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‣ SF2/ASF e SRPK2 : relação entre a maquinaria de splicing alternativo e o desenvolvimento da leucemia; SF2/ASF and SRPK2 : correlation of alternative splicing machinery and leucemia development

Germanna Lima Righetto
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 20/03/2013 Português
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O processo de splicing do RNAm é responsável por orquestrar a junção de exons, criando uma grande diversidade de isoformas gênicas. Alterações nos componentes da maquinaria de splicing e, consequentemente, no processamento do pré-RNAm podem causar ou contribuir para uma infinidade de doenças, dentre elas o câncer. A proteína SF2/ASF foi o primeiro fator de splicing a ser caracterizado como proto-oncogênico, estando superexpresso em diferentes tipos de neoplasias. Sabe-se que a ativação desse fator é, principalmente, mediada por SR quinases conhecidas como splicing quinases e pertencentes à família das SRPKs. A quinase SRPK1, responsável pela fosforilação de SF2/ASF no citoplasma, tem conhecida superexpressão em leucemias. Já a quinase SRPK2, paráloga a SRPK1, possui relação já demonstrada com a proliferação de células leucêmicas e com sua diferenciação. Diante desse quadro, buscamos nesse estudo possíveis correlações entre a maquinaria de splicing e o câncer, dando enfoque à relação entre essa maquinaria e a leucemia. Para tanto, buscamos alterações no cDNA de SRPK2 e quantificamos a expressão das SR quinases SRPK1, SRPK2 e CLK1 em diferentes linhagens de leucemia. Além disso, avaliamos, usando o sistema de Exon Array (Affymetrix)...

‣ G-quadruplex formation enhances splicing efficiency of PAX9 intron 1; Formação de G-quadruplex aumenta eficiência de splicing do íntron 1 do gene PAX9

Mariana Martins Ribeiro
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 27/02/2014 Português
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G-Quadruplexes são estruturas secundárias presentes nas moléculas de DNA e RNA, os quais são formados pelo empilhamento de G-quartetos (interação de quatro guaninas (G-tratos) delimitadas por ligações de hidrogênio do tipo Hoogsteen. O intron 1 do gene PAX9 humano tem um G-quadruplex formado na região localizada perto do exon 1, que é conservada entre os mamíferos placentários. Análises de Dicroísmo Circular (CD), e CD melting mostraram que estas sequências são capazes de formar estruturas quadruplex altamente estáveis. Devido à proximidade da estrutura quadruplex ao limite éxon-íntron foi utilizado um ensaio validado de splicing duplo repórter e PCR em tempo real para analisar o seu papel na eficiência de splicing. O quadruplex humano mostrou ter um papel chave na eficiência de splicing do íntron 1 do gene PAX9, já que uma mutação que aboliu a formação do quadruplex diminuiu drasticamente a eficiência de splicing. O quadruplex de rato, menos estável, mostrou menor eficiência quando comparado com sequências humanas. Além disso, o tratamento com 360A, um forte ligante que estabiliza estruturas quadruplex, aumentou ainda mais a eficiência de splicing do íntron 1 do PAX9 humano. Em conjunto estes resultados fornecem evidências de que as estruturas de G-quadruplex estão envolvidas na eficiência de splicing do intron 1 do gene PAX9.; G-Quadruplex are secondary structures present in DNA and RNA molecules...

‣ An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus

McNally, Lisa M.; McNally, Mark T.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/1998 Português
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The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3′ region resembles a minor class 5′ splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5′ half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case...

‣ RNA Splicing at Human Immunodeficiency Virus Type 1 3′ Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element

Bilodeau, Patricia S.; Domsic, Jeffrey K.; Mayeda, Akila; Krainer, Adrian R.; Stoltzfus, C. Martin
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2001 Português
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The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1B, A2...

‣ Regulation of RNA splicing in gag-deficient mutants of Moloney murine sarcoma virus MuSVts110.

De Mars, M; Sterner, D A; Chiocca, S M; Biggart, N W; Murphy, E C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1990 Português
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We investigated whether the MuSVts110 gag gene product (P58gag) can regulate the novel growth temperature dependence of MuSVts110 RNA splicing. MuSVts110 mutants with either frameshifts or deletions in the gag gene were tested for their ability to maintain the MuSVts110 splicing phenotype. Only small decreases in splicing efficiency and no changes in the thermosensitivity of viral RNA splicing were observed in MuSVts110 gag gene frameshift mutants. Deletions within the gag gene, however, variably decreased MuSVts110 splicing efficiency but had no effect on its thermosensitivity. Another class of MuSVts110 splicing mutants generated by treatment of MuSVts110-infected cells with NiCl2 was also examined. In these "nickel revertants," P58gag is made, but splicing of the viral transcript is nearly complete at all growth temperatures. The splicing of "tagged" viral RNA transcribed from a modified MuSVts110 DNA introduced into nickel revertant cells remained thermosensitive, arguing against trans effects of viral gene products on splicing efficiency. These experiments indicated that neither the MuSVts110 P58gag protein nor any other viral gene product acts in trans to regulate MuSVts110 splicing.

‣ The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.

Petersen-Mahrt, S K; Estmer, C; Ohrmalm, C; Matthews, D A; Russell, W C; Akusjärvi, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/1999 Português
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The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

‣ Two reactions of Haloferax volcanii RNA splicing enzymes: Joining of exons and circularization of introns

SALGIA, SHILPA R.; SINGH, SANJAY K.; GURHA, PRIYATANSH; GUPTA, RAMESH
Fonte: Copyright 2003 by RNA Society Publicador: Copyright 2003 by RNA Society
Tipo: Artigo de Revista Científica
Publicado em /03/2003 Português
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Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon–intron junctions, producing 2‘,3′-cyclic phosphate and 5′-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3′ terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5′ and 3′ flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3′,5′-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall...

‣ Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Zheng, Zhi-Ming
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
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Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two major cis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 21 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3′ splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on the cis-element’s identity and changes in cellular splicing factors under physiological or pathological conditions.

‣ Altered RNA splicing contributes to skeletal muscle pathology in Kennedy disease knock-in mice

Yu, Zhigang; Wang, Adrienne M.; Robins, Diane M.; Lieberman, Andrew P.
Fonte: The Company of Biologists Limited Publicador: The Company of Biologists Limited
Tipo: Artigo de Revista Científica
Português
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Here, we used a mouse model of Kennedy disease, a degenerative disorder caused by an expanded CAG repeat in the androgen receptor (AR) gene, to explore pathways leading to cellular dysfunction. We demonstrate that male mice containing a targeted Ar allele with 113 CAG repeats (AR113Q mice) exhibit hormone- and glutamine length-dependent missplicing of Clcn1 RNA in skeletal muscle. Changes in RNA splicing are associated with increased expression of the RNA-binding protein CUGBP1. Furthermore, we show that skeletal muscle denervation in the absence of a repeat expansion leads to increased CUGBP1 expression. However, this induction of CUGBP1 is not sufficient to alter Clcn1 RNA splicing, indicating that changes mediated by both denervation and AR113Q toxicity contribute to altered RNA processing. To test this notion directly, we exogenously expressed the AR in vitro and observed hormone-dependent changes in the splicing of pre-mRNAs from a human cardiac troponin T minigene. These effects were notably similar to changes mediated by RNA with expanded CUG tracts, but not CAG tracts, highlighting unanticipated similarities between CAG and CUG repeat diseases. The expanded glutamine AR also altered hormone-dependent splicing of a calcitonin/calcitonin gene-related peptide minigene...

‣ Excessive RNA Splicing and Inhibition of HIV-1 Replication Induced by Modified U1 Small Nuclear RNAs▿

Mandal, Dibyakanti; Feng, Zehua; Stoltzfus, C. Martin
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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HIV-1 RNA undergoes a complex splicing process whereby over 40 different mRNA species are produced by alternative splicing. In addition, approximately half of the RNA transcripts remain unspliced and either are used to encode Gag and Gag-Pol proteins or are packaged into virions as genomic RNA. It has previously been shown that HIV-1 splicing is regulated by cis elements that bind to cellular factors. These factors either enhance or repress definition of exons that are flanked by the HIV-1 3′ splice sites. Here we report that expression of modified U1 snRNPs with increased affinity to HIV-1 downstream 5′ splice sites and to sequences within the first tat coding exon act to selectively increase splicing at the upstream 3′ splice sites in cotransfected 293T cells. This results in a decrease of unspliced viral RNA levels and an approximately 10-fold decrease in virus production. In addition, excessive splicing of viral RNA is concomitant with a striking reduction in the relative amounts of Gag processing intermediates and products. We also show that T cell lines expressing modified U1 snRNAs exhibit reduced HIV-1 replication. Our results suggest that induction of excessive HIV-1 RNA splicing may be a novel strategy to inhibit virus replication in human patients.

‣ Understanding splicing regulation through RNA splicing maps

Witten, Joshua T.; Ule, Jernej
Fonte: Elsevier Trends Journals Publicador: Elsevier Trends Journals
Tipo: Artigo de Revista Científica
Publicado em /03/2011 Português
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Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein–RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.

‣ Incorporating Evolutionary Information and Functional Domains for Identifying RNA Splicing Factors in Humans

Hsu, Justin Bo-Kai; Bretaña, Neil Arvin; Lee, Tzong-Yi; Huang, Hsien-Da
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 16/11/2011 Português
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Regulation of pre-mRNA splicing is achieved through the interaction of RNA sequence elements and a variety of RNA-splicing related proteins (splicing factors). The splicing machinery in humans is not yet fully elucidated, partly because splicing factors in humans have not been exhaustively identified. Furthermore, experimental methods for splicing factor identification are time-consuming and lab-intensive. Although many computational methods have been proposed for the identification of RNA-binding proteins, there exists no development that focuses on the identification of RNA-splicing related proteins so far. Therefore, we are motivated to design a method that focuses on the identification of human splicing factors using experimentally verified splicing factors. The investigation of amino acid composition reveals that there are remarkable differences between splicing factors and non-splicing proteins. A support vector machine (SVM) is utilized to construct a predictive model, and the five-fold cross-validation evaluation indicates that the SVM model trained with amino acid composition could provide a promising accuracy (80.22%). Another basic feature, amino acid dipeptide composition, is also examined to yield a similar predictive performance to amino acid composition. In addition...

‣ SpliceDisease database: linking RNA splicing and disease

Wang, Juan; Zhang, Jie; Li, Kaibo; Zhao, Wei; Cui, Qinghua
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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RNA splicing is an important aspect of gene regulation in many organisms. Splicing of RNA is regulated by complicated mechanisms involving numerous RNA-binding proteins and the intricate network of interactions among them. Mutations in cis-acting splicing elements or its regulatory proteins have been shown to be involved in human diseases. Defects in pre-mRNA splicing process have emerged as a common disease-causing mechanism. Therefore, a database integrating RNA splicing and disease associations would be helpful for understanding not only the RNA splicing but also its contribution to disease. In SpliceDisease database, we manually curated 2337 splicing mutation disease entries involving 303 genes and 370 diseases, which have been supported experimentally in 898 publications. The SpliceDisease database provides information including the change of the nucleotide in the sequence, the location of the mutation on the gene, the reference Pubmed ID and detailed description for the relationship among gene mutations, splicing defects and diseases. We standardized the names of the diseases and genes and provided links for these genes to NCBI and UCSC genome browser for further annotation and genomic sequences. For the location of the mutation...

‣ Global impact of RNA splicing on transcriptome remodeling in the heart *

GAO, Chen; Wang, Yibin
Fonte: Zhejiang University Press Publicador: Zhejiang University Press
Tipo: Artigo de Revista Científica
Publicado em /08/2012 Português
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In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30 000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

‣ Modulation of dendritic AMPA receptor mRNA trafficking by RNA splicing and editing

La Via, Luca; Bonini, Daniela; Russo, Isabella; Orlandi, Cesare; Barlati, Sergio; Barbon, Alessandro
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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RNA trafficking to dendrites and local translation are crucial processes for superior neuronal functions. To date, several α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) mRNAs have been detected in dendrites and are subject to local protein synthesis. Here, we report the presence of all AMPAR GluA1-4 mRNAs in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. In particular, we showed that dendritic AMPAR mRNAs are present in the Flip versions in the cortex and hippocampus. To further confirm these data, we demonstrate, using in situ hybridization, the dendritic localization of the GluA2 Flip isoform in vitro and in vivo, whereas the Flop variant is restricted mainly to the soma. In addition, we report that dendritic AMPA mRNAs are edited at low levels at their R/G sites; this result was also supported with transfection experiments using chimeric GluA2 DNA vectors, showing that transcripts carrying an unedited nucleotide at the R/G site, in combination with the Flip exon, are more efficiently targeted to dendrites when compared with the edited-Flip versions. Our data show that post-transcriptional regulations such as RNA splicing, editing and trafficking might be mutually coordinated and that the localization of different AMPAR isoforms in dendrites might play a functional role in the regulation of neuronal transmission.

‣ Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /11/2014 Português
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Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

‣ Neuronal elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability

Ince-Dunn, G.; Okano, H.J.; Jensen, K.B.; Park, W.Y.; Zhong, R.; Ule, J.; Mele, A.; Fak, J.J.; Yang, C.; Zhang, C.; Yoo, J.; Herre, M.; Okano, H.; Noebels, J.L.; Darnell, R.B.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2012 Português
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The paraneoplastic neurologic disorders target several families of neuron-specific RNA binding proteins (RNABPs), revealing that there are unique aspects of gene expression regulation in the mammalian brain. Here, we used HITS-CLIP to deter- mine robust binding sites targeted by the neuronal Elav-like (nElavl) RNABPs. Surprisingly, nElav protein binds preferentially to GU-rich sequences in vivo and in vitro, with secondary binding to AU-rich sequences. nElavl null mice were used to validate the consequence of these binding events in the brain, demonstrating that they bind intronic sequences in a position dependent manner to regulate alternative splicing and to 30 UTR sequences to regulate mRNA levels. These controls converge on the glutamate synthesis pathway in neurons; nElavl proteins are required to maintain neurotransmitter glutamate levels, and the lack of nElavl leads to spontaneous epileptic seizure activity. The genome-wide anal- ysis of nElavl targets reveals that one function of neuron-specific RNABPs is to control excitation-inhibition balance in the brain.; Gulayse Ince-Dunn, Hirotaka J. Okano, Kirk B. Jensen, Woong-Yang Park, Ru Zhong, Jernej Ule, Aldo Mele, John J. Fak, ChingWen Yang, Chaolin Zhang, Jong Yoo, Margaret Herre...

‣ Control of retroviral RNA splicing through maintenance of suboptimal processing signals.

Katz, R A; Skalka, A M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1990 Português
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The full-length retroviral transcript serves as genomic RNA for progeny virions, as an mRNA for structural proteins and enzymes, and as a pre-mRNA substrate for splicing that yields subgenomic mRNAs that encode other essential proteins. Thus, RNA splicing to form subgenomic mRNAs must be incomplete or regulated in order to preserve some of the full-length transcripts. We have used the avian sarcoma virus system to delineate the viral functions that are required in the regulation of the splicing event that forms the envelope glycoprotein (env) subgenomic mRNA. We observed previously that a specific insertion mutation just 5' of the env splice acceptor site resulted in nearly complete splicing to form env mRNA and a concomitant replication defect which is presumably due to a deficit of the full-length transcript. Replication-competent pseudorevertants contained second-site mutations that restored splicing control, and these mapped either just upstream or downstream of the env splice acceptor site. In this report, we show that splicing control at this site does not require expression of any known viral replication protein(s), nor does it appear to require the viral splice donor site. From these results and analysis of additional splicing mutations obtained by in vivo selection...

‣ Correct in vivo RNA splicing of a mitochondrial intron in algal chloroplasts.

Herdenberger, F; Holländer, V; Kück, U
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/08/1994 Português
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The self-splicing group II intron (rl1) from Scenedesmus obliquus mitochondria together with its 6 bp intron binding site (IBS1) were inserted in the correct and inverse orientation into the chloroplast tscA gene from C.reinhardtii. Precursor RNA derived from the chimeric tscA-rl1 gene can be used to demonstrate in vitro self-splicing of the rl1 intron RNA. Using the particle bombardment technique, the tscA-rl1 construct was transferred into the chloroplast of the unicellular alga Chlamydomonas reinhardtii. We recovered transformants which contain the chimeric tscA-rl1 gene as shown by Southern analysis. Hybridization and PCR analysis of transcripts confirmed that the heterologous intron is correctly spliced in vivo. From sequencing of cDNA clones we conclude that the IBS1 sequence is sufficient for correct splicing of the mitochondrial intron in C. reinhardtii chloroplasts. Using specific probes, we demonstrate by Northern hybridization that the mature RNA, as well as an intron-3' exon intermediate, accumulate in transformants containing the rl1 intron, correctly inserted into the tscA gene. As expected, no RNA splicing at all was observed when the intron had an inverted orientation within the tscA gene. In addition, a mutated intron RNA with an altered 3' terminal nucleotide was tested in vivo. In contrast to similar mutants examined in vitro...

‣ Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing▿

Majerciak, Vladimir; Yamanegi, Koji; Allemand, Eric; Kruhlak, Michael; Krainer, Adrian R.; Zheng, Zhi-Ming
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 facilitates the expression of both intronless viral ORF59 genes and intron-containing viral K8 and K8.1 genes (V. Majerciak, N. Pripuzova, J. P. McCoy, S. J. Gao, and Z. M. Zheng, J. Virol. 81:1062-1071, 2007). In this study, we showed that disruption of ORF57 in a KSHV genome led to increased accumulation of ORF50 and K8 pre-mRNAs and reduced expression of ORF50 and K-bZIP proteins but had no effect on latency-associated nuclear antigen (LANA). Cotransfection of ORF57 and K8β cDNA, which retains a suboptimal intron of K8 pre-mRNA due to alternative splicing, promoted RNA splicing of K8β and production of K8α (K-bZIP). Although Epstein-Barr virus EB2, a closely related homolog of ORF57, had a similar activity in the cotransfection assays, herpes simplex virus type 1 ICP27 was inactive. This enhancement of RNA splicing by ORF57 correlates with the intact N-terminal nuclear localization signal motifs of ORF57 and takes place in the absence of other viral proteins. In activated KSHV-infected B cells, KSHV ORF57 partially colocalizes with splicing factors in nuclear speckles and assembles into spliceosomal complexes in association with low-abundance viral ORF50 and K8 pre-mRNAs and essential splicing components. The association of ORF57 with snRNAs occurs by ORF57-Sm protein interaction. We also found that ORF57 binds K8β pre-mRNAs in vitro in the presence of nuclear extracts. Collectively our data indicate that KSHV ORF57 functions as a novel splicing factor in the spliceosome-mediated splicing of viral RNA transcripts.