Tuneable endogenous mammalian target complementation via multiplexed plasmidbased recombineering

Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce ‘TEMTAC’, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2

Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering

Includes supplementary materials for the online appendix.Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce ‘TEMTAC’, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS and SHP2.We thank all members of our laboratories for their contributions and helpful discussions. We thank the CRG Genomics Unit and the Biomolecular Screening & Protein Technologies Unit. We acknowledge help in the quantifications of the Western blots by Dina Cramer and assistance in RNA sequencing analysis by Javier Delgado. Armelle Yart provided the HEK293 cells stably expressing GH receptor. This work was funded by the European Commission (EC) Framework programme (FP) 7 projects PRIMES (contract nr. 278568), ComplexINC (contract nr. 279039) and SynSignal (contract nr. 613879). LS is supported by the Spanish Ministerio de Economía y Competitividad, Plan Nacional BIO2012-39754 and the European Fund for Economic and Regional Development. We are particularly grateful for the support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017′ (SEV-2012-0208)

rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions

Knowing which proteins and RNAs directly interact is essential for understanding cellular mechanisms. Unfortunately, discovering such interactions is costly and often unreliable. To overcome these limitations, we developed rec-YnH, a new yeast two and three-hybrid-based screening pipeline capable of detecting interactions within protein libraries or between protein libraries and RNA fragment pools. rec-YnH combines batch cloning and transformation with intracellular homologous recombination to generate bait–prey fusion libraries. By developing interaction selection in liquid–gels and using an ORF sequence-based readout of interactions via next-generation sequencing, we eliminate laborious plating and barcoding steps required by existing methods. We use rec-Y2H to simultaneously map interactions of protein domains and reveal novel putative interactors of PAR proteins. We further employ rec-Y2H to predict the architecture of published coprecipitated complexes. Finally, we use rec-Y3H to map interactions between multiple RNA-binding proteins and RNAs—the first time interactions between protein and RNA pools are simultaneously detected.We also acknowledge the support from the Spanish Ministry of Economy and Competitiveness (MINECO) for Juan de la Cierva-Incorporación Programme (IJCI‐2014‐22070) to J.-S.Y., L.S. (BFU2015-63571-P), and S.M. (BFU2014-54278-P and BFU2015-62550-ERC). We further acknowledge support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013-2017”, SEV-2012-0208 and the CERCA Programme/Generalitat de Catalunya. This work was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) reference MINECO PE 2013-2016 PN FEDER and the European Regional Development Fund (ERDF). All sequencing was done in the CRG Genomics Core Facility

Silencing of SRRM4 suppresses microexon inclusion and promotes tumor growth across cancers

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.Funding: This project was funded in part by a grant from the Plan Estatal de Investigación Científica y Técnica y de Innovación to L.S. (PGC2018-101271-B-I00, http://www.ciencia.gob.es). S.A.H. is supported by a Marie Skłodowska-Curie Individual Fellowship from the European Union’s Horizon 2020 research and innovation programme (MSCA-IF-2017-794629, http://ec.europa.eu/). X.H. is supported by a PhD fellowship from the Fundación Ramón Areces (http://www.fundacionareces.es). We acknowledge the support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa’, the CERCA Programme / Generalitat de Catalunya, and the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnershi