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 Hfq Globally Binds and Destabilizes sRNAs and mRNAs in Yersinia pestis

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Yanping Han ◽  
Dong Chen ◽  
Yanfeng Yan ◽  
Xiaofang Gao ◽  
Zizhong Liu ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Small Rnas ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cross Linking ◽  
Base Pairing ◽  
Binding Motifs ◽  
Content Type ◽  
Rna Interaction

ABSTRACT Hfq is a ubiquitous Sm-like RNA-binding protein in bacteria involved in physiological fitness and pathogenesis, while its in vivo binding nature remains elusive. Here we reported genome-wide Hfq-bound RNAs in Yersinia pestis, a causative agent of plague, by using cross-linking immunoprecipitation coupled with deep sequencing (CLIP-seq) approach. We show that the Hfq binding density is enriched in more than 80% mRNAs of Y. pestis and that Hfq also globally binds noncoding small RNAs (sRNAs) encoded by the intergenic, antisense, and 3′ regions of mRNAs. An 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, which is consistent with a model in which Hfq facilitates 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. IMPORTANCE Discovered 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. With the assistance of Hfq, small RNAs in bacteria play important roles 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 cDNAs converted from the Hfq-bound RNA fragments using UV cross-linking coupled immunoprecipitation technology. We demonstrate that Hfq could bind to hundreds of sRNAs and the majority of mRNAs in Y. pestis. The enriched binding motifs in sRNAs and mRNAs are 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 homeostasis of most mRNAs in bacteria.

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 The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2020 ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

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.

scholarly journals Loss of Quaking RNA binding protein disrupts the expression of genes associated with astrocyte maturation in mouse brain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kristina Sakers ◽  
Yating Liu ◽  
Lorida Llaci ◽  
Scott M. Lee ◽  
Michael J. Vasek ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Specific Gene ◽  
Binding Motifs ◽  
Expression Of Genes ◽  
Transcriptional Changes ◽  
Oligodendrocyte Development

AbstractQuaking RNA binding protein (QKI) is essential for oligodendrocyte development as myelination requires myelin basic protein mRNA regulation and localization by the cytoplasmic isoforms (e.g., QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we define the targets of QKI in the mouse brain via CLIPseq and we show that QKI-6 binds 3′UTRs of a subset of astrocytic mRNAs. Binding is also enriched near stop codons, mediated partially by QKI-binding motifs (QBMs), yet spreads to adjacent sequences. Using a viral approach for mosaic, astrocyte-specific gene mutation with simultaneous translating RNA sequencing (CRISPR-TRAPseq), we profile ribosome associated mRNA from QKI-null astrocytes in the mouse brain. This demonstrates a role for QKI in stabilizing CLIP-defined direct targets in astrocytes in vivo and further shows that QKI mutation disrupts the transcriptional changes for a discrete subset of genes associated with astrocyte maturation.

The properties of the RNA-binding protein NF90 are considerably modulated by complex formation with NF45

2017 ◽  
Vol 474 (2) ◽  
pp. 259-280 ◽  
Author(s):  
Tobias Schmidt ◽  
Paul Knick ◽  
Hauke Lilie ◽  
Susann Friedrich ◽  
Ralph Peter Golbik ◽  
...  
Keyword(s):  
Complex Formation ◽  
Rna Binding ◽  
Binding Capacity ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cell Types ◽  
Binding Mode ◽  
Binding Motifs ◽  
C Terminus ◽  
Thermodynamic Stabilization

Nuclear factor 90 (NF90) is an RNA-binding protein (RBP) that regulates post-transcriptionally the expression of various mRNAs. NF90 was recently shown to be capable of discriminating between different RNA substrates. This is mediated by an adaptive and co-operative interplay between three RNA-binding motifs (RBMs) in the protein's C-terminus. In many cell types, NF90 exists predominantly in a complex with NF45. Here, we compared the RNA-binding properties of the purified NF90 monomer and the NF90–NF45 heterodimer by biophysical and biochemical means, and demonstrate that the interaction with NF45 considerably affects the characteristics of NF90. Along with a thermodynamic stabilization, complex formation substantially improves the RNA-binding capacity of NF90 by modulating its binding mode and by enhancing its affinity for single- and double-stranded RNA substrates. Our data suggest that features of both the N- and C-termini of NF90 participate in the heterodimerization with NF45 and that the formation of NF90–NF45 changes the conformation of NF90's RBMs to a status in which the co-operative interplay of the RBMs is optimal. NF45 is considered to act as a conformational scaffold for NF90's RBMs, which alters the RNA-binding specificity of NF90. Accordingly, the monomeric NF90 and the NF90–NF45 heterodimer may exert different functions in the cell.

scholarly journals The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

scholarly journals The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction

2020 ◽  
Author(s):  
Ewan M Smith ◽  
Nour El Houda Benbahouche ◽  
Katherine Morris ◽  
Ania Wilczynska ◽  
Sarah Gillen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Binding ◽  
Binding Protein ◽  
Mammalian Target Of Rapamycin ◽  
Rna Binding Protein ◽  
Mtor Inhibition ◽  
Growth And Survival ◽  
Mrna Targets ◽  
Terminal Oligopyrimidine ◽  
Rna Interaction

Abstract The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5′ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.

Sign in / Sign up

Export Citation Format

Share Document