RESUMO
Chromosomal duplication requires de novo assembly of nucleosomes from newly synthesized histones, and the process involves a dynamic network of interactions between histones and histone chaperones. sNASP and ASF1 are two major histone H3-H4 chaperones found in distinct and common complexes, yet how sNASP binds H3-H4 in the presence and absence of ASF1 remains unclear. Here we show that, in the presence of ASF1, sNASP principally recognizes a partially unfolded Nα region of histone H3, and in the absence of ASF1, an additional sNASP binding site becomes available in the core domain of the H3-H4 complex. Our study also implicates a critical role of the C-terminal tail of H4 in the transfer of H3-H4 between sNASP and ASF1 and the coiled-coil domain of sNASP in nucleosome assembly. These findings provide mechanistic insights into coordinated histone binding and transfer by histone chaperones.
Assuntos
Chaperonas de Histonas , Histonas , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Nucleossomos , Ligação ProteicaRESUMO
A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.
Assuntos
MicroRNAs/química , MicroRNAs/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Animais , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Spodoptera/citologiaRESUMO
Assembly of the spliceosomal small nuclear ribonucleoparticle (snRNP) core requires the participation of the multisubunit SMN (survival of motor neuron) complex, which contains SMN and several Gemin proteins. The SMN and Gemin2 subunits directly bind Sm proteins, and Gemin5 is required for snRNP biogenesis and has been implicated in snRNA recognition. The RNA sequence required for snRNP assembly includes the Sm site and an adjacent 3' stem-loop, but a precise understanding of Gemin5's RNA-binding specificity is lacking. Here we show that the N-terminal half of Gemin5, which is composed of two juxtaposed seven-bladed WD40 repeat domains, recognizes the Sm site. The tandem WD40 repeat domains are rigidly held together to form a contiguous RNA-binding surface. RNA-contacting residues are located mostly on loops between ß strands on the apical surface of the WD40 domains. Structural and biochemical analyses show that base-stacking interactions involving four aromatic residues and hydrogen bonding by a pair of arginines are crucial for specific recognition of the Sm sequence. We also show that an adenine immediately 5' to the Sm site is required for efficient binding and that Gemin5 can bind short RNA oligos in an alternative mode. Our results provide mechanistic understandings of Gemin5's snRNA-binding specificity as well as valuable insights into the molecular mechanism of RNA binding by WD40 repeat proteins in general.
Assuntos
Modelos Moleculares , RNA Nuclear Pequeno/metabolismo , Proteínas do Complexo SMN/química , Proteínas do Complexo SMN/metabolismo , Repetições WD40/fisiologia , Cristalização , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , RNA Nuclear Pequeno/químicaRESUMO
We propose an approach to directly measure the bending effect on individual modes in few-mode fibers using a wavelength-scanning spatially and spectrally resolved imaging technique. By collecting a fiber output beam profile with a scanning wavelength at different bending diameters and analyzing the peaks of its Fourier transformation, we have distinguished higher-order modes (HOMs) of the fiber and investigated their group delay, beam profile, bend loss, and mode splitting individually. We have experimentally verified with a multilayer core fiber at 1-8 cm bending diameters that its HOMs experience more loss compared to lower-order modes, but delay at approximately the same speed as the bending diameter decreases. Mode splitting of the LP11 mode at a small bending diameter due to bending-induced birefringence is also observed and discussed. This method provides an efficient tool to study the bending effect on individual HOMs in fibers and could be extended to studying fiber stretching and twisting.
RESUMO
We demonstrate the two-dimensional tunable orbital angular momentum (OAM) generation in a ring-core (vortex) fiber. The LP11 mode generated by an all fiber fused coupler is coupled into a vortex fiber. Because the vector modes of the LP11 mode group in the vortex fiber are no longer degenerate, the mode status will change between linearly polarized modes (LPMs) and complex OAM modes periodically during propagation. The generated OAM can be tuned smoothly by filtering the mixed mode with different polarization directions or changing the wavelength at a certain polarization directions. The two-dimensional tuning of OAM from l=-1 to l=+1 is experimentally demonstrated in an all fiber OAM generator.
RESUMO
We present a method in this Letter to generate optical vortices with tunable orbital angular momentum (OAM) in optical fibers. The tunable OAM optical vortex is produced by combining different vector modes HE2,meven (HE2,modd) and TE0,m (TM0,m) when l=1 or combining HEl+1,meven (HEl+1,modd) and EHl-1,modd (EHl-1,meven) when l>1 with a π/2 phase shift. The vortex can be regarded as a result of overlapping two orthogonal optical vortex beams of equal helicity but opposite chirality with a π/2 phase shift. We have experimentally demonstrated the smooth variation of OAM from l=-1 to l=+1 by adjusting a polarizer at the output end of the fiber.
RESUMO
To achieve economical and efficient decolorization, two novel flocculants, weakly hydrophobic comb-like chitosan-graft-poly (N, N-Dimethylacrylamide) (CSPD) and strongly hydrophobic chain-like chitosan-graft-L-Cyclohexylglycine (CSLC) were synthesized in this study. To assess the effectiveness and application of CSPD and CSLC, the impacts of factors, including flocculant dosages, initial pH, initial dye concentrations, co-existing inorganic ions and turbidities, on the decolorization performance were explored. The results suggested that the optimum decolorizing efficiencies of the five anionic dyes ranged from 83.17% to 99.40%. Moreover, for accurately controlling flocculation performance, the responses to flocculant molecular structures and hydrophobicity in flocculation using CSPD and CSLC were studied. The Comb-like structure gives CSPD a wider dosage range for effective decolorization and better efficiencies with large molecule dyes under weak alkaline conditions. The strong hydrophobicity makes CSLC more effective in decolorization and more suitable for removing small molecule dyes under weak alkaline conditions. Meanwhile, the responses of removal efficiency and floc size to flocculant hydrophobicity are more sensitive. Mechanism studies revealed that charge neutralization, hydrogen bonding and hydrophobic association worked together in the decolorization of CSPD and CSLC. This study has provided meaningful guidance for developing flocculants in the treatment of diverse printing and dyeing wastewater.
RESUMO
BACKGROUND: Effective targeted therapy for lung adenocarcinoma (LUAD), the number one cancer killer worldwide, continues to be a difficult problem because of the limitation of number of applicable patients and acquired resistance. Identifying more promising drug targets for LUAD treatment holds immense clinical significance. Recent studies have revealed that the U2 auxiliary factor (U2AF) homology motif kinase 1 (UHMK1) is a robust pro-oncogenic factor in many cancers. However, its biological functions and the underlying molecular mechanisms in LUAD have not been investigated. METHODS: The UHMK1 expression in LUAD cells and tissues was evaluated by bioinformatics analysis, immunohistochemistry (IHC), western blotting (WB), and real time quantitative polymerase chain reaction (RT-qPCR) assays. A series of gain- and loss-of-function experiments for UHMK1 were carried out to investigate its biological functions in LUAD in vitro and in vivo. The mechanisms underlying UHMK1's effects in LUAD were analyzed by transcriptome sequencing and WB assays. RESULTS: UHMK1 expression was aberrantly elevated in LUAD tumors and cell lines and positively correlated with tumor size and unfavorable patient prognosis. Functionally, UHMK1 displayed robust pro-oncogenic capacity in LUAD and mechanistically exerted its biological effects via the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. CONCLUSION: UHMK1 is a potent oncogene in LUAD. Targeting UHMK1 may significantly improve the effect of LUAD treatment via inhibiting multiple biological ways of LUAD progression.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Adenocarcinoma de Pulmão/patologia , Transdução de Sinais , Neoplasias Pulmonares/patologia , Transformação Celular Neoplásica , Carcinogênese , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Nucleolar and spindle associated protein 1 (NUSAP1), an indispensable mitotic regulator, has been reported to be involved in the development, progression, and metastasis of several types of cancer. Here, we investigated the expression and biological function of NUSAP1 in human glioblastoma (GBM), an aggressive brain tumor type with largely ineffective treatment options. Analysis of the molecular data in CGGA, TCGA and Rembrandt datasets demonstrated that NUSAP1 was significantly upregulated in GBM relative to low grade gliomas and non-neoplastic brain tissue samples. Kaplan-Meier analysis indicated that patients with tumors showing high NUSAP1 expression exhibited significantly poorer survival in both CGGA (P = 0.002) and Rembrandt cohorts (P = 0.017). Analysis of RNA sequencing data from P3-cells with stable knockdown of NUSAP1 revealed topoisomerase 2A (TOP2A) as a possible molecule downregulated by the loss of NUSAP1. Molecular analysis of the CGGA data revealed a strong correlation between NUSAP1 and TOP2A expression in primary gliomas and recurrent gliomas samples. SiRNA knockdown of either NUSAP1 or TOP2A in U251, T98 and GBM derived patient P3 cells inhibited GBM cell proliferation and invasion, and induced cell apoptosis. Finally, stable knockdown of NUSAP1 with shRNA led to decreased tumor growth in an orthotopic xenograft model of GBM in mice. Taken together, NUSAP1 gene silencing induced apoptosis possibly through the downregulation of the candidate downstream molecule TOP2A. Interference with the expression of NUSAP1 might therefore inhibit malignant progression in GBM, and NUSAP1 might thus serve as a promising molecular target for GBM treatment.
Assuntos
Neoplasias Encefálicas , DNA Topoisomerases Tipo II , Glioblastoma , Glioma , Proteínas Associadas aos Microtúbulos , Proteínas de Ligação a Poli-ADP-Ribose , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo II/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Glioma/genética , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Interferente Pequeno/genéticaRESUMO
Alternative splicing is involved in functional regulation of the mutagenic enzyme activation-induced cytidine deaminase (AID). However, the molecular basis for AID splicing regulation remains undefined. Using a mini-gene-based screen in HeLa cells, we found that overexpression of RNA binding motif protein 5 (RBM5, or LUCA-15/H37) significantly promoted AID exon 4 skipping by suppressing the splicing of intron 3. The inhibitive effect of RBM5 on intron 3 splicing required a weak 3'-splice site (ss). Indicative of the underlying mechanism, RBM5 interfered with the binding of U2AF65 to the polypyrimidine tract at the 3'-ss in vitro. Our findings thus not only shed lights on the regulatory mechanism of AID exon 4 skipping, but also provide new insights into how RBM5 functions in splicing regulation.
Assuntos
Processamento Alternativo , Proteínas de Ciclo Celular/genética , Citidina Desaminase/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Proteínas Supressoras de Tumor/genética , Regiões 3' não Traduzidas , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Citidina Desaminase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Éxons , Regulação da Expressão Gênica , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AF , Proteínas Supressoras de Tumor/metabolismoRESUMO
RNA binding motif protein 5 (RBM5) is a candidate tumor suppressor gene. Recent studies showed that RBM5 functions as an alternative splicing regulator of apoptosis-related genes. Here, we identify DHX15 and PRP19, two spliceosome components, as novel RBM5-interacting partners. We then show that the G-patch domain of RBM5 is indispensable for its ability to interact with DHX15. Strikingly, we find that RBM5 stimulates the helicase activity of DHX15 in a G patch domain-dependent manner in vitro. Helicase activities play critical roles in modulating pre-mRNA splicing. Our findings thus suggest a new mechanism underlying the regulatory roles of RBM5 in pre-mRNA splicing.