scholarly journals α-Actinin TvACTN3 ofTrichomonas vaginalisIs an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism

2014 ◽  
Vol 2014 ◽  
pp. 1-20 ◽  
Author(s):  
Jaeson Santos Calla-Choque ◽  
Elisa Elvira Figueroa-Angulo ◽  
Leticia Ávila-González ◽  
Rossana Arroyo
Keyword(s):  
Trichomonas Vaginalis ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
De Novo ◽  
Binding Protein ◽  
Regulation Of Gene Expression ◽  
Iron Concentration ◽  
Rna Binding Protein ◽  
Cross Linking ◽  
Cytoplasmic Protein

Trichomonas vaginalisis a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration inT. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein inT. vaginalisthat interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts fromT. vaginaliswith the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays asα-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS andde novosequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron inT. vaginalisthrough a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors

2002 ◽  
Vol 115 (5) ◽  
pp. 1027-1039 ◽  
Author(s):  
Mark P. Sowden ◽  
Nazzareno Ballatori ◽  
Karen L. de Mesy Jensen ◽  
Lakesha Hamilton Reed ◽  
Harold C. Smith
Keyword(s):  
Apolipoprotein B ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Catalytic Subunit ◽  
Cross Linking ◽  
Primary Hepatocytes ◽  
Mrna Editing ◽  
Apob Mrna ◽  
Rat Primary Hepatocytes

Apolipoprotein B mRNA cytidine to uridine editing requires the assembly of a multiprotein editosome comprised minimally of the catalytic subunit,apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1), and an RNA-binding protein, APOBEC-1 complementation factor (ACF). A rat homolog has been cloned with 93.5% identity to human ACF (huACF). Peptide-specific antibodies prepared against huACF immunoprecipitated a rat protein of similar mass as huACF bound to apolipoprotein B (apoB) RNA in UV cross-linking reactions, thereby providing evidence that the p66, mooring sequence-selective, RNA-binding protein identified previously in rat liver by UV cross-linking and implicated in editosome assembly is a functional homolog of huACF. The rat protein (p66/ACF) was distributed in both the nucleus and cytoplasm of rat primary hepatocytes. Within a thin section, a significant amount of total cellular p66/ACF was cytoplasmic, with a concentration at the outer surface of the endoplasmic reticulum. Native APOBEC-1 co-fractionated with p66/ACF in the cytoplasm as 60S complexes. In the nucleus, the biological site of apoB mRNA editing, native p66/ACF, was localized to heterochromatin and fractionated with APOBEC-1 as 27S editosomes. When apoB mRNA editing was stimulated in rat primary hepatocytes with ethanol or insulin, the abundance of p66/ACF in the nucleus markedly increased. It is proposed that the heterogeneity in size of complexes containing editing factors is functionally significant and reflects functionally engaged editosomes in the nucleus and an inactive cytoplasmic pool of factors.

scholarly journals Targeting the RNA-Binding Protein HuR Alleviates Neuroinflammation in Experimental Autoimmune Encephalomyelitis: Potential Therapy for Multiple Sclerosis

2020 ◽  
Author(s):  
Vittoria Borgonetti ◽  
Maria Domenica Sanna ◽  
Laura Lucarini ◽  
Nicoletta Galeotti
Keyword(s):  
Multiple Sclerosis ◽  
Experimental Autoimmune Encephalomyelitis ◽  
Rna Binding ◽  
Binding Protein ◽  
Regulation Of Gene Expression ◽  
Rna Binding Protein ◽  
Motor Dysfunction ◽  
Autoimmune Encephalomyelitis ◽  
Activated Microglia ◽  
Experimental Autoimmune

AbstractMultiple sclerosis (MS) is a chronic autoimmune inflammatory and neurodegenerative disease of the central nervous system characterized by demyelination, axonal loss, and motor dysfunction. Activated microglia are associated with the destruction of myelin in the CNS. Activated microglia produce cytokines and proinflammatory factors, favoring neuroinflammation, myelin damage, and neuronal loss, and it is thought to be involved in the disease pathogenesis. The present study investigated the role of post-transcriptional regulation of gene expression on the neuroinflammation related to experimental autoimmune encephalomyelitis (EAE) in mice, by focusing on HuR, an RNA-binding protein involved in inflammatory and immune phenomena. Spinal cord sections of EAE mice showed an increased HuR immunostaining that was abundantly detected in the cytoplasm of activated microglia, a pattern associated with its increased activity. Intrathecal administration of an anti-HuR antisense oligonucleotide (ASO) decreased the proinflammatory activated microglia, inflammatory infiltrates, and the expression of the proinflammatory cytokines IL-1β, TNF-α, and IL-17, and inhibited the activation of the NF-κB pathway. The beneficial effect of anti-HuR ASO in EAE mice corresponded also to a decreased permeability of the blood–brain barrier. EAE mice showed a reduced spinal CD206 immunostaining that was restored by anti-HuR ASO, indicating that HuR silencing promotes a shift to the anti-inflammatory and regenerative microglia phenotype. Mice that received anti-HuR ASO exhibited improved EAE-related motor dysfunction, pain hypersensitivity, and body weight loss. Targeting HuR might represent an innovative and promising perspective to control neurological disturbances in MS patients.

scholarly journals Pleiotropic Roles for the Plasmodium berghei RNA Binding Protein UIS12 in Transmission and Oocyst Maturation

2020 ◽  
Author(s):  
Katja Müller ◽  
Olivier Silvie ◽  
Hans-Joachim Mollenkopf ◽  
Kai Matuschewski
Keyword(s):  
Plasmodium Berghei ◽  
Rna Binding ◽  
Binding Protein ◽  
Regulation Of Gene Expression ◽  
Rna Binding Protein ◽  
Early Time ◽  
Transcript Abundance ◽  
Mosquito Vector ◽  
Targeted Gene Deletion ◽  
Complete Life Cycle

AbstractColonization of the mosquito host by Plasmodium parasites is achieved by sexually differentiated gametocytes. Gametocytogenesis, gamete formation and fertilization are tightly regulated processes, and translational repression is a major regulatory mechanism for stage conversion. Here, we present a characterization of a Plasmodium berghei RNA binding protein, UIS12, that contains two conserved eukaryotic RNA recognition motifs (RRM). Targeted gene deletion resulted in viable parasites that replicate normally during blood infection, but form fewer gametocytes. Upon transmission to Anopheles stephensi mosquitoes, both numbers and size of midgut-associated oocysts were reduced and their development stopped at an early time point. As a consequence, no salivary gland sporozoites were formed indicative of a complete life cycle arrest in the mosquito vector. Comparative transcript profiling in mutant and wild-type infected red blood cells revealed a decrease in transcript abundance of mRNAs coding for signature gamete-, ookinete- and oocyst-specific proteins in uis12(-) parasites. Together, our findings indicate multiple roles for UIS12 in regulation of gene expression after blood infection in good agreement with the pleiotropic defects that terminate successful sporogony and onward transmission to a new vertebrate host.

scholarly journals Hfq Globally Binds and Destabilizes the bound sRNAs and mRNAs in Yersinia pestis

2018 ◽  
Author(s):  
Yanping Han ◽  
Dong Chen ◽  
Yanfeng Yan ◽  
Hongduo Wang ◽  
Zizhong Liu ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Rna Binding ◽  
Binding Protein ◽  
Large Fraction ◽  
Rna Binding Protein ◽  
Cross Linking ◽  
Base Pairing ◽  
Binding Motifs ◽  
Interaction Sites ◽  
Rna Interaction

ABSTRACTHfq is a ubiquitous Sm-like RNA binding protein in bacteria involved in physiological fitness and pathogenesis, while its in vivo binding natures still remain elusive. Here we reported the first study of the Hfq-bound RNAs map in Yersinia pestis, the causative agent of a kind of plague, by using Cross-Linking Immunoprecipitation coupled with deep sequencing (CLIP-Seq) approach. We show that Hfq binds over 80% mRNAs of Y. pestis, and also globally binds non-coding sRNAs encoded by the intergenic, antisense, and the 3’ regions of mRNAs. Hfq U-rich stretch is highly enriched in sRNAs, while motifs partially complementary to AGAAUAA and GGGGAUUA are enriched in both mRNAs and sRNAs. Hfq binding motifs are enriched at both terminal sites and in the gene body of mRNAs. Surprisingly, a large fraction of the sRNA and mRNA regions bound by Hfq and those downstream are destabilized, likely via a 5’P-activated RNase E degradation pathway and consistent with Hfq-facilitated sRNA-mRNA base-pairing and the coupled degradation in Y. pestis. These results together have presented a high-quality Hfq-RNA interaction map in Y. pestis, which should be important for further deciphering the regulatory role of Hfq-sRNAs in Y. pestis.AUTHOR SUMMARYDiscovered in 1968 as an Escherichia coli host factor that was essential for replication of the bacteriophage Qβ, the Hfq protein is a ubiquitous and highly abundant RNA binding protein in many bacteria. Under the assistance of Hfq, small RNAs in bacteria play important role in regulating the stability and translation of mRNAs by base-pairing. In this study, we want to elucidate the Hfq assisted sRNA-mRNA regulation in Yersinia pestis. A global map of Hfq interaction sites in Y. pestis was obtained by sequencing of cDNAs converted from the Hfq-bound RNA fragments using UV cross-linking coupled immunoprecipitation technology. We demonstrate that Hfq could hundreds of sRNAs and the majority of mRNAs in living Y. pestis. The enriched binding motifs in sRNAs and mRNA are significantly complementary to each other, suggesting a general base-pairing mechanism for sRNA-mRNA interaction. The Hfq-bound sRNA and mRNA regions were both destabilized. The results suggest that Hfq binding facilitates sRNA-mRNA base-pairing and coordinates their degradation, which might enable Hfq to surveil the hemostasis of most mRNAs in bacteria.

scholarly journals C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis

2019 ◽  
Vol 47 (17) ◽  
pp. 9386-9399 ◽  
Author(s):  
Shreekara Gopalakrishna ◽  
Sarah F Pearce ◽  
Adam M Dinan ◽  
Florian A Schober ◽  
Miriam Cipullo ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Large Subunit ◽  
Regulation Of Gene Expression ◽  
Mitochondrial Protein ◽  
Rna Binding Protein ◽  
Mitochondrial Rna ◽  
Mitochondrial Translation

Abstract In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain—an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.

scholarly journals The Fission Yeast RNA-Binding Protein Meu5 Is Involved in Outer Forespore Membrane Breakdown during Spore Formation

2020 ◽  
Vol 6 (4) ◽  
pp. 284
Author(s):  
Bowen Zhang ◽  
Erika Teraguchi ◽  
Kazuki Imada ◽  
Yuhei O. Tahara ◽  
Shuko Nakamura ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Target Genes ◽  
Rna Binding ◽  
De Novo ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Biological Significance ◽  
Spore Wall ◽  
Spore Formation ◽  
Membrane Breakdown

In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Dongcheng Liu ◽  
Yan Wang ◽  
Yan Yuan
Keyword(s):  
Dna Replication ◽  
Noncoding Rna ◽  
Rna Binding ◽  
De Novo ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Noncoding Rnas ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation andde novoinfection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UVcross-linking andimmunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication duringde novoinfection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication duringde novoinfection.IMPORTANCEGenomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.

scholarly journals Regulation of Klf16 by Double Strand RNA-binding Protein STAU1 in Adipocyte Differentiation in vitro

2020 ◽  
Author(s):  
Ya Qun Guan ◽  
Xuan Yu Meng ◽  
Xiao Di Liang ◽  
Ting Ting Hu ◽  
Nurbierye Nuermamati ◽  
...  
Keyword(s):  
Rna Binding ◽  
Adipocyte Differentiation ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Adipogenic Differentiation ◽  
Regulation Of Gene Expression ◽  
Rna Binding Protein ◽  
Negative Regulator ◽  
Transcriptional Level

Abstract Background: Adipogenesis is an essential process in organismal development and plays a significant role in adipose tissue homeostasis. Post-transcriptional regulation of gene expression plays a key role in adipogenesis and involves many RNA-binding proteins (RBPs). In mammals, Staufen1 (STAU1) is a conserved RBP(RNA Binding Protein )consisting of several dsRBP (double strand RNA). STAU1 plays an important role in the Stau1-mediated mRNA decay (SMD) pathway, which is related to adipocyte formation, myocyte development, and neural differentiation. Klf16 (Kruppel like transcription factor 16) is a negative regulator that inhibits adipocyte differentiation. AIM:This study was conducted to determine the role of Klf16 in adipocyte differentiation in the context of the SMD pathway.Methods: 3T3-L1 cells were induced and cultured in vitro by cocktail method, Knockdown and Overexpression of STAU1 and KLF16. Then, adipocyte differentiation andexpression of adipogenic-related genes (STAU1, KLF16, PPARγ, and Lipin1) were measured by RT-qPCR and Western blot.RNA immunoprecipitation (RIP) method verified that STAU1 protein can bind to KLF16.Results: The results revealed that STAU1 regulates Klf16 expression at the post-transcriptional level during the adipogenic differentiation of 3T3-L1 cells.STAU1 candirectly bind the 3′UTR of Klf16 mRNA. Klf16 mRNA was found to be degraded through the SMD pathway, thus promoting adipocyte differentiation.Conclusions: In this study, the mechanism of adipocyte differentiation regulation at the post-transcriptional level is demonstrated, and Klf16 is shown as a substrate of the SMD pathway, thus providing new insights into adipogenesis.

scholarly journals Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

2017 ◽  
Author(s):  
Jahan-Yar Parsa ◽  
Selim Boudoukha ◽  
Jordan Burke ◽  
Christina Homer ◽  
Hiten D. Madhani
Keyword(s):  
Genome Stability ◽  
Rna Binding ◽  
De Novo ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Heterochromatin Formation ◽  
Repressor Complex ◽  
Polymerase Pausing ◽  
Heterochromatin Assembly ◽  
Dependent Mechanism

Packaging of pericentromeric DNA into heterochromatin is crucial for genome stability, development and health, yet its endogenous triggers remain poorly understood1. A defining feature of pericentromeric heterochromatin is histone H3 lysine 9 methylation (H3K9me)2–4. In S. pombe, transcripts derived from the pericentromeric dg and dh repeat during S phase5–7 promote heterochromatin formation through two pathways: an RNAi-dependent mechanism involving recruitment of the Clr4 H3K9 methyltransferase complex (CLR-C) via the RITS complex8–13, and RNAi-independent mechanism involving an RNAPII-associated RNA-binding protein Seb1, the repressor complex SHREC, and RNA processing activities14–19. We show here that Seb1 promotes long-lived RNAPII pausing. Pause sites associated with sequence-specific Seb1 RNA binding events are significantly enriched in pericentromeric repeat regions and their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Remarkably, globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking functional RITS, demonstrating that RNAPII pausing can be sufficient to trigger de novo heterochromatin independently of RNAi. These results uncover Seb1-mediated polymerase stalling as a new signal for nucleating heterochromatin assembly in repetitive DNA.

scholarly journals Corrigendum to “α-Actinin TvACTN3 ofTrichomonas vaginalisIs an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism”

2016 ◽  
Vol 2016 ◽  
pp. 1-2
Author(s):  
Jaeson Santos Calla-Choque ◽  
Elisa Elvira Figueroa-Angulo ◽  
Leticia Ávila-González ◽  
Rossana Arroyo
Keyword(s):  
Rna Binding ◽  
Regulatory Mechanism ◽  
Binding Protein ◽  
Rna Binding Protein
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