scholarly journals Leigh Syndrome-inducing Mutations Affect LRPPRC / SLIRP Complex Formation

2020 ◽  
Author(s):  
Sandrine Coquille ◽  
Stéphane Thore
Keyword(s):  
Complex Formation ◽  
Rna Binding ◽  
Leigh Syndrome ◽  
Stable Complex ◽  
Specific Gene ◽  
Functional Domain ◽  
Independent Manner ◽  
Stem Loop

ABSTRACTMitochondria are essential organelles carrying their own genetic information which require specific gene expression processes. The leucine rich pentatricopeptide protein (LRPPRC) and its partner the SRA stem-loop interacting RNA binding protein (SLIRP) form a stable complex implicated in mRNA stability and polyadenylation. LRPPRC/SLIRP complex formation is still poorly characterized. We demonstrate that SLIRP interacts with the N-terminal region of LRPPRC in a RNA independent manner. We further show that the complex is stable in presence of high salt concentration. Point mutation and deletions found in the LRPPRC protein and responsible for the French-Canadian Leigh Syndrome (LSFC) are shown to affect complex formation in vitro. Our data are identifying the key region of LRPPRC involved in SLIRP association and showing the direct consequence of various LSFC mutations on the complex formation. Further experiments aiming at deciphering LRPPRC/SLIRP function(s) in vivo will benefit from our functional domain characterization.

scholarly journals Dissecting the Fine Details of Assembly of aT = 3 Phage Capsid

2005 ◽  
Vol 6 (2) ◽  
pp. 119-125 ◽  
Author(s):  
P. G. Stockley ◽  
A. E. Ashcroft ◽  
S. Francese ◽  
G. S. Thompson ◽  
N. A. Ranson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Model Systems ◽  
Replicase Gene ◽  
Nmr Studies ◽  
Partial Explanation ◽  
Stem Loop ◽  
Assembly Pathway

The RNA bacteriophages represent ideal model systems in which to probe the detailed assembly pathway for the formation of aT = 3 quasi-equivalent capsid. For MS2, the assembly reaction can be probedin vitrousing acid disassembled coat protein subunits and a short (19 nt) RNA stem-loop that acts as the translational operator of the replicase gene and leads to sequence-specific sequestration and packaging of the cognate phage RNAin vivo. Reassembly reactions can be initiated by mixing these components at neutral pH. The molecular basis of the sequence-specific RNA–protein interaction is now well understood. Recent NMR studies on the protein demonstrate extensive mobility in the loops of the polypeptide that alter their conformations to form the quasi-equivalent conformers of the final capsid. It seems reasonable to assume that RNA binding results in reduction of this flexibility. However, mass spectrometry suggests that these RNA–protein complexes may only provide one type of quasi-equivalent capsid building block competent to form five-fold axes but not the full shell. Work with longer RNAs suggests that the RNA may actively template the assembly pathway providing a partial explanation of how conformers are selected in the growing shell.

scholarly journals Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse

2018 ◽  
Vol 115 (47) ◽  
pp. E11061-E11070 ◽  
Author(s):  
Kyu-Hyeon Yeom ◽  
Simon Mitchell ◽  
Anthony J. Linares ◽  
Sika Zheng ◽  
Chia-Ho Lin ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Rna Binding ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Stem Loop ◽  
Polypyrimidine Tract Binding Protein ◽  
Precursor Rna

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem–loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

scholarly journals Transcriptional Pausing in the Bacillus subtilis pyr Operon In Vitro: a Role in Transcriptional Attenuation?

2003 ◽  
Vol 185 (16) ◽  
pp. 4764-4771 ◽  
Author(s):  
Hesheng Zhang ◽  
Robert L. Switzer
Keyword(s):  
Bacillus Subtilis ◽  
Rna Binding ◽  
Bacterial Species ◽  
Regulatory Protein ◽  
Stem Loop ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Transcriptional Pausing

ABSTRACT The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro. Using templates with each of the three known B. subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo. With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

scholarly journals Drosophila Stem-Loop Binding Protein Intracellular Localization Is Mediated by Phosphorylation and Is Required for Cell Cycle-regulated Histone mRNA Expression

2004 ◽  
Vol 15 (3) ◽  
pp. 1112-1123 ◽  
Author(s):  
David J. Lanzotti ◽  
Jeremy M. Kupsco ◽  
Xiao-Cui Yang ◽  
Zbigniew Dominski ◽  
William F. Marzluff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Binding ◽  
Binding Protein ◽  
Intracellular Localization ◽  
Mrna Processing ◽  
Nuclear Targeting ◽  
Histone Mrna ◽  
Stem Loop

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.

scholarly journals Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

2007 ◽  
Vol 18 (10) ◽  
pp. 4119-4128 ◽  
Author(s):  
Huaiping Zhu ◽  
Guangyu Zhu ◽  
Jay Liu ◽  
Zhimin Liang ◽  
Xuejun C. Zhang ◽  
...  
Keyword(s):  
Membrane Targeting ◽  
Binding Motif ◽  
Functional Domain ◽  
Nucleotide Exchange ◽  
Independent Manner ◽  
Nucleotide Exchange Factor ◽  
Early Endosomes ◽  
Necessary And Sufficient

Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135–399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81–135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135–230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5–independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals A novel BRD4 inhibitor suppresses osteoclastogenesis and ovariectomized osteoporosis by blocking RANKL-mediated MAPK and NF-κB pathways

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Ying Liu ◽  
Wenjie Liu ◽  
Ziqiang Yu ◽  
Yan Zhang ◽  
Yinghua Li ◽  
...  
Keyword(s):  
Bone Loss ◽  
Osteoporosis Treatment ◽  
Inhibitory Effect ◽  
Specific Gene ◽  
Actin Ring ◽  
Treatment Target ◽  
Significant Inhibitory Effect ◽  
Promising Treatment

AbstractBromodomain-containing protein 4 (BRD4) has emerged as a promising treatment target for bone-related disorders. (+)-JQ1, a thienotriazolodiazepine compound, has been shown to inhibit pro-osteoclastic activity in a BRD4-dependent approach and impede bone loss caused by ovariectomy (OVX) in vivo. However, clinical trials of (+)-JQ1 are limited because of its poor druggability. In this study, we synthesized a new (+)-JQ1 derivative differing in structure and chirality. One such derivative, (+)-ND, exhibited higher solubility and excellent inhibitory activity against BRD4 compared with its analogue (+)-JQ1. Interestingly, (-)-JQ1 and (-)-ND exhibited low anti-proliferative activity and had no significant inhibitory effect on RANKL-induced osteoclastogenesis as compared with (+)-JQ1 and (+)-ND, suggesting the importance of chirality in the biological activity of compounds. Among these compounds, (+)-ND displayed the most prominent inhibitory effect on RANKL-induced osteoclastogenesis. Moreover, (+)-ND could inhibit osteoclast-specific gene expression, F‐actin ring generation, and bone resorption in vitro and prevent bone loss in OVX mice. Collectively, these findings indicated that (+)-ND represses RANKL‐stimulated osteoclastogenesis and averts OVX-triggered osteoporosis by suppressing MAPK and NF-κB signalling cascades, suggesting that it may be a prospective candidate for osteoporosis treatment.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

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