RNA regulons in Hox 5 UTRs confer ribosome specificity to gene regula=on

RNA regulons in Hox 5 UTRs confer ribosome specificity to gene regula=on Shifeng Xue, Siqi Tian, Kotaro Fujii, Wipapat Kladwang, Rhiju Das & Maria Barna Nature, Volume 157, Pages 33-38 (1 st of January 2015) 12 th of may 2015 Presented by Pascal Gasser Mus Musculus, Moisés Mallo, and Claudio R. Alonso Development, 2013

Ribosome- mediated transla=onal specificity Ribosomal Protein Expression during Organogenesis Anterior Posterior Summary I Ribosomal proteins show Gssue- specific expression MutaGons in ribosomal protein Rpl38 display palerning defects RPL38 regulates the translagon of Hox mrnas Kondrashov et al., Cell 2011 Copyright 2011 Elsevier Inc

A) How is this Ribosome- mediated transla=onal specificity in certain Hox genes regulated? Cap- dependent ini=a=on vs. Cap- independent ini=a=on DefiniEon...an IRES can be defined as a RNA element or (...) capable of recruigng a ribosome... Sunnie R. Thompson, WIREs RNA 2012

Func=onal assay to evaluate the transla=onal ini=a=on process Bicistronic reporter assay RPL38- Hox mrnas (Firefly luciferase) (Renilla luciferase) RPL38- regulated Hox mrnas possess IRES elements Controls: i) RNA levels of reporter ii) short hairpin RNA (shrna) against Rluc iii) no crypgc splice sites and no ribosome read- through

B) Are the cap- independent transla=on controlled from the Hox 5 UTR by RPL38? Eengineering of TALEN (TranscripGon acgvator- like effector nucleases) Disruption of Rpl38 in C3H10T1/2 Conclusion IRES- dependent translagon of Hox target mrnas that are regulated by RPL38 Sanjana et al., Nature Protocols 2012

C) Are there addi=onal element (s) in the Hoxa9 5 UTR? Monoscistronic Reporter assay Hox9a 5 UTRs are cloned into pgl3 vector à transfected in C3H10T1/2 cells (capped) Conclusion nt 1 342 of the Hoxa9 5 UTR strongly inhibit cap- dependent translagon à 5 UTR mrna element the translaeon inhibitory element (TIE).

D) Does allow the 5 UTR topology specialized transla=on? β- globin (Hbb) 5 UTR Conclusion 5 UTR topology of both a TIE close to the 5 end of the mrna and an IRES element is a mechanism of translagonal regulagon

Hoxa9 IRES is essen=al for transla=on in vivo Conclusion HomeoGc transformagon of thoracic vertebra to the first lumbar vertebrate Hoxa9 IRES/ΔIRES embryos do not show any major change in Hoxa9 transcript boundaries or expression levels

Take- home message RNA regulons in Hox 5 UTR confer ribosome specificity to gene regula=on () Bicistronic Reporters assay offers a tool to evaluate translagonal inigagon TALEN: tool for specific delegons of genes- sequences Ribosomal protein L38 (not essengal in yeast) facilitate specifically translagon inigagon of a subset of Hox mrna (i.e. HoxA Cluster) The combina=on of TIEs and IRES mogfs seem to impede scanning by the PIC (Pre- inigagon complex) during mammalian development Dinman., Nature News & Views 2015

QUESTIONS?? Crystal Structure of the EukaryoGc 60S Ribosomal Subunit (Klinge et al., Science, 2011)

Model for how ribosome- mediated regula=on of gene expression encoded within the mrna sequence fail- safe mechanism of IRES elements stress condigons, e.g. apoptosis, hypoxia à Down regulagons of cap- dependent translagon Cis- ac>ng regulons in 5 UTR Specific L38 ribosomal proteins à TIE inhibits cap- dependent translagon

SHAPE and mutate- and map analysis Conclusion The Hoxa9 IRES is an RNA structure that facilitates recruitment of the ribosome and is required for normal translagon

RPL38 Controls 80S Complex Forma=on on Selec=ve Hox mrnas from the Ribosome Kondrashov et al., Cell 2011 Copyright 2011 Elsevier Inc

Transcrip=on ac=vator- like effector nucleases (TALENs), Sanjana et al., Nature Protocols 2012

B) Are the cap- independent transla=on controlled from the Hox 5 UTR by RPL38? Transcrip=on ac=vator- like effector nucleases (TALENs) Cap- dependent Disruption of Rpl38 in C3H10T1/2 Cap- independent Sanjana et al., Nature Protocols 2012

Evalua=ng IRES - Ac=vity Sunnie R. Thompson, WIREs RNA 2012

Func=onal assay to evaluate the transla=onal ini=a=on process Bicistronic reporter assay Controls Controls: i) RNA levels of reporter

Func=onal assay to evaluate the transla=onal ini=a=on process Bicistronic reporter assay Controls Controls: i) RNA levels of reporter ii) short hairpin RNA (shrna) against Rluc

Func=onal assay to evaluate the transla=onal ini=a=on process Bicistronic reporter assay Controls Controls: i) RNA levels of reporter ii) short hairpin RNA (shrna) against Rluc iii) no crypgc splice sites and no ribosome read- through

Are addi=onal element (s) in the Hoxa9 5 UTR may intrinsically inhibit cap- dependent transla=on? Monoscistronic Reporter assay Hox9a 5 UTRs are cloned into pgl3 vector (all uorf ATGs are mutated to TTA) à transfected in C3H10T1/2 cells (capped) Conclusion upstream open reading frames (uorfs) do not inhibit the translagon of the main open reading frame

C) Is Hoxa9 IRES an RNA structure that recruits ribosomes? In- vitro transcripgon of Hoxa9 5 UTR Pull down ribosomal proteins Pull down rrna 18S / 28S Conclusion the 80S ribosome is able to form on the uncapped Hoxa9 5 UTR.

Conserva=on of Hoxa9 5 UTR Sequence Alignment Conclusion Minimum fragment for RPL38- dependent IRES acgvity to nucleogdes (nt) 944 1,266 (IRES- Element) High conservagon of Hoxa9- element from fish to mammals IRES - funcgon is conserved

High Conserva=on of the IRES func=on 450 ± 36 Myr Numbers, NCBI EMBO Rep. 2003 EvoluGonary relagonship adapted h_ps://cgwb.nci.nih.gov/ (Access 09 /05/ 15)

Hoxa9 IRES is essen=al for transla=on in vivo Xue et al., Nature 2015

Hoxa9 IRES Knockout in mice Nega=ve Selec=on, Phosphoglycerate Kinase 1 (PGK) promoter, Diptheria Toxin A (DTA) cassele: PGK promoter / DTA- A

Localisa=on of L38 on the surface of the ribosome Armache et al., PNAS 2010

RPS25 is essen=al for transla=on ini=a=on in Dicistroviridae and hepa=ts c viral IRES Dicistroviridae Landry et al., Genes and Development 2010 hepa=ts c viral IRES