RESUMEN
BACKGROUND: DYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and DCDC2 interact on the genetic and the protein level. RESULTS: Here, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b in zebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among DYX1C1 and DCDC2 in a cellular model. CONCLUSIONS: In summary, we describe the physical and functional interaction between the two genes DYX1C1 and DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set the stage for future functional studies.
Asunto(s)
Cilios , Chaperonas Moleculares , Proteínas de Pez Cebra , Pez Cebra , Animales , Movimiento Celular/genética , Regulación de la Expresión Génica , Fenotipo , Pez Cebra/genética , Chaperonas Moleculares/genética , Proteínas de Pez Cebra/genética , Cilios/patologíaRESUMEN
The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed. By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.
RESUMEN
The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/biosíntesis , Homólogo de la Proteína 1 Relacionada con la Autofagia/antagonistas & inhibidores , Homólogo de la Proteína 1 Relacionada con la Autofagia/fisiología , Línea Celular , Cuerpos Enrollados , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/fisiología , Canales Iónicos/metabolismo , Fosforilación , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Factores de Empalme de ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Animales , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mutación , Estabilidad Proteica , Empalme del ARN , TranscriptomaRESUMEN
Recently, by combining transcriptomics with functional splicing reporter assays we were able to identify GT > GC > TT as the three highest ranked dinucleotides of human 5' splice sites (5'ss). Here, we have extended our investigations to the proteomic characterization of nuclear proteins that bind to canonical and noncanonical 5'ss. Surprisingly, we found that U1 snRNP binding to functional 5'ss sequences prevented components of the DNA damage response (DDR) from binding to the RNA, suggesting a close link between spliceosome arrangement and genome stability. We demonstrate that all tested noncanonical 5'ss sequences are bona-fide targets of the U2-type spliceosome and are bound by U1 snRNP, including U1-C, in the presence of splicing enhancers. The quantity of precipitated U1-C protein was similar for all noncanonical 5'ss dinucleotides, so that the highly different 5'ss usage was likely due to a later step after early U1 snRNP binding. In addition, we show that an internal GT at positions +5/+6 can be advantageous for splicing at position +1 of noncanonical splice sites. Likewise, and in agreement with previous observations, splicing inactive U1 snRNP binding sites could serve as splicing enhancers, which may also explain the higher abundance of U1 snRNPs compared to other U snRNPs. Finally, we observe that an arginine-serine (RS)-rich domain recruitment to stem loop I of the U1 snRNA is functionally sufficient to promote exon-definition and upstream 3'ss activation.
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Sitios de Unión , Sitios de Empalme de ARN , Empalme del ARN , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Daño del ADN , Elementos de Facilitación Genéticos , Exones , Humanos , Intrones , Unión Proteica , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Empalmosomas/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Jumonji-domain-containing protein 6 (JMJD6) is a Fe(II) and 2-oxogluterate (2OG) dependent oxygenase involved in gene regulation through post-translationally modifying nuclear proteins. It is highly expressed in many cancer types and linked to tumor progression and metastasis. Four alternatively-spliced jmjd6 transcripts were annotated. Here, we focus on the two most abundantly expressed ones, which we call jmjd6-2 and jmjd6-Ex5. TCGA SpliceSeq data revealed a significant decrease of jmjd6-Ex5 transcripts in patients and postmortem tissue of several tumors. The two protein isoforms are distinguished by their C-terminal sequences, which include a serine-rich region (polyS-domain) in JMJD6-2 that is not present in JMJD6-Ex5. Immunoprecipitation followed by LC-MS/MS for JMJD6-Ex5 shows that different sets of proteins interact with JMJD6-2 and JMJD6-Ex5 with only a few overlaps. In particular, we found TFIIF-associating CTD phosphatase (FCP1), proteins of the survival of motor neurons (SMN) complex, heterogeneous nuclear ribonucleoproteins (hnRNPs) and upstream binding factor (UBF) to interact with JMJD6-Ex5. Like JMJD6-2, both UBF and FCP1 comprise a polyS-domain. The polyS domain of JMJD6-2 might block the interaction with polyS-domains of other proteins. In contrast, JMJD6-2 interacts with many SR-like proteins with arginine/serine-rich (RS)-domains, including several splicing factors. In an HIV-based splicing reporter assay, co-expression of JMJD6-2 inhibited exon inclusion, whereas JMJD6-Ex5 did not have any effect. Furthermore, the silencing of jmjd6 by siRNAs favored jmjd6-Ex5 transcripts, suggesting that JMJD6 controls splicing of its own pre-mRNA. The distinct molecular properties of JMJD6-2 and JMJD6-Ex5 open a lead into the functional implications of the variations of their relative abundance in tumors.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/metabolismo , Empalme del ARN , Células HEK293 , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Neoplasias/metabolismo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers "nakedly", without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.
Asunto(s)
VIH-1/genética , Oligonucleótidos/farmacología , Empalme del ARN , Estabilidad del ARN , VIH-1/efectos de los fármacos , Células HeLa , Humanos , Células Jurkat , ARN Mensajero/genética , ARN Mensajero/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Most human pathogenic mutations in 5' splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in â¼1% of all human 5'ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5'ss are used. In this work, we systematically examined noncanonical 5' splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5'ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5'ss. In particular, we could confirm splicing at different positions (i.e., -1, +1, +5) of a splice site for all noncanonical dinucleotides "weaker" than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5'ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5'ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking.
Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , Estudio de Asociación del Genoma Completo , Sitios de Empalme de ARN , Empalme del ARN , Adolescente , Adulto , Anciano , Empalme Alternativo , Secuencia de Bases , Línea Celular , Elementos de Facilitación Genéticos , Exones , Femenino , Estudio de Asociación del Genoma Completo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN , Adulto JovenRESUMEN
Alternative splicing plays a key role in the HIV-1 life cycle and is essential to maintain an equilibrium of mRNAs that encode viral proteins and polyprotein-isoforms. In particular, since all early HIV-1 proteins are expressed from spliced intronless and late enzymatic and structural proteins from intron containing, i.e. splicing repressed viral mRNAs, cellular splicing factors and splicing regulatory proteins are crucial for the replication capacity. In this review, we will describe the complex network of cis-acting splicing regulatory elements (SREs), which are mainly localized in the neighbourhoods of all HIV-1 splice sites and warrant the proper ratio of individual transcript isoforms. Since SREs represent binding sites for trans-acting cellular splicing factors interacting with the cellular spliceosomal apparatus we will review the current knowledge of interactions between viral RNA and cellular proteins as well as their impact on viral replication. Finally, we will discuss potential therapeutic approaches targeting HIV-1 alternative splicing.
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Empalme Alternativo , Infecciones por VIH/virología , VIH-1/genética , Replicación Viral , Animales , Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , VIH-1/fisiología , Humanos , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
BACKGROUND: The foamy viral genome encodes four central purine-rich elements localized in the integrase-coding region of pol. Previously, we have shown that the first two of these RNA elements (A and B) are required for protease dimerization and activation. The D element functions as internal polypurine tract during reverse transcription. Peters et al., described the third element (C) as essential for gag expression suggesting that it might serve as an RNA export element for the unspliced genomic transcript. RESULTS: Here, we analysed env splicing and demonstrate that the described C element composed of three GAA repeats known to bind SR proteins regulates env splicing, thus balancing the amount of gag/pol mRNAs. Deletion of the C element effectively promotes a splice site switch from a newly identified env splice acceptor to the intrinsically strong downstream localised env 3' splice acceptor permitting complete splicing of almost all LTR derived transcripts. We provide evidence that repression of this env splice acceptor is a prerequisite for gag expression. This repression is achieved by the C element, resulting in impaired branch point recognition and SF1/mBBP binding. Separating the branch point from the overlapping purine-rich C element, by insertion of only 20 nucleotides, liberated repression and fully restored splicing to the intrinsically strong env 3' splice site. This indicated that the cis-acting element might repress splicing by blocking the recognition of essential splice site signals. CONCLUSIONS: The foamy viral purine-rich C element regulates splicing by suppressing the branch point recognition of the strongest env splice acceptor. It is essential for the formation of unspliced gag and singly spliced pol transcripts.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen gag/genética , Genes env , Genes pol , Purinas/química , Spumavirus/genética , Genoma Viral , Humanos , Empalme del ARN , ARN Viral/genéticaRESUMEN
Even though splicing repression by hnRNP complexes bound to exonic sequences is well-documented, the responsible effector domains of hnRNP proteins have been described for only a select number of hnRNP constituents. Thus, there is only limited information available for possible varying silencer activities amongst different hnRNP proteins and composition changes within possible hnRNP complex assemblies. In this study, we identified the glycine-rich domain (GRD) of hnRNP proteins as a unifying feature in splice site repression. We also show that all four hnRNP D isoforms can act as genuine splicing repressors when bound to exonic positions. The presence of an extended GRD, however, seemed to potentiate the hnRNP D silencer activity of isoforms p42 and p45. Moreover, we demonstrate that hnRNP D proteins associate with the HIV-1 ESSV silencer complex, probably through direct recognition of "UUAG" sequences overlapping with the previously described "UAGG" motifs bound by hnRNP A1. Consequently, this spatial proximity seems to cause mutual interference between hnRNP A1 and hnRNP D. This interplay between hnRNP A1 and D facilitates a dynamic regulation of the repressive state of HIV-1 exon 3 which manifests as fluctuating relative levels of spliced vpr- and unspliced gag/pol-mRNAs.
Asunto(s)
Represión Epigenética/genética , Exones/genética , VIH-1/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Isoformas de Proteínas/genética , Línea Celular , Glicina/genética , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Empalme del ARN/genética , ARN Viral/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Central to the pathogenesis of malaria is the proliferation of Plasmodium falciparum parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and host cell. One key ligand, Apical Membrane Antigen 1 (AMA1), is a leading blood-stage vaccine and previous work indicates that phosphorylation of its cytoplasmic domain (CPD) is important to its function during invasion. Here we investigate the significance of each of the six available phospho-sites in the CPD. We confirm that the cyclic AMP/protein kinase A (PKA) signalling pathway elicits a phospho-priming step upon serine 610 (S610), which enables subsequent phosphorylation in vitro of a conserved, downstream threonine residue (T613) by glycogen synthase kinase 3 (GSK3). Both phosphorylation steps are required for AMA1 to function efficiently during invasion. This provides the first evidence that the functions of key invasion ligands of the malaria parasite are regulated by sequential phosphorylation steps.
Asunto(s)
Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Malaria Falciparum/metabolismo , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Sistemas de Mensajero Secundario , Antígenos de Protozoos/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Eritrocitos/metabolismo , Humanos , Malaria Falciparum/genética , Malaria Falciparum/patología , Proteínas de la Membrana/genética , Fosforilación/genética , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Dominios Proteicos , Proteínas Protozoarias/genéticaRESUMEN
BACKGROUND: The viral regulatory protein Tat is essential for establishing a productive transcription from the 5'-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3'ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE(5807-5838), henceforth referred to as ESE tat ) located between ESS2p and ESE2/ESS2. Here we show that ESE tat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3'ss A3 usage. RESULTS: Mutational inactivation or locked nucleic acid (LNA)-directed masking of the ESE tat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3'ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESE tat and ESE2 for 3'ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESE tat -negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable. CONCLUSIONS: Based on our results, we propose that splicing at 3'ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESE tat and ESE2 sequence. Mutational inactivation or interference specifically with ESE tat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.
Asunto(s)
VIH-1/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Ribonucleoproteínas/metabolismo , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesis , Línea Celular , Análisis Mutacional de ADN , Expresión Génica , VIH-1/genética , Humanos , Unión Proteica , Factores de Empalme Serina-Arginina , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: The HIV-1 accessory proteins, Viral Infectivity Factor (Vif) and the pleiotropic Viral Protein R (Vpr) are important for efficient virus replication. While in non-permissive cells an appropriate amount of Vif is critical to counteract APOBEC3G-mediated host restriction, the Vpr-induced G2 arrest sets the stage for highest transcriptional activity of the HIV-1 long terminal repeat. RESULTS: We identified a G run localized deep in the vpr AUG containing intron 3 (GI3-2), which was critical for balanced splicing of both vif and vpr non-coding leader exons. Inactivation of GI3-2 resulted in excessive exon 3 splicing as well as exon-definition mediated vpr mRNA formation. However, in an apparently mutually exclusive manner this was incompatible with recognition of upstream exon 2 and vif mRNA processing. As a consequence, inactivation of GI3-2 led to accumulation of Vpr protein with a concomitant reduction in Vif protein. We further demonstrate that preventing hnRNP binding to intron 3 by GI3-2 mutation diminished levels of vif mRNA. In APOBEC3G-expressing but not in APOBEC3G-deficient T cell lines, mutation of GI3-2 led to a considerable replication defect. Moreover, in HIV-1 isolates carrying an inactivating mutation in GI3-2, we identified an adjacent G-rich sequence (GI3-1), which was able to substitute for the inactivated GI3-2. CONCLUSIONS: The functionally conserved intronic G run in HIV-1 intron 3 plays a major role in the apparently mutually exclusive exon selection of vif and vpr leader exons and hence in vif and vpr mRNA formation. The competition between these exons determines the ability to evade APOBEC3G-mediated antiviral effects due to optimal vif expression.
Asunto(s)
Citidina Desaminasa/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Especificidad del Huésped/genética , Intrones , Desaminasa APOBEC-3G , Línea Celular , Línea Celular Tumoral , Citidina Desaminasa/genética , Productos del Gen vpr/genética , Células HEK293 , Infecciones por VIH/metabolismo , Células HeLa , Humanos , Mutación/genética , Empalme del ARN/genética , ARN Mensajero/genética , Linfocitos T/metabolismo , Linfocitos T/virología , Replicación Viral/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based 'HEXplorer score' as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide.
Asunto(s)
Empalme Alternativo , Genómica/métodos , Secuencias Reguladoras de Ácido Ribonucleico , Algoritmos , Simulación por Computador , Exones , Células HEK293 , VIH/genética , Células HeLa , Humanos , Mutación , Mutación Puntual , Precursores del ARN/química , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Viral/química , ARN Viral/metabolismoRESUMEN
HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , Interacciones Huésped-Patógeno , Fosfatidilinositol 3-Quinasas/metabolismo , Precursores del ARN/metabolismo , Empalme del ARNRESUMEN
This study focuses on the long stretch of highly conserved amino acids in the membrane proximal part of the HIV-1 cytoplasmic tail (Env amino acids (aa) 706-718) upstream of the overlap with the tat and rev second coding exons. Changes in Env aa 713 and 715, although they did not affect Env function, abrogated replicative spread. Other amino acid substitutions, i.e., 706-712, 714 and 716, despite their conservation, did not result in defective replicative phenotypes even in primary peripheral blood lymphocytes. Our results point to their involvement in presently unrecognized essential Env functions pertinent only in in vivo. Interestingly, changes in the codons for residues 717-718 as well as some mutations in residues 714-716 abrogated Gag expression but still allowed expression of functional Env in a rev-independent manner. This could be due to the inactivation of a rev-regulated negative element within the respective nucleotide sequence (8354-8368).
Asunto(s)
VIH/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia Conservada , Análisis Mutacional de ADN , Regulación Viral de la Expresión Génica/fisiología , VIH/genética , Mutación , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8(+) T-cell responses.
Asunto(s)
Antígeno HLA-B44/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Empalme Alternativo , Presentación de Antígeno/genética , Exones/genética , Antígeno HLA-B44/genética , Células HeLa , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica , Proteína Disulfuro Isomerasas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Eliminación de Secuencia/genética , Transgenes/genéticaRESUMEN
Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.
Asunto(s)
Citidina Desaminasa/metabolismo , VIH-1/genética , Intrones/genética , Empalme del ARN , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Desaminasa APOBEC-3G , Secuencia de Aminoácidos , Línea Celular , Citidina Desaminasa/genética , Células HEK293 , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/fisiología , Células HeLa , Humanos , Células Jurkat , Datos de Secuencia Molecular , Mutación , Sitios de Empalme de ARN , ARN Mensajero/genética , ARN Viral/genética , Alineación de Secuencia , Análisis de Secuencia de ARN , Linfocitos T/inmunología , Linfocitos T/virología , Regulación hacia Arriba , Replicación Viral , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/biosíntesisRESUMEN
Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3' splice site (3'ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5'ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE(vpr)) localized between exonic splicing silencer ESSV and 5'ss D3. The ESE(vpr) sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE(vpr). Further analyses revealed that ESE(vpr) supports the binding of U1 snRNA at 5'ss D3, allowing bridging interactions across the upstream exon with 3'ss A2. In line with this, an increase or decrease in the complementarity of 5'ss D3 to the 5' end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3'ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5'ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3'ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.