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Becerra S.,CSIC - Biological Research Center | Montes M.,CSIC - Biological Research Center | Montes M.,Copenhagen University | Hernandez-Munain C.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC | Sune C.,CSIC - Biological Research Center
RNA | Year: 2015

The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5' splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3' splice site. The 5' and 3' splice site complexes are thought to be joined together by protein- protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF65. Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5' and 3' splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival. © 2015 Becerra et al.

Hernandez-Munain C.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC
Transcription | Year: 2015

Tcra/Tcrd includes 2 genes with distinct developmental programs controlled by 2 distant enhancers, Eα and Eδ. These enhancers work as a developmental switch during thymocyte development and they are essential for generation of αβ and γδ T-lymphocytes. Tcra and Tcrd transit from an unrearranged configuration to a rearranged configuration during T-cell development. Eα and Eδ are responsible for transcription of their respective unrearranged genes in thymocytes but are dispensable for such functions in the context of the rearranged genes in mature T-cells. Interestingly, Eα activates transcription of the rearranged Tcrd in γδ T-lymphocytes but it is inactive in αβ T-lymphocytes. © 2015 Taylor & Francis Group, LLC.

Montes M.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC | Becerra S.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC | Sanchez-Alvarez M.,The Institute of Cancer Research | Sune C.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC
Gene | Year: 2012

The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing is a key mechanism to increase the number and complexity of proteins encoded by the genome. Evidence gathered in recent years has established that transcription and splicing are physically and functionally coupled and that this coupling may be an essential aspect of the regulation of splicing and alternative splicing. Recent advances in our understanding of transcription and of splicing regulation have uncovered the multiple interactions between components from both types of machinery. These interactions help to explain the functional coupling of RNAPII transcription and pre-mRNA alternative splicing for efficient and regulated gene expression at the molecular level. Recent technological advances, in addition to novel cell and molecular biology approaches, have led to the development of new tools for addressing mechanistic questions to achieve an integrated and global understanding of the functional coupling of RNAPII transcription and pre-mRNA alternative splicing. Here, we review major milestones and insights into RNA polymerase II transcription and pre-mRNA alternative splicing as well as new concepts and challenges that have arisen from multiple genome-wide approaches and analyses at the single-cell resolution. © 2012 Elsevier B.V.

Diaz-Toledano R.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC | Diaz-Toledano R.,Research Center Biologica En Red Of Enfermedades Hepaticas gestivas | Gomez J.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC | Gomez J.,Research Center Biologica En Red Of Enfermedades Hepaticas gestivas
Cellular and Molecular Life Sciences | Year: 2015

The purpose of this work was to ascertain whether liver mRNA species share common structural features with hepatitis C virus (HCV) mRNA that allow them to support the RNase-P (pre-tRNA/processing enzyme) cleavage reaction in vitro. The presence of RNase-P competitive elements in the liver mRNA population was determined by means of biochemical techniques, and a set of sensitive mRNA species were identified through microarray screening. Cleavage specificity and substrate length requirement of around 200 nts, were determined for three mRNA species. One of these cleavage sites was found in interferon-alpha 5 (IFNA5) mRNA between specific base positions and with the characteristic RNase-P chemistry of cleavage. It was mapped within a cloverleaf-like structure revealed by a comparative structural analysis based on several direct enzymes and chemical probing methods of three RNA fragments of increasing size, and subsequently contrasted against site-directed mutants. The core region was coincident with the reported signal for the cytoplasmic accumulation region (CAR) in IFNAs. Striking similarities with the tRNA-like element of the antagonist HCV mRNA were found. In general, this study provides a new way of looking at a variety of viral tRNA-like motifs as this type of structural mimicry might be related to specific host mRNA species rather than, or in addition to, tRNA itself. © 2015 The Author(s).

Abengozar M.A.,CSIC - Biological Research Center | Bustos L.A.,University of Salamanca | Bustos L.A.,Catolica del Norte University | Garcia-Hernandez R.,Institute Parasitologia y Biomedicina Lopez Neyra IPBLN CSIC | And 6 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2015

Leishmaniasis is the protozoan disease second in importance for human health, superseded only by malaria; however, the options for chemotherapeutic treatment are increasingly limited due to drug resistance and toxicity. Under this perspective, a quest for new chemical compounds is urgently needed. An N-substituted 2-aminoalkan-1-ol scaffold has been shown to be a versatile scaffold for antiparasitic activity. Knowledge about its mechanism of action is still rather limited. In this work, we endeavored to define the leishmanicidal profile of such β-amino alkanol derivatives using a set of 15 N-mono- and disubstituted surrogates, tested on Leishmania donovani promastigotes and intracellular amastigotes. The best compound (compound 5), 2-ethylamino-dodecan-1-ol, had a 50% effective concentration (EC50) of 0.3 μM and a selectivity index of 72 for infected THP-1 cells and was selected for further elucidation of its leishmanicidal mechanism. It induced fast depletion of intracellular ATP content in promastigotes in the absence of vital dye intracellular entry, ruling out plasma membrane permeabilization as its origin. Confocal and transmission electron microscopy analyses showed that compound 5 induced severe mitochondrial swelling and vesiculation. Polarographic analysis using an oxygen electrode demonstrated that complex II of the respiratory chain (succinate reductase) was strongly inhibited by compound 5, identifying this complex as one of the primary targets. Furthermore, for other β-amino alkanols whose structures differed subtly from that of compound 5, plasma membrane permeabilization or interference with membrane traffic was also observed. In all, N-substituted β-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration. Copyright © 2015 American Society for Microbiology. All Rights Reserved.

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