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1.
Nucleic Acids Res ; 51(7): 3357-3374, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-36869663

RESUMO

The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.


Assuntos
Proteômica , Ribonucleoproteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Ligação a RNA , RNA/genética
2.
Nucleic Acids Res ; 50(4): 2172-2189, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-35150569

RESUMO

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.


Assuntos
MicroRNAs , Complexo de Inativação Induzido por RNA , Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Coativadores de Receptor Nuclear/química , Ribonuclease III/genética , Ribonuclease III/metabolismo
3.
Nucleic Acids Res ; 49(2): 1094-1113, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367824

RESUMO

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Trifosfato de Adenosina/metabolismo , Proteínas do Olho/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Mol Cell ; 39(4): 618-31, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20797632

RESUMO

Posttranslational SUMO modification is an important mechanism of regulating protein function, especially in the cell nucleus. The nucleolus is the subnuclear organelle responsible for rRNA synthesis, processing, and assembly of the large and small ribosome subunits. Here, we have used SILAC-based quantitative proteomics to identify nucleolar SUMOylated proteins. This reveals a role for SUMOylation in the biogenesis and/or function of small nucleolar ribonucleoprotein complexes (snoRNPs) via the targeting of Nhp2 and Nop58. Using combined in vitro and in vivo approaches, both Nhp2 and Nop58 (also known as Nop5) are shown to be substrates for SUMOylation. Mutational analyses revealed the sites of modification on Nhp2 as K5, and on Nop58 as K467 and K497. Unlike Nop58 and Nhp2, the closely related Nop56 and 15.5K proteins appear not to be SUMO targets. SUMOylation is essential for high-affinity Nop58 binding to snoRNAs. This study provides direct evidence linking SUMO modification with snoRNP function.


Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Humanos , Lisina , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteômica/métodos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Proteína SUMO-1/metabolismo , Transfecção , Ubiquitinas/metabolismo
5.
Mol Cell ; 39(6): 912-924, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20864038

RESUMO

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.


Assuntos
Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Multimerização Proteica/fisiologia , RNA Polimerase II/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Alfa-Amanitina/farmacologia , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Genes Reporter/genética , HIV-1/genética , Humanos , Complexos Multiproteicos/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteômica , RNA Polimerase I/metabolismo , RNA Polimerase II/genética , RNA Interferente Pequeno
6.
RNA Biol ; 14(6): 680-692, 2017 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27715451

RESUMO

Box C/D and box H/ACA snoRNAs are abundant non-coding RNAs that localize in the nucleolus and mostly function as guides for nucleotide modifications. While a large pool of snoRNAs modifies rRNAs, an increasing number of snoRNAs could also potentially target mRNAs. ScaRNAs belong to a family of specific RNAs that localize in Cajal bodies and that are structurally similar to snoRNAs. Most scaRNAs are involved in snRNA modification, while telomerase RNA, which contains H/ACA motifs, functions in telomeric DNA synthesis. In this review, we describe how box C/D and H/ACA snoRNAs are processed and assembled with core proteins to form functional RNP particles. Their biogenesis involve several transport factors that first direct pre-snoRNPs to Cajal bodies, where some processing steps are believed to take place, and then to nucleoli. Assembly of core proteins involves the HSP90/R2TP chaperone-cochaperone system for both box C/D and H/ACA RNAs, but also several factors specific for each family. These assembly factors chaperone unassembled core proteins, regulate the formation and disassembly of pre-snoRNP intermediates, and control the activity of immature particles. The AAA+ ATPase RUVBL1 and RUVBL2 belong to the R2TP co-chaperones and play essential roles in snoRNP biogenesis, as well as in the formation of other macro-molecular complexes. Despite intensive research, their mechanisms of action are still incompletely understood.


Assuntos
RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Animais , Proteínas de Transporte , Corpos Enovelados/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico , Processamento Pós-Transcricional do RNA , RNA Nucleolar Pequeno/química , Transdução de Sinais , Transcrição Gênica
7.
Nucleic Acids Res ; 43(18): 8973-89, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26275778

RESUMO

The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins.


Assuntos
Proteínas do Olho/metabolismo , Chaperonas Moleculares/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas do Complexo SMN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Linhagem Celular , Corpos Enovelados/metabolismo , Citoplasma/metabolismo , Proteínas do Olho/química , Proteínas do Olho/genética , Células HeLa , Humanos , Mutagênese Insercional , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética
8.
Nucleic Acids Res ; 42(13): 8663-77, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25013170

RESUMO

Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5'-end m(7)G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5'-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.


Assuntos
Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Selenoproteínas/genética , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/metabolismo , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/genética , Humanos , Metilação , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Polirribossomos/química , Biossíntese de Proteínas , RNA Mensageiro/análise , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas do Complexo SMN/metabolismo , Selenoproteínas/biossíntese , Selenoproteínas/metabolismo , Glutationa Peroxidase GPX1
9.
EMBO J ; 30(11): 2205-18, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21522132

RESUMO

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre-snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine-rich nuclear export signal, shuttles in a CRM1-dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/metabolismo , RNA Nucleolar Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Humanos , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteína Exportina 1
10.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38858088

RESUMO

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.


Assuntos
Nucléolo Celular , Proteômica , Ribossomos , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/metabolismo , Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Humanos , Proteômica/métodos , Proteínas Nucleares/metabolismo , Corpos Enovelados/metabolismo , Células HeLa , Retículo Endoplasmático/metabolismo
11.
Hum Mol Genet ; 20(10): 1873-85, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21330300

RESUMO

The AFF (AF4/FMR2) family of genes includes four members: AFF1/AF4, AFF2/FMR2, AFF3/LAF4 and AFF4/AF5q31. AFF2/FMR2 is silenced in FRAXE intellectual disability, while the other three members have been reported to form fusion genes as a consequence of chromosome translocations with the myeloid/lymphoid or mixed lineage leukemia (MLL) gene in acute lymphoblastic leukemias (ALLs). All AFF proteins are localized in the nucleus and their role as transcriptional activators with a positive action on RNA elongation was primarily studied. We have recently shown that AFF2/FMR2 localizes to nuclear speckles, subnuclear structures considered as storage/modification sites of pre-mRNA splicing factors, and modulates alternative splicing via the interaction with the G-quadruplex RNA-forming structure. We show here that similarly to AFF2/FMR2, AFF3/LAF4 and AFF4/AF5q31 localize to nuclear speckles and are able to bind RNA, having a high apparent affinity for the G-quadruplex structure. Interestingly, AFF3/LAF4 and AFF4/AF5q31, like AFF2/FMR2, modulate, in vivo, the splicing efficiency of a mini-gene containing a G-quadruplex structure in one alternatively spliced exon. Furthermore, we observed that the overexpression of AFF2/3/4 interferes with the organization and/or biogenesis of nuclear speckles. These findings fit well with our observation that enlarged nuclear speckles are present in FRAXE fibroblasts. Furthermore, our findings suggest functional redundancy among the AFF family members in the regulation of splicing and transcription. It is possible that other members of the AFF family compensate for the loss of AFF2/FMR2 activity and as such explain the relatively mild to borderline phenotype observed in FRAXE patients.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Fibroblastos/metabolismo , Expressão Gênica/genética , Ordem dos Genes , Genes Reporter/genética , Células HeLa , Humanos , Espaço Intranuclear/metabolismo , Dados de Sequência Molecular , Transporte Proteico , Splicing de RNA/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
12.
Mol Cell Proteomics ; 9(5): 861-79, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20023298

RESUMO

The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.


Assuntos
Biblioteca de Peptídeos , Mapeamento de Interação de Proteínas/métodos , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Humanos , Marcação por Isótopo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Reprodutibilidade dos Testes
13.
J Mol Biol ; 434(19): 167760, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35901867

RESUMO

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built a 3D model of DPCD. In addition, we used a variety of orthogonal biophysical techniques including small-angle X-ray scattering, structural mass spectrometry and electron microscopy to assess the molecular determinants of DPCD interaction with R1R2. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Transporte , DNA Helicases , Complexos Multiproteicos , Proteínas , ATPases Associadas a Diversas Atividades Celulares/química , Proteínas de Transporte/química , DNA Helicases/química , Humanos , Complexos Multiproteicos/química , Proteínas/química
14.
Nat Commun ; 12(1): 3646, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131137

RESUMO

U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.


Assuntos
Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fatores de Alongamento de Peptídeos , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
15.
Nat Commun ; 12(1): 4810, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376666

RESUMO

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.


Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mucosa Intestinal/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Humanos , Mucosa Intestinal/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo
16.
Structure ; 26(9): 1196-1209.e8, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30033218

RESUMO

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90. We also established the 3D structure of an RPAP3:PIH1D1 sub-complex demonstrating the need for a 34-residue insertion, specific of RPAP3 isoform 1, for the tight binding of PIH1D1. We also confirm the existence of a complex lacking PIH1D1 in human cells (R2T), which shows differential binding to certain clients. These results highlight similarities and differences between the yeast and human R2TP complexes, and document the diversification of this family of co-chaperone complexes in human.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Sítios de Ligação , Linhagem Celular , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerização Proteica
17.
Nat Commun ; 9(1): 2093, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29844425

RESUMO

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Testículo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/genética , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de Membrana/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Transdução de Sinais
18.
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
19.
Cell Rep ; 18(11): 2635-2650, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28297668

RESUMO

The nuclear cap-binding complex (CBC) stimulates processing reactions of capped RNAs, including their splicing, 3'-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3'-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA) transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.


Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , RNA/metabolismo , Células HEK293 , Células HeLa , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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