RESUMO
U7 snRNP is a multisubunit endonuclease required for 3' end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B, and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50, and pICln known to methylate arginines in the carboxy-terminal regions of the Sm proteins B, D1, and D3 during the spliceosomal Sm ring assembly. Both biochemical and cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the amino-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an amino-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.
Assuntos
Ribonucleoproteína Nuclear Pequena U7 , Ribonucleoproteínas Nucleares Pequenas , Animais , Ribonucleoproteína Nuclear Pequena U7/química , Metilação , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Histonas/metabolismo , Arginina/químicaRESUMO
MicroRNAs play a critical role in bone marrow mesenchymal stem cell (MSC) chondrogenesis and regulate the progression of joint regeneration in osteoarthritis. Our previous research confirmed that miR146a relieves osteoarthritis by modulating cartilage homeostasis. However, few studies have revealed the relationship between miR146a and the chondrogenesis of MSCs, and the exact mechanisms remain unclear. This study aimed to determine the function of miR146a in the chondrogenic differentiation of MSCs and the potential mechanisms involved. MiR146a expression increased during chondrogenesis. MiR146a knockout (KO) led to the increased chondrogenesis of MSCs compared to that in wild-type (WT) MSCs, whereas the overexpression of miR146a by mimics resulted in the decreased chondrogenesis of MSCs, as determined by the mRNA expression of collagen, type II, alpha 1 (COL2A1), aggrecan, cartilage oligomeric matrix protein (COMP), and matrix metallopeptidase 13 (MMP13). Furthermore, cartilage defects could be treated better when injected with spheres induced from miR146aKO MSCs than from WT MSCs, indicating that miR146a inhibits chondrogenesis in vivo. In addition, based on miRNA-mRNA prediction analysis and a dual-luciferase reporter assay, we observed that the deletion of miR146a led to the increased expression of Lsm11 during chondrogenesis and demonstrated that miR146a targeted Lsm11 by binding to its 3'-untranslated region (UTR) and inhibited its translation. The inhibition of Lsm11 by silencing RNA (siRNA) reversed the increased ability of chondrogenesis by knocking out miR146a both in vivo and in vitro, suggesting that miR146a inhibits chondrogenesis by directly inhibiting Lsm11 in MSCs, which may be a novel target for treating osteoarthritis.
Assuntos
Células-Tronco Mesenquimais , MicroRNAs , Osteoartrite , Humanos , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Cleavage of histone pre-mRNAs at the 3' end requires stem-loop binding protein (SLBP) and U7 snRNP that consists of U7 snRNA and a unique Sm ring containing two U7-specific proteins: Lsm10 and Lsm11. Lsm11 interacts with FLASH and together they bring a subset of polyadenylation factors to U7 snRNP, including the CPSF73 endonuclease that cleaves histone pre-mRNA. SLBP binds to a conserved stem-loop structure upstream of the cleavage site and acts by promoting an interaction between the U7 snRNP and a sequence element located downstream from the cleavage site. We show that both human and Drosophila SLBPs stabilize U7 snRNP on histone pre-mRNA via two regions that are not directly involved in recognizing the stem-loop structure: helix B of the RNA binding domain and the C-terminal region that follows the RNA binding domain. Stabilization of U7 snRNP binding to histone pre-mRNA by SLBP requires FLASH but not the polyadenylation factors. Thus, FLASH plays two roles in 3' end processing of histone pre-mRNAs: It interacts with Lsm11 to form a docking platform for the polyadenylation factors, and it cooperates with SLBP to recruit U7 snRNP to histone pre-mRNA.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Histonas/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
3'-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3'-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3'-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem-loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation-CPSF, CstF, and CF Im-are present in Drosophila nuclear extracts in a stable supercomplex.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/genética , Processamento de Terminações 3' de RNA , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Drosophila melanogaster , Histonas/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Subunidades Proteicas/metabolismo , Clivagem do RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Faithful DNA replication requires that cells fine-tune their histone pool in coordination with cell-cycle progression. Replication-dependent histone biosynthesis is initiated at a low level upon cell-cycle commitment, followed by a burst at the G1/S transition, but it remains unclear how exactly the cell regulates this burst in histone biosynthesis as DNA replication begins. Here, we use single-cell time-lapse imaging to elucidate the mechanisms by which cells modulate histone production during different phases of the cell cycle. We find that CDK2-mediated phosphorylation of NPAT at the restriction point triggers histone transcription, which results in a burst of histone mRNA precisely at the G1/S phase boundary. Excess soluble histone protein further modulates histone abundance by promoting the degradation of histone mRNA for the duration of S phase. Thus, cells regulate their histone production in strict coordination with cell-cycle progression by two distinct mechanisms acting in concert.
Assuntos
Ciclina E , Histonas , Histonas/metabolismo , Fase S , Ciclina E/genética , Ciclina E/metabolismo , Proteínas Nucleares/metabolismo , Retroalimentação , Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Ciclo Celular , RNA MensageiroRESUMO
BACKGROUND: Brain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen. RESULTS: The knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers. CONCLUSIONS: The screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED) were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.
Assuntos
Sistemas CRISPR-Cas , Glioma , Humanos , Criança , Metilação de DNA , Glioma/genética , Genes Reguladores , Histonas/genéticaRESUMO
Histone pre-mRNAs are cleaved at the 3' end by a complex that contains U7 snRNP, the FLICE-associated huge protein (FLASH) and histone pre-mRNA cleavage complex (HCC) consisting of several polyadenylation factors. Within the complex, the N terminus of FLASH interacts with the N terminus of the U7 snRNP protein Lsm11, and together they recruit the HCC. FLASH through its distant C terminus independently interacts with the C-terminal SANT/Myb-like domain of nuclear protein, ataxia-telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both hydrogen/deuterium exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo.