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MOLECULAR CLONING, CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE RBM20, A NOVEL RIBONUCLEOPROTEIN GENE ASSOCIATED TO FAMILIAL DILATED CARDIOMYOPATHY.
No full textLo splicing alternativo del pre-RNA messaggero (pre-mRNA) è un processo strettamente regolato che coinvolge lo spliceosoma e proteine di legame all’RNA che possono reprimere o attivare i siti di splicing selezionati. Oltre il 15% delle malattie genetiche umane sono state associate a mutazioni negli elementi di splicing agenti in cis, ma poche tra queste sono attribuibili a fattori agenti in trans che controllano lo splicing alternativo. Gli effetti di queste mutazioni che coinvolgono il macchinario di splicing basale e i regolatori dello splicing alternativo sono stati associati all’autismo, alla sclerosi laterale amiotrofica e al cancro. Nel cuore e nel muscolo scheletrico, lo splicing alternativo svolge un ruolo critico per la funzione muscolare. In entrambi i sistemi, le transizioni di splicing avvengono secondo delle vie temporali strettamente raggruppate, in cui sono coinvolte le stesse proteine. Un ruolo diretto dei regolatori dello splicing nelle patologie cardiache è stato dimostrato recentemente per due proteine con un motivo di legame all’RNA (RBM), RBM25 e RBM20.
Lo studio sperimentale presentato in questa tesi è focalizzato sulla proteina RBM20.
RBM20, proteina con un motivo di legame all’RNA di tipo 20, è un fattore agente in trans espresso preferenzialmente nel tessuto cardiaco, che regola lo splicing alternativo di geni aventi un ruolo chiave nella funzionalità cardiaca, compresi quelli che regolano l’omeostasi ionica, la biologia del sarcomero e la trasduzione del segnale. Mutazioni del gene RBM20 sono associate a cardiomiopatia dilatativa familiare e la maggior parte di queste altera i residui amminoacidici presenti nel dominio ricco in arginine e serine della proteina. I motivi funzionali della proteina RBM20 sono stati predetti attraverso l’omologia di sequenza, ma sono stati poco caratterizzati a livello funzionale. In questo studio è stato clonato il cDNA della isoforma cardiaca della proteina RBM20 umana e della corrispondente proteina murina. L’utilizzo di vettori di espressione della proteina RBM20 prodotti nel corso dello studio ha contribuito alla caratterizzazione funzionale dei domini richiesti per la localizzazione nucleare di RBM20. Analisi di microscopia confocale hanno mostrato che RBM20 ha una localizzazione nucleare con distribuzione punteggiata a livello di strutture denominate “nuclear speckles”, questa distribuzione è tipica dei fattori di splicing con un dominio ricco in arginine e serine (RS). Attraverso la produzione di vettori che esprimono forme tronche della proteina e comparando la loro distribuzione subcellulare abbiamo identificato le sequenze necessarie alla localizzazione nucleare di RBM20. Questa regione comprende il dominio di legame all’RNA e il dominio ricco in arginine e serine. La sequenza è conservata in molte specie che appartengono solo alle proteine ortologhe di RBM20.
Questi studi dimostrano l’esistenza di una selezione specifica durante l’evoluzione nella regolazione post-trascrizionale del cuore, indicando RBM20 come un fattore chiave negli eventi di regolazione dello splicing richiesti per la funzione cardiaca.Alternative splicing of pre-messenger RNA is a tightly regulated process that involves the spliceosome and additional RNA binding proteins that can repress or activate splice site selection. More than 15% of human genetic diseases have been associated to mutations in cis-acting splice elements, but very few of them are attributable to trans-factors that control alternative splicing. The effects of these mutations that involve the basal splicing machinery and regulators of alternative splicing have been associated with autism spectrum disorders, amyotrophic lateral sclerosis and cancer. In heart and skeletal muscle, alternative splicing plays a critical role in muscle function. In both systems, splicing transition occurs in tightly grouped temporal ways, partially involving the same proteins. A direct involvement of splicing regulators to cardiac diseases has been proven recently for two RNA binding motif (RBM) protein, the RBM25 and the RBM20.
This experimental study is focused on the RBM20 protein.
RNA binding motif protein type 20 (RBM20) is a trans-acting factor expressed preferentially in heart tissue, which regulates alternative splicing of gene that have a key role in cardiac function, including ion homeostasis, sarcomere biology and signal transduction. Mutations in the RBM20 gene are linked to familial dilated cardiomyopathy and the most of them alters residues in the arginine-serine domain of the protein. The functional motifs of the RBM20 protein have been predicted by sequence homology, but poorly functionally characterized. In the present study by cloning the cardiac isoform of human RBM20 and the mouse RBM20 full length cDNA I provided functional annotations of structural domains required for the RBM20 nuclear localization. Confocal microscopy analysis showed that RBM20 had a punctuate localization in nuclear speckles, which is typical of RS splicing factors. By producing expressing vectors for truncated proteins and comparing their subcellular distribution we identified the sequences necessary for RBM20 full nuclear retention. The region overlaps both RNA binding motif and arginine-serine domain. The sequence is conserved in many species but belongs only to RBM20 protein orthologs.
These studies demonstrate a specific evolutionary selection for post-transcriptional regulation in heart, highlighting RBM20 as a key factor for regulation of splicing events required for cardiac function
Identification of nuclear retention domains in the RBM20 protein
No full textAbstractRBM20 is a nuclear protein which regulates alternative splicing of expressed genes that have a key role in cardiac function. By cloning the human and mouse RBM20 cDNA, producing expressing vectors for truncated proteins, and comparing their sub-cellular distribution in transfected cells, we have identified the sequences necessary for RBM20 full nuclear retention. The region overlaps an RNA binding motif and a serine–arginine domain. The sequence is conserved in many species but belongs only to RBM20 orthologs. The RMB20 tissue specificity, together with the properties of its nuclear localization determinant, demonstrates a specific evolutionary selection of post-transcriptional regulation factors
Identification of nuclear retention domains in the alternative splicing regulator RBM20
No full textRNA binding motif protein type 20 (RBM20) is a nuclear protein which regulates alternative splicing of expressed genes that have a key role in cardiac functions. Mutations in the RBM20 gene are linked to familial dilated cardiomyopathy and most of them alter residues in the RS domain of the protein. Functional motifs in the RBM20 protein have been poorly characterized. Our study provides functional annotations to structural domains within the RBM20 protein required for its nuclear localization. By cloning the human and mouse RBM20 genes, producing expressing vectors for truncated proteins and comparing their sub-cellular distribution in transfected cells, we have identified the sequences necessary for RBM20 full nuclear retention. The region overlaps both an RNA binding motif and a serine-arginine domain. The sequence is conserved in many species but belongs only to RBM20 protein orthologs. The RMB20 tissue specificity, together with the properties of its nuclear localization determinant, demonstrates a specific evolutionary selection for post-transcriptional regulation in the heart, highlighting RBM20 as a key factor for regulation of splicing events required for cardiac function
Molecular cloning and expression analyses of the RBM20 gene that is associated to familial dilated cardiomyopathy
No full textMolecular characterization of the RNA binding motif protein 20: determination of nuclear localization signals.
No full textRNA splicing is a tightly regulated process that involves the spliceosome and additional RNA binding proteins that can repress or activate splice sites selection. Recent studies have indicated that mutations in RBM20, a gene encoding a novel ribonucleic acid - binding protein, are associated to human dilated cardiomyopathy (DCM). RBM20 regulates alternative splicing of expressed genes that have a key role in cardiac function, including ion homeostasis, sarcomere biology, and signal transduction. The functional motifs of the RBM20 protein have been poorly investigated.The focus of this study is to characterize the protein domains that contribute to the nuclear function of RBM20. Predictive in silico analysis of the translated RBM20 gene identifies an RNA recognition motif (RRM motif), a serine /arginine (RS) domain and Zn2+ finger domains. We have produced GFP-RBM20 fusion proteins in order to map the functional domains of the protein that contribute to subcellular distribution. We have produced truncated mutants of the RBM20 proteins and analyzed separately in immunofluorescence assays in transfected cells. We identified a region necessary and sufficient to nuclear localization of RBM20 protein that maps between the RRM and the RS domain. Actually we are producing RBM20 mutant proteins in order to characterize the nuclear localization signal (NLS). Further structural and functional characterization of RBM20 may contribute to understand the molecular pathogenesis of familiar DCM
Expression, localization and functional role of ribonucleoproteins in myoblasts differentiation
No full textLarge-scale expressed sequence tag and genome-wide analyses estimate that the majority of human genes undergo alternative splicing with a differential tissue distribution. More than 15% of human genetic diseases are associated to mutations in the consensus splice sites and disruption of splicing regulatory networks contributes to various diseases. Alternative splicing of pre-messenger RNA represents, consequently, an intensive post-transcriptional regulatory activity that involves several RNA binding proteins and splicing regulators. They belong principally to SR (serine/arginine rich proteins) and hnRNP (heterogeneous nuclear ribonucleoprotein particles) proteins. Both types can act as enhancers or repressors of alternative splicing helping in defining cis-regulatory elements positioned either in the exon or in the intron sequences. Dynamic antagonism between members of the SR and hnRNP protein families is demonstrated to be important for determining a number of alternative splicing patterns. Among the hnRNPs that have been implicated in regulating the alternative splicing events with tissue-specificity, polypyrimidine tract binding protein (PTB) and its paralog nPTB play a well established role as negative splicing regulators. We have previously characterized PTB, its paralog nPTB and Raver1 (a PTB co-repressor factor) gene expression in human tissues and cell lines (1-3). We showed that alternative spliced nPTB transcripts are distributed with a tissue specificity involving brain and skeletal muscle. During muscle development PTB regulates exons splicing in several specific transcribed genes. In the present study we are investigating the contribution of ribonucleoproteins to splicing events occurring during myoblasts differentiation. We are analyzing the expression, subcellular localization and functional role of PTB, Raver1 and Rbm20 (a SR protein which mutations are associated to familial dilated cardiomyopathy) during C2C12 myoblast differentiation. Preliminary results show that Raver1 expression is reduced both at transcriptional and translational levels during differentiation and that its overexpression can affect exon inclusion in calpain 3 (CAPN3) and myotubularin-related protein 1 (MTMR1). Confocal microscopy analyses show that Raver1 diffuses in the cytoplasm from the nucleus, localizing in polarized cytoplasmic area during myoblasts differentiation. These studies may shed new light on the role of ribonucleopreoteins in the post-transcriptional regulation events that occur during muscle development in both nuclear and cytoplasmic compartments. 1. Romanelli et al. Biochim Biophys Acta; 2001, 1520: 85 – 88. 2. Romanelli et al. Gene; 2005, 356:11-18. 3 Romanelli et al. Gene; 2007, 405: 79 – 87
Transcriptional regulation of the human Raver2 ribonucleoprotein gene.
No full textRaver2 is a putative modulator of the activity of the polypyrimidine-tract binding protein (PTB), one of the most intensively studied splicing repressors. Little is known about Raver2 expression, and all current data is from mice where it shows tissue specificity. In the present study, by comparing Raver2 transcript expression in human and mouse tissues, we found that human Raver2 is ubiquitously expressed in adult tissues. In order to investigate human Raver2 transcription regulation, we identified and characterized a putative promoter region in a 1000bp region upstream of the transcription starting site of the gene. Dual luciferase reporter assays demonstrated that this region had promoter activity conferred by the first 160bp. By mutagenic analyses of putative cis-acting regulatory sequences, we identified an individual site that decreased the promoter activity by up to 40% when mutated. Together, our results suggest that regulation of human Raver2 expression involves TATA-less transcriptional activity
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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