makes additi onal contacts with the nucleic acid. In doing so, La protects RNA transcripts from exonuclease acti vity and assists in folding and maturati on.

Structural analyses using X-ray crystallography and Nuclear Magneti c Resonance were essenti al in resolving the domain structure of the La protein. More recently, structural work has revealed the precise mode of binding of 3´-oligo(U) sequences by the concerted acti on of the La moti f and RRM1, which together comprise a functi onal unit within the N-terminal porti on of the protein that we have dubbed the ‘La module’. This module displays surprising plasti city in its interacti ons with RNA 3´-ends and is an important conserved component of La-related proteins (LARPs).

We will review the current state of structural analysis of La and look forward to the most immediate remaining challenges in examining the structural biology of this important and fascinati ng protein.

S04A functi onal overview of the LARP family of proteinsDr Richard J. Maraia

Intramural Research Program on Genomics of Diff erenti ati on, Eunice Kennedy Shriver Nati onal Insti tute of Child Health and Human Development, Nati onal Insti tutes of Health, Bethesda, MD USA

Genuine La proteins bind a large variety of RNAs and exhibit acti viti es in two broad categories: the intranuclear maturati on of nascent pol III transcripts and some other snRNAs by binding to their common UUU-3’OH ends, and the translati on of an assortment of mRNAs, most notably those with internal ribosome entry sites (IRES) or other control elements. Recent phylogeneti c sequence analyses indicate La-related protein families, LARPs 1, 4, 6 & 7, with LARP7 most closely related to La protein and LARP4 most distant. While genuine La binds the full spectrum of pol III transcripts, LARP7 is highly specifi c for the pol III transcript, 7SK snRNA, which controls the pol II transcripti on acti vity of P-TEFb. Intricate use of adjacent RNA binding moti fs by La protein has been shown to provide RNA chaperone-like acti vity that also appears to contribute to a functi on of some LARP7 members.

Although it might have been predicted that the other LARPs would bind a specifi c pol III transcript, this has not been found. LARPs 1, 4 & 6 appear to be specialized for cytoplasmic mRNA-related functi ons, which will be reviewed. Recent data suggest that LARPs 6 & 4 are diverged in RNA binding acti vity. Thus it appears that while genuine La proteins exhibit broad involvement in snRNA-related and mRNA-related functi ons, diff erent LARP families may have evolved acti viti es in either snRNA or mRNA related functi ons. Where possible, emphasis will be placed on RNP assembly and the possible contributi on of RNA chaperone-like acti viti es for some of the LARPs.

Speakers’ Abstracts

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S057SK RNA, a non-coding RNA that controls elongati on of transcripti on elongati on Professor Olivier Bensaude, Gaelle Diribarne, Nina Verstraete

IBENS - CNRS UMR 8197

In metazoans, transcripti on is oft en controlled at the transiti on between initi ati on and elongati on of transcripti on steps. Negati ve elongati on factors (NELF and DSIF) are responsible for RNA polymerase II pausing aft er initi ati on. Positi ve transcripti on factor acti vity (P-TEFb) is then required to resume transcripti on. P-TEFb consists in a kinase (CDK9) associated with a Cyclin (T1 or T2). It phosphorylates NELF, DSIF as well as the repeats in the C-terminal domain (CTD) of RNA polymerase II.

P-TEFb acti vity is inhibited by the HEXIM (1 and 2) proteins bound to 7SK snRNP. This snRNP comprises 7SK RNA, a conserved class III transcript (331 nt in humans) that is monomethylated at its 5’ end by a specifi c capping enzyme MePCE and is protected at its 3’ end by a specifi c La-related protein, LARP7. The 7SK snRNP also binds heterogenous nuclear ribonucleoproteins (hnRNP). When transcripti on is arrested, hnRNPs are released from pre-mRNAs and trap the 7SK snRNP which is no more available for HEXIM binding and hence P-TEFb inhibiti on. Truncated LARP7 variants are found in some tumours that contain low levels of 7SK RNA. The resulti ng high P-TEFb acti vity might contribute to tumorigenesis.

S06The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and aff ects transcripti on of cellular and viral polymerase II genesProfessor Utz Fischer¹, Dr Michael Grimm¹, Dr Andreas Markert¹, Javier Marti nez², Julia Wiesner¹, Professor Andreas Meyerhans², Professor Oded Meyuhas³, Professor Albert Sickmann¹

¹University of Würzburg, Germany, ² University of Saarland, Germany, ³ University of Jerusalem, Israel

The positi ve transcripti on elongati on factor b (P-TEFb) is a heterodimeric complex composed of cyclin-dependent kinase 9 and its regulator cyclin T1/2. It sti mulates transcripti on elongati on by phosphorylati on of serine 2 residues in the carboxy-terminal domain of polymerase II. 7SK RNA and HEXIM proteins can antagonize transcripti onal sti mulati on by sequestering P-TEFb in a catalyti cally inacti ve ribonucleoprotein (RNP). We will present data indicati ng that the previously uncharacterized La-related protein 7 (LARP7) has a role in 7SK-mediated regulati on of transcripti on. LARP7 binds to the highly conserved 3’-terminal U-rich stretch of 7SK RNA and is an integral part of the 7SK RNP. On sti mulati on, LARP7 remains associated with 7SK RNA, whereas P-TEFb is released. Interesti ngly, reducti on of LARP7 by RNA interference enhances transcripti on from cellular polymerase II promoters, as well as a TAT-dependent HIV-1 promoter. Thus, LARP7 is a negati ve transcripti onal regulator of polymerase II genes, acti ng by means of the 7SK RNP system.

Speakers’ Abstracts

20 21

S07Regulati on of translati on under conditi ons of cell stressProfessor Anne Willis, Laura Cobbold, Helen King, Kirsty Sawicka, Amandine Basti de, Ruth Spriggs, Tim Gant, Marti n Bushell

MRC Toxicology Unit, Leicester

Under conditi ons of patho-physiological cell stress post transcripti onal regulati on of gene expression is achieved by re-programming protein synthesis. Following exposure of cells to nutrient stress, temperature shock, DNA damage and under hypoxic conditi ons there is a general shutdown of protein synthesis. However, under each of these situati ons there is selecti ve recruitment, to the ribosomes, of mRNAs whose protein products are required as part of the stress response and this recruitment is regulated by elements within their 5’ and 3’ untranslated regions. These elements include internal ribosome entry segments, upstream open reading frames and microRNA target sites. These can act either singly or in combinati on and are themselves regulated by trans-acti ng factors. Inducti on of stress pathways that lead to translati onal re-programming can result in increases in lifespan, and conversely deregulati on of these processes is associated with disease including cancer.

S08LARP1: a role in EMT and cancer progression?Dr Sarah Blagden, Dr Manuela Mura, Miss Normala Abd-Lati p, Miss Theodora Michael, Mr Xun Yu Choong, Dr Sarah-Jane Lam, Dr Francesco Mauri, Dr Charles Jameson, Miss Helen McDill

Imperial College, London

Human LARP1 is an RNA-binding protein required for translati on initi ati on and the synthesis of 15% proteins overall. LARP1 interacts with PABP and exists in complexes with eIF4E and cytoskeletal proteins such as acti n and myosin. Here we show that LARP1 is required to enable cells to transiti on from an epithelial to mesenchymal-like morphology, a process that equips the cell with invasive and migratory properti es. In cervical cancer, levels of cytoplasmic LARP1 correlate with invasiveness of tumour and, in ovarian cancer, higher LARP1 is associated with increased plati num resistance and poorer overall survival. This suggests that, through its role in epithelial-mesenchymal transiti on, LARP1 is involved in cancer progression and chemotherapy resistance.

Speakers’ Abstracts

22 23

S09The La-Moti f containing proteins of the model plant Arabidopsis thaliana: functi onal study of the AtLARP1a proteinDr Cécile Bousquet-Antonelli, Miss Julie Descombin, Professor Jean-Marc Deragon

Université de Perpignan via Domiti a / CNRS, France

A. thaliana encodes eight La-Moti f containing proteins: two of which (AtLa1 and AtLa2), display evoluti ve and conserved domains features of genuine La proteins. This duplicity is extremely unusual and consistently we demonstrated that only the AtLa1 protein is a functi onal La autoanti gen; the functi onality of AtLa2 remaining uncertain so far.

Arabidopsis LARP proteins are from the LARP1 or 6 families. All three AtLARP6 proteins display canonical features of this sub-family with the LAM-RRML3a and C-terminal LSA conserved domains. In additi on AtLARP6b and c but not AtLARP6a, bear a PABP interacti ng PAM2 moti f at their NH2 extremity. Microarray data suggest that AtLARP6a is ubiquitously expressed in plant ti ssues and at diff erent developmental stages but with striking overaccumulati on in mature pollen. AtLARP6b and c mRNAs profi les are opposite with 6b present in most but male reproducti ve ti ssues where 6c is solely accumulati ng.

The AtLARP1 cluster has three members but only AtLARP1a, has the RRM-L5 and DM15 canonical boxes. We showed that AtLARP1a protein accumulates ubiquitously throughout development except for embryos and mature seeds. A yeast two-hybrid screen suggests that AtLARP1 interacts with several proteins involved in mRNA metabolism regulati on such as AtXRN4 (XRN1) and the poly(A) Binding protein (AtPAB2). Consistently, upon stress, AtLARP1a massively aggregates to stress granules where it co-localizes with AtPAB2 and several of its yeast two-hybrid partners. Preliminary results suggest that in vivo AtLARP1-AtPAB2 interacti on only takes place in stress conditi ons. Our present working model is that AtLARP1 might be required for AtPAB2 addressing and / or maintenance in stress granules.

Speakers’ Abstracts

P01A loss of LARP1 results in multi ple phenotypes of spermatogenesis in DrosophilaDr Yoshihiro H. Inoue, Miss Keiko Ichihara, Mrs Hanako Shimizu

Kyoto Insti tute of Technology, Japan

We isolated novel Drosophila sterile mutati ons, meteor (mtr) that turned out to be mutati ons for larp gene encoding the LARP1 with a domain homologous to a La type RNA-binding protein. The larp mutant males exhibited multi ple meioti c phenotypes including defects in chromosome segregati on, cytokinesis and mitochondrial parti ti on. Our cytological examinati on revealed that these meioti c abnormaliti es are associated with defects in spindle pole organizati on and spindle formati on. We speculate that the LARP1 plays a direct or indirect role(s) in multi steps of spermatogenesis in Drosophila. The meioti c phenotypes of the larp are quite similar to those of mutants for poly(A) binding protein. A loss of single copy of the larp gene considerably elevated a frequency of abnormal meioti c fi gures in pAbp mutants. These geneti c data suggest a close relati onship between two gene products. 16 of spermatocytes form a cell unit and all cells within a unit perform synchronous cell cycle progression and they initi ate meioti c divisions at a same ti me. We found that spermatocytes from the larp mutants also showed a perturbati on of cell cycle progression at pre-meioti c stages. It was reported that the Drosophila LARP7 orthologue, Mxc (multi ple sex combs) plays a role in a silencing of a Hox gene through an interacti on with Pc group genes. The larp was originally identi fi ed as one of Hox target genes. We have recently obtained geneti c evidence that a larp mutati on interacts with Pc group genes. We will discuss a possible biochemical functi on of the LARP1 in Drosophila.

24 25

S11A sti mulatory role for the la-related protein 4B in translati onProfessor Utz Fischer¹, Katrin Schäffl er¹, Kristi na Schulz¹, Anja Hirmer¹, Julia Wiesner², Dr Michael Grimm¹, Professor Albert Sickmann²

¹Department of Biochemistry at the Theodor Boveri-Insti tute, University of Würzburg, Würzburg, Germany.² ISAS - Insti tute for Analyti cal Sciences, Dortmund, Germany

La-related proteins (LARPs) belong to an evoluti onarily conserved family of factors with predicted roles in RNA metabolism. In our research, we focused on investi gati ons concerning the cellular interacti ons and functi on of LARP4B, a thus far uncharacterized member of the LARP family. At this we show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interacti on of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for acti vated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditi ons, LARP4B co-sedimented with polysomes in cellular extracts, suggesti ng a role in translati on. In agreement with this noti on, over-expression of LARP4B sti mulated protein synthesis whereas knockdown of the factor by RNA-interference impaired translati on of a large number of cellular mRNAs. In summery, we identi fi ed LARP4B as a sti mulatory factor of translati on. We speculate that LARP4B exerts its functi on by bridging mRNA factors of the 3’ end with initi ati ng ribosomes.

Speakers’ Abstracts

S10Binding of LARP6 to the 5’ stem-loop of collagen mRNAs and regulati on of translati onDr Branko Stefanovic, Lela Stefanovic, Le Cai

College of Medicine, Florida State University, USA

Type I collagen is the most abundant protein in human body, composed of two α1(I) and one α2(I) polypepti des. Collagen mRNAs have a unique 5’ stem-loop (5’SL) that regulates their stability and translati on. LARP6 binds the 5’SL of collagen mRNAs with high affi nity and specifi city. LARP6 interacts with fi ve nucleoti des within the single-stranded bulge of the 5’SL; the F135 of the La domain of LARP6 and E257 of the RBD are needed for these interacti ons. Knock-down of LARP6 by siRNA or mutati on of 5’SL inhibits collagen synthesis. Cells derived from knock in mice with mutati on of the 5’SL in the endogenous collagen α1(I) gene express less collagen and develop less fi brosis. Overexpression of LARP6 blocks ribosomal loading on collagen mRNAs, suggesti ng that the protein must be ti ghtly regulated and that it must dissociate from mRNAs for translati on initi ati on. S251 in the RBD modulates the binding affi nity of LARP6, its mutati on into asparti c acid drasti cally reduces the binding, while its change into alanine has no eff ect. LARP6 interacts with its C-terminal domain with nonmuscle myosin and associates collagen mRNAs with nonmuscle myosin fi laments. When the fi laments are disrupted co-localizati on of collagen α1(I) and α2(I) polypepti des, required for assembly of the heterotrimer, is diminished and the cells failed to secrete type I collagen or secreted only homotrimers of α1(I) polypepti des. We postulate that LARP6 prevents premature translati on of collagen mRNAs and associates them with nonmuscle myosin fi laments, which coordinate translati on of the mRNAs to facilitate the folding of collagen heterotrimer.

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P02The interacti on of La protein with the loop IV of the HCV IRES domain reveals a new RNA recogniti on mechanismDr Luigi Marti no¹, Simon Spennell², Stephen Smerdon², Geoff Kelly², Olga Koti k-Kogan³, Stephen Curry³, Maria R Conte¹

¹King’s College London, ²Nati onal Insti tute for Medical Research, London, ³Imperial College, London

The human La protein is required for the correct processing, maturati on, cellular localisati on and folding of nascent RNA Pol III transcripts. La binds specifi cally to the 3’ poly (U) ends characteristi c of most Pol III precursors and this associati on has been shown to prevent their degradati on. La is a monomeric modular protein containing a La moti f and two RNA recogniti on moti fs, RRM1 and RRM2. The so-called ‘La module’, containing the La moti f and the RRM1, is highly conserved across species and it contains the determinants for 3’ poly (U) RNA binding. The C-terminal domain varies greatly between diff erent eukaryotes; it is involved in nuclear retenti on but its role is not fully understood.

La is also reported to bind to several viral RNAs in the cytoplasm, enhancing their translati on and replicati on effi ciency. One of the most prominent examples is the biding to the Hepati ti s C Virus (HCV), genome on the stem-loop IV of the IRES domain (Internal Ribosomal Entry Site). The latt er RNA target possesses very diff erent structural characteristi cs from Pol III precursors, emphasising the remarkable versati lity of the La protein in RNA recogniti on.

Here, we summarise the results from our characterisati on of the interacti on between the La protein and the loop IV of the HCV IRES domain. Using a variety of biophysical techniques we have shown a strong RNA interacti on is only achieved by deleti on mutants containing the La moti f, RRM1 and RRM2. This study provides the fi rst evidence of a new RNA recogniti on mechanism by La that also involves the RRM2.

Poster Abstracts

S12La-related protein-4 (LARP4) binds poly(A) and interacts with PABP on polysomes to promote gene expression by stabilizing mRNARichard J. Maraia1,3, Ruiqing Yang1, Sergei A. Gaidamakov1, Amanda K. Crawford1, Sridar Chitt ur2, Frank Doyle2, Scott A. Tenenbaum2

1Intramural Research Program on Genomics of Diff erenti ati on, Eunice Kennedy Shriver Nati onal Insti tute of Child Health and Human Development, Nati onal Insti tutes of Health, Bethesda, MD USA, 2University at Albany-SUNY, Rensselaer, NY, USA 3Commissioned Corps, U.S. Public Health Service, USA

Conserved La proteins contain a La domain comprised of two separate RNA binding moti fs uniquely arranged to bind UUU-3’OH and stabilize RNAs from 3’ decay. Of four recently disti nguished La-related protein (LARP) sequence families, LARP4 has a most diverged RNA binding pocket. We show that the La and LARP4 La domains exhibit multi ple qualitati ve diff erences in RNA binding, including sequence specifi city. LARP4 binds poly(A) via its La domain and interacts with poly(A) binding protein (PABP) via a variant PABP interacti on moti f-2 (vPAM2), and both contribute to associati on with polyribosomes. Consistent with this, LARP4 binds a large number of mRNAs. Yeast two-hybrid screening revealed that LARP4 also interacts with RACK1, a ribosome- and mRNA-associated protein, and the interacti on was confi rmed by reciprocal immunoprecipitati ons from HeLa cells. We show that LARP4 promotes gene expression by stabilizing mRNA. LARP4 acti vity correlates with its ability to interact with PABP and polysomes. The data indicate that LARP4 is a factor involved in the metabolism of poly-A mRNAs that are used for translati on.

Speakers’ Abstracts

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P03Similariti es and diff erences of the RNA recogniti on mechanisms by LARPs: a preliminary studyDr Luigi Marti no¹, Reneè Tata¹, Paul Brown¹, Stephen Curry², Maria R Conte¹

¹King’s College London, ²Imperial College, London

The La-related proteins (LARPs) family is divided into fi ve major groups: genuine La, LARP 1, LARP4, LARP6 and LARP7. LARPs have important roles in diff erent aspects of RNA metabolism, including transcripti onal regulati on, mRNA stabilisati on and translati on. They are characterised by a conserved two-domain RNA-binding module, termed the “La module” that was fi rstly identi fi ed in the genuine La protein. The La module contains a La moti f and an RRM domain working in synergy to recognize RNA targets. Though investi gati ons are in their infancy, it is already clear that the RNA ligands recognised by LARPs diff er in sequence and shape, which are likely to be related to structural diff erences between the LARPs.

The Conte lab is focusing its eff orts on the structural characterisati on of the human LARPs and the study of their interacti ons with diff erent RNA targets. Combining together several biophysical techniques such as calorimetry, NMR and CD spectroscopy, we have been able to collect exciti ng preliminary results towards the molecular understanding of the adaptability of the La module in RNA recogniti on.

P04Interacti on of 7SK with LARP7Dr Anne-Catherine Dock-Bregeon¹, Emiko Uchikawa¹, Denise Martyinez-Zapien¹, Alexandre Durand¹

IGBMC, Illkirch, France

Human 7SK RNA functi ons as a repressor of transcripti on elongati on1;2. 7SK binds protein HEXIM1 (or HEXIM2), and the resulti ng complex sequester the Positi ve Elongati on Factor PTEFb (comprised of Cyclin T1 or T2 and CDK9) in an inacti ve form. On another hand, when not involved in PTEFb repression, 7SK has been shown to be bound by hnRNPs3-5. It seems that the minimal 7SK snRNP contains LARP76-8, a La-related protein and MePCE9, a specifi c capping enzyme. These proteins maintain the stability of 7SK, and help recruitment of the other partners in the regulati on mechanism10.

In vivo studies with deleti on mutants of 7SK indicated that LARP7 binds the U-rich sequence at the 3’end of 7SK6. In order to understand the basis of the specifi c recogniti on of 7SK by LARP7 we use a multi ple approach, combining biochemical (EMSA, gel permeati on) and biophysical (fl uorescence, mass spectrometry) methods. Our fi rst results will be presented.

1. V. T. Nguyen, et al. Nature 414, 322-325 (2001).

2. Z. Yang, et al, Nature 414, 317-322 (2001).

3. E. Van Herreweghe et al., EMBO J. 26, 3570-3580 (2007).

4. C. Barrandon, et al, Mol.Cell Biol. 27, 6996-7006 (2007).

5. J. R. Hogg and K. Collins, RNA. 13, 868-880 (2007).

6. N. He et al., Mol.Cell 29, 588-599 (2008).

7. B. J. Krueger et al., Nucleic Acids Res. 36, 2219-2229 (2008).

8. A. Markert et al., EMBO Rep. 9, 569-575 (2008).

9. C. Jeronimo et al., Mol.Cell 27, 262-274 (2007).

10. Y. Xue, et al Nucleic Acids Res. 38, 360-369 (2010).

Poster Abstracts

30 31

P05 Parti cipati on of ZBP1 in cytoplasmic Y-RNPsMarcel Köhn¹, Stefan Hütt elmaier¹

¹Marti n-Luther-University, Halle, Germany

The RNA-binding protein ZBP1 (Zipcode Binding Protein 1) is a member of the oncofetal VICKZ-(Vg1RBP, IMP-1,2,3, CRD-BP, KOC, ZBP1)-protein family. Proteins of the VICKZ family are essenti al for the control of turnover or translati on of specifi c target RNAs. They exhibit a conserved domain structure consisti ng of two N-terminal RRM-(RNA recogniti on moti ve)-domains and four C-terminal KH-(hnRNP K-homology)-domains. At steady state all members of the VICKZ family show a mainly cytoplasmic localizati on although they are capable to shutt le between nucleus and cytoplasm.

Some of the proteins identi fi ed to associate with VICKZ proteins (Ro60, PTB, hnRNP K) bind small non coding RNAs termed Y-RNAs. So far four diff erent Y-RNAs (Y1, Y3, Y4, Y5) have been identi fi ed in humans. Therefore we analyzed, if ZBP1 like its ligands is capable to bind these RNAs as well. To investi gate associati on of ZBP1 with Y-RNAs we used randomly NIR-dye labeled RNA probes. In vitro studies identi fi ed Y3 and Y1 as potenti al new target RNAs of ZBP1. Whereas binding of ZBP1 to the β-acti n zipcode (mRNA target of ZBP1) and Y3 was exclusive, the protein formed a trimeric complex with the La-protein and Y3. This was dissociated in the presence of Y5 RNA resulti ng in the formati on of disti nct ZBP1/Y3 and La/Y5 complexes. These fi ndings suggest that in cells, ZBP1 is comprised in at least two disti nct RNPs, mRNPs containing mRNAs or yRNPs comprising Y-RNAs. The compositi on and cellular localizati on of ZBP1 containing yRNPs is now under investi gati on.

Poster Abstracts Delegate List

Name Insti tuti on EmailAbd Lati p, Normala Imperial College, London n.abd-lati p07@ic.ac.ukBousquet-Antonelli, C. Université de Perpignan, cecile.antonelli@univ-perp.frBayfi eld, Mark York University, Toronto bayfi eld@yorku.caBensaude, Olivier IBENS, Paris, France bensaude@biologie.ens.fr Blagden, Sarah Imperial College, London s.blagden@imperial.ac.ukBrown, Paul Kings College London paul.brown@kcl.ac.ukConte, Sasi (Maria R) King’s College London sasi.conte@kcl.ac.ukCummaro, Annunziata King’s College London nunziacummaro@libero.itCurry, Stephen Imperial College, London s.curry@imperial.ac.ukDeragon, Jean-Marc Université de Perpignan jean-marc.deragon@univ-perp.frDock-Bregeon, A.C. IGBMC, Illkirch, France dock@igbmc.fr Fischer, Utz University of Würzburg utz.fi scher@biozentrum.uni-wuerzburg.de Inoue, Yoshihiro Kyoto Insti tute of Technology yhinoue@kit.ac.jpKöhn, Marcel Marti -Luther-University, Halle marcel.koehn@medizin.uni-halle.deMaraia, Richard NIH, Bethesda, USA maraiar@mail.nih.govMarti no, Luigi King’s College London luigi.marti no@kcl.ac.ukMura, Manuela Imperial College, London manuela.mura1@imperial.ac.uk Pelz, Jann-Patrick University of Würzburg jann-patrick.pelz@stud-mail.uni-wuerzburg.deSchäffl er, Katrin University of Würzburg katrin.schaeffl er@biozentrum.uni-wuerzburg.de Stefanovic, Branko Florida State University, USA branko.stefanovic@med.fsu.eduStefanovic, Lela Florida State University, USA lela.stefanovic@med.fsu.eduWillis, Anne MRC Toxicology, Leicester aew5@le.ac.uk

Author Index

Name Abstract Number Page Number Bousquet-Antonelli, C. S02, S09 15, 22Bensaude, Olivier S05 18Blagden, Sarah S08 21Brown, Paul P03 28Conte, Sasi (Maria R) S01, S03, P02, P03 14, 16, 27, 28Curry, Stephen S01, S03, P02, P03 14, 16, 27, 28Deragon, Jean-Marc S02, S09 15, 22Dock-Bregeon, A.C. P04 29Fischer, Utz S06, S11 19, 25Inoue Yoshihiro P01 23Köhn, Marcel P05 30Maraia, Richard S04, S12 17, 26Marti no, Luigi S01, P02, P03 14, 27, 28Schäffl er, Katrin S11 25Stefanovic, Branko S10 24Stefanovic, Lela S10 24Willis, Anne S07 20

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