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1.
Hum Mutat ; 35(3): 308-17, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24302620

RESUMO

Mutations in SNRP200 gene cause autosomal-dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre-mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild-type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a ß-globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2-wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5'-splice-site recognition and that the RP-linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function.


Assuntos
Mutação , Sítios de Splice de RNA/genética , Retinose Pigmentar/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Processamento Alternativo , Clonagem Molecular , Genes Reporter , Células HeLa , Humanos , RNA Helicases/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Spliceossomos , Globinas beta/genética , Globinas beta/metabolismo
2.
Life Sci Alliance ; 6(6)2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37019475

RESUMO

A subset of patients with retinitis pigmentosa (RP) carry mutations in several spliceosomal components including the PRPF8 protein. Here, we established two alleles of murine Prpf8 that genocopy or mimic aberrant PRPF8 found in RP patients-the substitution p.Tyr2334Asn and an extended protein variant p.Glu2331ValfsX15. Homozygous mice expressing the aberrant Prpf8 variants developed within the first 2 mo progressive atrophy of the cerebellum because of extensive granule cell loss, whereas other cerebellar cells remained unaffected. We further show that a subset of circRNAs were deregulated in the cerebellum of both Prpf8-RP mouse strains. To identify potential risk factors that sensitize the cerebellum for Prpf8 mutations, we monitored the expression of several splicing proteins during the first 8 wk. We observed down-regulation of all selected splicing proteins in the WT cerebellum, which coincided with neurodegeneration onset. The decrease in splicing protein expression was further pronounced in mouse strains expressing mutated Prpf8. Collectively, we propose a model where physiological reduction in spliceosomal components during postnatal tissue maturation sensitizes cells to the expression of aberrant Prpf8 and the subsequent deregulation of circRNAs triggers neuronal death.


Assuntos
Proteínas de Ligação a RNA , Retinose Pigmentar , Animais , Camundongos , Proteínas de Ligação a RNA/genética , RNA Circular , Mutação , Cerebelo
3.
PLoS One ; 17(4): e0265742, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35385551

RESUMO

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5' splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity.


Assuntos
RNA Helicases DEAD-box , Fatores de Processamento de RNA , Retinose Pigmentar , RNA Helicases DEAD-box/genética , Humanos , Mutação , Sítios de Splice de RNA , Splicing de RNA , Fatores de Processamento de RNA/genética , Retinose Pigmentar/genética , Spliceossomos/metabolismo
4.
Hum Mol Genet ; 18(11): 2014-23, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19293337

RESUMO

The AD29 mutation in HPRP31 belongs to a series of mutations that were initially linked with the autosomal dominant disorder retinitis pigmentosa (RP) type 11. The HPRP31 gene encodes the hPrp31 protein that specifically associates with spliceosomal small nuclear ribonucleoprotein particles (snRNPs). Despite intensive research, it is still unclear how the AD29 (Ala216Pro) mutation causes RP. In this study, we report that the expression of this mutant protein affects cell proliferation and alters the structure of nuclear Cajal bodies that are connected with snRNP metabolism. Interestingly, these effects can be reversed by the over-expression of the hPrp6 protein, a binding partner of hPrp31. Although Ala216 is not contained within the U4 or U5 snRNP interacting domains, we present several lines of evidence that demonstrate that the association between the AD29 mutant and snRNPs in the cell nucleus is significantly reduced. Finally, we show that the stability of the AD29 mutant is severely affected resulting in its rapid degradation. Taken together, our results indicate that the Ala216Pro mutation destabilizes the hPrp31 protein structure in turn reducing its interaction with snRNP binding partners and leading to its rapid degradation. These findings significantly impact our understanding of the molecular mechanisms underlying RP and suggest that the insufficiency of the functional hPrp31 protein combined with the potential cytotoxicity associated with the expression the AD29 mutant are at least partially causative of the RP phenotype.


Assuntos
Proteínas do Olho/química , Proteínas do Olho/genética , Mutação de Sentido Incorreto , Retinose Pigmentar/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Corpos Enovelados/genética , Corpos Enovelados/metabolismo , Proteínas do Olho/metabolismo , Células HeLa , Humanos , Ligação Proteica , Estabilidade Proteica , Retinose Pigmentar/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética
5.
J Struct Biol ; 165(2): 107-17, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19056497

RESUMO

Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories/chromatin is maintained in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of nucleolus organizer region-bearing chromosomes. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non-random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome organization, there is also a significant stochastic component associated with reassortment of chromosome territories/chromatin that results in their positional rearrangements.


Assuntos
Cromatina/química , Linhagem Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromossomos/ultraestrutura , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Fatores de Tempo
6.
Chromosoma ; 117(5): 487-97, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18548265

RESUMO

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis.


Assuntos
Proteínas de Transporte/metabolismo , Nucléolo Celular/enzimologia , DNA Helicases/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Nucléolo Celular/ultraestrutura , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/biossíntese , Transcrição Gênica
7.
J Cell Biol ; 216(6): 1579-1596, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28515276

RESUMO

Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteômica/métodos , Interferência de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Transfecção
8.
J Cell Biochem ; 94(5): 899-916, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15714458

RESUMO

Evidence is presented for the reversible, cold-dependent immunofluorescence detection of the epitope (hereafter referred to as epiC), recognized by a monoclonal anti-actin antibody in diploid human fibroblast cell nuclei and mitotic chromosomes. The nuclear/chromosomal epiC was detected in a cell cycle window beginning in early S phase and extending through S phase, G(2) phase, mitosis until early G(1) phase of the subsequent daughter cells. A small but significant level of co-localization was measured between the nuclear epiC and active sites of DNA replication in early S phase. The level of co-localization was strikingly enhanced beginning approximately 1 h after the initial labeling of early S phase replicating chromatin domains. In contrast, epiC did not co-localize with late S phase replicated chromatin either during DNA replication or at any other time in the cell cycle. We propose a replication-coupled modulation of early S phase replicated chromatin domains that is detected by the chromatin epiC positivity, persists on the chromatin domains from early S until early G(1) of the next cell generation, and may be involved in the regulation and/or coordination of replicational and transcriptional processes during the cell cycle. Further studies will be required to resolve the possible role of nuclear actin in this modulation process.


Assuntos
Actinas/imunologia , Cromatina/química , Replicação do DNA , Western Blotting , Células Cultivadas , Fase G1 , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Fase S
9.
J Cell Biochem ; 94(1): 126-38, 2005 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-15523671

RESUMO

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase.


Assuntos
Replicação do DNA , Fase S , Fluorescência , Células HeLa , Humanos , Cinética , Microscopia Eletrônica
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