scholarly journals The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by a RNA-Mediated Dimerization Mechanism

2017 ◽  
Author(s):  
Nadine M. Shaban ◽  
Ke Shi ◽  
Kate V. Lauer ◽  
Michael A. Carpenter ◽  
Christopher M. Richards ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Binding ◽  
Cytosine Deaminase ◽  
Rnase A ◽  
Binding Motif ◽  
Innate And Adaptive Immunity ◽  
Enzyme Preparations ◽  
Gene Diversification ◽  
Hiv 1

SUMMARYHuman APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA editing enzymes function in innate immunity by restricting the replication of viruses and transposons. Misregulated APOBEC3H also contributes to cancer mutagenesis. Here we address the role of RNA in APOBEC3H regulation. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment yields enzyme preparations with stronger DNA deaminase activity. RNA binding-defective mutants are DNA hypermutators. Chromatography profiles and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. RNA binding is required for APOBEC3H cytoplasmic localization and for packaging into HIV-1 particles and antiviral activity. Related DNA deaminases including other APOBEC3 family members and the antibody gene diversification enzyme AID also bind RNA and are predicted to have a similar RNA binding motif suggesting mechanistic conservation and relevance to innate and adaptive immunity and to multiple diseases.HIGHLIGHTSRNA inhibits human APOBEC3H DNA cytosine deaminase activityRNA binding mutants are DNA hypermutatorsX-ray structure demonstrates an RNA duplex-mediated APOBEC3H dimerization mechanismRNA binding is required for packaging into HIV-1 particles and antiviral activity

169. THE MECHANISM OF SPERMATID MATURATION - A LINK TO TUMOUR SUPPRESSION

2010 ◽  
Vol 22 (9) ◽  
pp. 87
Author(s):  
D. Jamsai ◽  
S. J. Smith ◽  
A. E. O'Connor ◽  
D. J. Merriner ◽  
C. Borg ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Male Fertility ◽  
Rna Binding ◽  
Ex Vivo ◽  
Round Spermatid ◽  
Null Alleles ◽  
Compound Heterozygous ◽  
Binding Motif ◽  
Rna Recognition Motif

To comprehensively uncover novel male fertility regulators, we utilised an unbiased forward genetic screen, ENU mutagenesis. Using this approach, we have identified several novel infertile mouse lines including a male-specific infertile line that we designated ‘Joey’. The mutant Joey mice produced no sperm due to an arrest of male germ cells at the round spermatid stage. The mutation was identified in the RNA binding motif 5 (Rbm5) gene that resulted in an arginine to proline substitution within a highly conserved RNA recognition motif of the protein. The substitution of proline is likely to interfere with RNA binding and/or recognition. In humans, the RBM5 gene maps to a region that is frequently deleted in lung cancers. Ex vivo studies have suggested that RBM5 is a tumour suppressor, apoptosis modulator and RNA splicing regulator. To date, the role of Rbm5 has never been liked to male fertility and the Joey line is the only mouse model of Rbm5 dysfunction. Using our RBM5-specific antibody, we showed that RBM5 is expressed in pachytene spermatocytes and round spermatids. Based on the protein localisation, the proposed role of RBM5 in mRNA processing, the onset of the Joey phenotype, and the site of the identified mutation, we hypothesise that the Rbm5 mutant allele results in a hypomorphic protein, and that RBM5 has an essential role in regulating male germ cell mRNA storage, transport and/or translational regulation of mRNAs that are critical for spermatid maturation. Further, we generated mice compound heterozygous of the Joey Rbm5 mutation and Rbm5 null alleles. We showed that the compound heterozygous males are infertile due to spermatid maturation arrest resembling the Joey mutant males. This result further confirmed the identification of the Rbm5 mutation as a cause of infertility in the Joey mice and a crucial role of Rbm5 in male fertility.

scholarly journals The mechanism by which molecules containing the HIV gp41 core-binding motif HXXNPF inhibit HIV-1 envelope glycoprotein-mediated syncytium formation

2007 ◽  
Vol 403 (3) ◽  
pp. 565-571 ◽  
Author(s):  
Jing-He Huang ◽  
Heng-Wen Yang ◽  
Shuwen Liu ◽  
Jing Li ◽  
Shibo Jiang ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Critical Role ◽  
Syncytium Formation ◽  
Phage Clone ◽  
Fusion Process ◽  
Binding Motif ◽  
Active Core ◽  
Different Temperatures ◽  
Hiv 1

The HIV-1 gp41 (glycoprotein 41) core plays a critical role in fusion between the viral and target cell membranes. We previously identified a gp41 core-binding motif, HXXNPF, by screening the phage display peptide libraries. In the present study, we elucidated the mechanism of action of HXXNPF motif-containing molecules of different sizes, including the phage clone L7.8 (a selected positive phage clone), L7.8-g3p* (a 10–kDa fragment of the gene 3 protein) and JCH-4 (a peptide containing 13 residues of L7.8-g3p*), regarding their respective binding abilities to the six-helix bundle and inhibition on syncytium formation at different temperatures. We found that all of the HXXNPF motif-containing molecules could bind to the gp41 core, and that their binding sites may be located in the N-helix domain. L7.8-g3p* and JCH-4 effectively inhibited HIV-1 Env (envelope glycoprotein)-mediated syncytium formation at 37 °C, while the phage clone L7.8 showed no inhibition under the same conditions. However, at suboptimal temperature (31.5 °C), all of these HXXNPF motif-containing molecules were capable of inhibiting syncytium formation. These results suggest that these HXXNPF motif-containing molecules mainly bind to the gp41 core and stop the fusion process mediated by the fusion-active core, resulting in inhibition of HIV-1 fusion and entry. The HXXNPF motif-containing molecules may be used as probes for studying the role of the HIV-1 gp41 core in the late stage of the membrane-fusion process.

scholarly journals New Insights on the Versatile Role of the Cholesterol Binding Motif of the HIV-1 Glycoprotein Gp41

2014 ◽  
Vol 106 (2) ◽  
pp. 62a
Author(s):  
Roland Schwarzer ◽  
Andreas Herrmann ◽  
Ilya Levental ◽  
Andrea Gramatica
Keyword(s):  
Binding Motif ◽  
Glycoprotein Gp41 ◽  
Hiv 1 ◽  
Cholesterol Binding

scholarly journals 7SL RNA Mediates Virion Packaging of the Antiviral Cytidine Deaminase APOBEC3G

2007 ◽  
Vol 81 (23) ◽  
pp. 13112-13124 ◽  
Author(s):  
Tao Wang ◽  
Chunjuan Tian ◽  
Wenyan Zhang ◽  
Kun Luo ◽  
Phuong Thi Nguyen Sarkis ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Binding ◽  
Cytidine Deaminase ◽  
Rna Packaging ◽  
Cellular Functions ◽  
7Sl Rna ◽  
Immunodeficiency Virus ◽  
Antiviral Function ◽  
Pol Iii ◽  
Hiv 1

ABSTRACT Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.

scholarly journals The Arginine-Rich RNA-Binding Motif of HIV-1 Rev Is Intrinsically Disordered and Folds upon RRE Binding

2013 ◽  
Vol 105 (4) ◽  
pp. 1004-1017 ◽  
Author(s):  
Fabio Casu ◽  
Brendan M. Duggan ◽  
Mirko Hennig
Keyword(s):  
Rna Binding ◽  
Binding Motif ◽  
Intrinsically Disordered ◽  
Hiv 1

scholarly journals Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Jennifer A. Bohn ◽  
Justin DaSilva ◽  
Siarhei Kharytonchyk ◽  
Maria Mercedes ◽  
Jennifer Vosters ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nucleic Acid ◽  
Antiviral Activity ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Viral Dna ◽  
Dna Substrates ◽  
Apobec3 Proteins ◽  
Rna Interaction ◽  
Hiv 1

ABSTRACT APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination. IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

scholarly journals Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

2019 ◽  
Author(s):  
Jennifer Elliott ◽  
Jenna E. Eschbach ◽  
Pratibha C. Koneru ◽  
Wen Li ◽  
Maritza Puray Chavez ◽  
...  
Keyword(s):  
Rna Binding ◽  
Critical Role ◽  
Underlying Mechanism ◽  
Class Ii ◽  
Viral Rna ◽  
Target Cells ◽  
Ribonucleoprotein Complexes ◽  
Virion Morphogenesis ◽  
Hiv 1

ABSTRACTA large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

scholarly journals Architectural RNA in chromatin organization

2020 ◽  
Vol 48 (5) ◽  
pp. 1967-1978
Author(s):  
Jitendra Thakur ◽  
Steven Henikoff
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ribonuclease A ◽  
Rnase A ◽  
Chromatin Organization ◽  
Nuclear Bodies ◽  
Non Coding Rnas ◽  
Rna Depletion

RNA plays a well-established architectural role in the formation of membraneless interchromatin nuclear bodies. However, a less well-known role of RNA is in organizing chromatin, whereby specific RNAs have been found to recruit chromatin modifier proteins. Whether or not RNA can act as an architectural molecule for chromatin remains unclear, partly because dissecting the architectural role of RNA from its regulatory role remains challenging. Studies that have addressed RNA's architectural role in chromatin organization rely on in situ RNA depletion using Ribonuclease A (RNase A) and suggest that RNA plays a major direct architectural role in chromatin organization. In this review, we will discuss these findings, candidate chromatin architectural long non-coding RNAs and possible mechanisms by which RNA, along with RNA binding proteins might be mediating chromatin organization.

scholarly journals Identification of protein-protein and ribonucleoprotein complexes containing Hfq

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joël Caillet ◽  
Bruno Baron ◽  
Irina V. Boni ◽  
Célia Caillet-Saguy ◽  
Eliane Hajnsdorf
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Direct Interaction ◽  
Rnase A ◽  
Rnase E ◽  
Protein Protein Interactions ◽  
Control Of Gene Expression ◽  
Ribonucleoprotein Complexes

Abstract Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.

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