Translational control by the RNA-binding protein CSDE1 : Insights into a stimulatory role in translation elongation

RNA-binding proteins are potent regulators of post-transcriptional gene expression. Among the repertoire of RNAs bound by a particular RNA-binding protein, networks of co-ordinately regulated RNAs, or ‘regulons’, are functionally related and their regulation in concert acts to drive cellular processes. In melanoma, the RNA-binding protein CSDE1 is highly upregulated. In this context, CSDE1 regulates the translation of mRNA targets in multiple regulons in a direction that is consistent with oncogenic progression, whereby, tumour-suppressors, such as PTEN, are downregulated and pro-metastatic factors, for example, the key epithelial-to-mesenchymal transition proteins, VIM and RAC1, are upregulated. Ribosome profiling studies indicate that CSDE1 enhances the translation of VIM and RAC1 at the level of translation elongation. Such a stimulatory role for an RNA-binding protein in translation elongation may represent a novel mechanism of translational regulation. Here, we seek to elucidate the protein partners of CSDE1 in inducing this unusual stimulatory effect on translation elongation, and to explore the relationship of CSDE1 to the translational machinery. We confirm that CSDE1 promotes the translation of RAC1 mRNA at the level of elongation, and further highlight a rapid adaptation of melanoma cells to CSDE1 depletion. We demonstrate extensive contact of CSDE1 with the translational machinery. CSDE1 is a ribosome-associated protein, interacting with the small ribosomal subunit, and is further observed to co-sediment with translating polysomes. Amongst 38 high-confidence CSDE1-interacting proteins that we identify in melanoma are 16 ribosomal proteins and a further 11 members of the ribo-interactome. Moreover, we show that CSDE1 associates to tRNAs in a manner dependent on both isodecoder and isoacceptor identity, and within these subgroups disparate patterns of CSDE1 affinity to the tRNA structure may be observed. The data lead us to propose a model whereby the stimulatory effect of CSDE1 on translation elongation may be underpinned by the binding of CSDE1 to both its target mRNAs and to tRNAs, thereby concentrating tRNAs towards the translating ribosome.Les proteïnes d’unió a l’ARN són uns potents reguladors de l’expressió gènica post-transcripcional. D’entre el conjunt d’ARN que es troben units a una proteïna d’unió a l’ARN concreta, podem distingir xarxes d’ARN que es regulen coordinadament, els regulons. Els ARN d’aquests regulons estan funcionalment relacionats i la seva regulació orquestrada promou processos cel·lulars. En el melanoma, la proteïna d’unió a l’ARN CSDE1 està altament sobreactivada. En aquest context, CSDE1 regula la traducció dels seus ARNm diana de manera consistent amb un programa de progressió oncogènica; així, el supressors tumorals com PTEN es troben regulats negativament, mentre que factors pro-metastàtics, per exemple les proteïnes claus en la transició epiteli-mesènquima VIM i RAC1, es regulen positivament. Estudis de ribosome profiling indiquen que CSDE1 promou la traducció de VIM i RAC1 a nivell de l’elongació. Aquesta funció estimulant, duta a terme per a una proteïna d’unió a l’ARN, podria representar un nou mecanisme de regulació de la traducció. En la present tesi busquem clarificar quines proteïnes acompanyen CSDE1 en la inducció de l’efecte estimulatori a nivell de l’elongació traduccional, així com explorar la implicació de CSDE1 en la maquinària traduccional. Hem confirmat que CSDE1 promou la traducció de l’ARNm de RAC1 a nivell de l’elongació, a més a més destaquem que les cèl·lules de melanoma s’adapten ràpidament a la depleció de CSDE1. Demostrem com aquesta proteïna contacta extensivament amb la maquinària traduccional, sent CSDE1 una proteïna associada al ribosoma mitjançant la subunitat petita. D’altra banda, hem observat que co-sedimenta amb polisomes que s’estan traduint. D’un total de 38 proteïnes caracteritzades amb un alt grau de confiança com a proteïnes que interaccionen amb CSDE1, 16 són proteïnes ribosomals i 11 altres són proteïnes que formen part del ribo-interactome. A més a més, CSDE1 s’associa al ARNt, un associació que depèn tant de la identitat dels iso-decodificadors com la dels iso-acceptors, tanmateix, dins d’aquests subgrups s’ha vist que l’afinitat de CSDE1 per a les diferents estructures dels ARNt segueix patrons diferenciats. Aquestes dades ens permeten proposar un model en què l’efecte estimulant de CSDE1 en l’elongació traduccional podria sostenir-se mitjançant l’unió de CSDE1 tant als ARNm diana com als ARNt, tot concentrant aquests últims cap als ribosomes que s’estan traduin

Analysis of polyQ aggregate load.

<p>(<b>A</b>) Exemplified filter retardation analysis to visualize polyQ aggregates. Decreasing amounts of loaded protein derived from fly heads of control (<i>GMR-GAL4</i>, top), <i>GMR>polyQ</i> (middle) or <i>GMR>polyQ</i> in combination with a candidate suppressor (bottom). (<b>B</b>) Densitometric measures of filter retardation analysis. Data depicted as fold change compared to control (<i>GMR>polyQ</i>) for suppressors and enhancers of polyQ-induced toxicity. Independent homogenates (if available) were used for repetitions. In case of none or only one independent repetition n≤2 is indicated. In all other cases, number of independent repetitions is n≥3. Significant changes are indicated * p<0.05; *** p<0.001.</p

Computational analysis of modifiers of polyQ-induced toxicity.

<p>(<b>A</b>) Meta-interaction network displaying modifiers of polyQ toxicity. Only candidates causing a robust modification of the REP (red) as well as directly interacting subtle modifiers (black) were retained from an initial network of more than 5 k genes with 20 k interactions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047452#pone.0047452-Costello1" target="_blank">[32]</a>. One local cluster of functionally interacting modifiers is highlighted. (<b>B</b>) Gene Ontology analysis of these candidate gene groups. Shown are -log₁₀(p-value) scores for GO term enrichment for candidate gene groups (horizontal axis, see inset for group identities) and GO term (vertical). The matrix incorporates the structure of the GO hierarchy and is based on the Topology Weighted Term-algorithm as implemented in Ontologizer (terms with a p-value<0.005 are shown).</p

List of unspecific modifiers of polyQ-induced toxicity.

<p>Table lists gene name (if applicable) and gene ID of all candidates identified to have a similar effect on polyQ- and Tau-induced REPs. Mode of modification is indicated (enhancement (E), suppression (S)). A brief summary of the molecular and biological functions assigned to the identified gene products is listed.</p

Screening for modifiers of polyQ-induced toxicity.

<p>(<b>A</b>) Rough eye phenotype (REP) used as a primary readout for screening. Compared to control (upper panels), eye-specific (<i>GMR-GAL4</i>) expression of polyQ (lower panels) induces disturbances of the external eye texture, e. g. depigmentation of the compound eye observed by light microscopy (left) and as depicted in scanning electron micrographs (middle). Toluidine blue-stained semi-thin eye sections reveal that the disturbance of external eye structures is accompanied by degeneration of retinal cells (right). (<b>B</b>) Modification of the polyQ-induced REP by enhancers and suppressors. VDRC transformants used to silence respective genes: <i>CG3284</i> (11219), <i>CG16807</i> (23843), <i>CG15399</i> (19450) and <i>CG7843</i> (22574). (<b>C</b>) Flow chart of the screening procedures to identify modifiers of polyQ-induced toxicity. (<b>D</b>) Brief summary of screen results. Scale bars represent either 200 µm in eye pictures or 50 µm in semi-thin eye sections.</p

Overlap between screens for genetic modifiers of polyQ-induced toxicity or aggregation.

<p>The Venn-like diagram displays only candidate genes shared by the different screens. Mode of modification (enhancement/suppression) is not addressed, due to the different readouts (aggregation/toxicity), model systems (<i>Drosophila</i>, insect cells, <i>C. elegans</i>) and elongated polyQ-containing proteins used in the diverse screening approaches.</p

From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.The ORFEU program was created by the Catalan Enterprise and Knowledge Department with the Department of Health and funded by the Government of Catalonia, who trusted the expertise of research institutes to add value to the health system during the pandemic. We also extend our thanks to the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme / Generalitat de Catalunya, the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement, and the co-financing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Program. We acknowledge support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, to the EMBL partnership and to the Co-financing with funds from the European Regional Development Fund corresponding to the Programa Operativo FEDER Plurirregional de España (POPE) 2014-2020. We acknowledge also support of the Centro de Excelencia Severo Ochoa and the Generalitat de Catalunya through the CERCA Programme, through Departament de Salut and Departament d’Empresa i Coneixement and the Co-financing with funds from the European Regional Development Fund by the Secretaria d’Universitats i Recerca corresponding to the Programa Operatiu FEDER de Catalunya 2014-202