Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

Abstract RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

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RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽

10.3390/v13030361 ◽

2021 ◽

Vol 13 (3) ◽

pp. 361

Author(s):

Rui-Zhu Shi ◽

Yuan-Qing Pan ◽

Li Xing

Keyword(s):

Virus Replication ◽

Rna Binding ◽

Rna Viruses ◽

Rna Helicase ◽

Rna Helicase A ◽

Double Stranded Rna ◽

Binding Domains ◽

N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

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Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

10.1101/466763 ◽

2018 ◽

Author(s):

Pravin Kumar Ankush Jagtap ◽

Marisa Müller ◽

Pawel Masiewicz ◽

Sören von Bülow ◽

Nele Merret Hollmann ◽

...

Keyword(s):

Rna Binding ◽

Binding Motifs ◽

Double Stranded Rna ◽

Binding Domains ◽

Dsrna Binding ◽

Rna Binding Domains ◽

Human Orthologue ◽

Rox Rna

ABSTRACTMaleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

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The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis

Science Advances ◽

10.1126/sciadv.abf3898 ◽

2021 ◽

Vol 7 (32) ◽

pp. eabf3898

Author(s):

Di Sun ◽

Yanjun Li ◽

Zeyang Ma ◽

Xingxing Yan ◽

Niankui Li ◽

...

Keyword(s):

Gene Silencing ◽

Small Interfering Rnas ◽

Loss Of Function ◽

Double Stranded Rna ◽

Posttranscriptional Gene Silencing ◽

Epigenetic Factor ◽

Dsrna Binding ◽

Dsrna Binding Protein

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.

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Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

10.1101/2021.06.14.448452 ◽

2021 ◽

Author(s):

Christine Roden ◽

Yifan Dai ◽

Ian Seim ◽

Myungwoon Lee ◽

Rachel Sealfon ◽

...

Keyword(s):

Phase Separation ◽

Rna Structure ◽

Nucleocapsid Protein ◽

Rna Binding ◽

Genome Packaging ◽

Rna Motifs ◽

Double Stranded Rna ◽

N Protein ◽

Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

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RNA-Regulated Interaction of Transportin-1 and Exportin-5 with the Double-Stranded RNA-Binding Domain Regulates Nucleocytoplasmic Shuttling of ADAR1

Molecular and Cellular Biology ◽

10.1128/mcb.01519-08 ◽

2009 ◽

Vol 29 (6) ◽

pp. 1487-1497 ◽

Cited By ~ 72

Author(s):

Jutta Fritz ◽

Alexander Strehblow ◽

Andreas Taschner ◽

Sandy Schopoff ◽

Pawel Pasierbek ◽

...

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Nucleocytoplasmic Shuttling ◽

Double Stranded Rna ◽

Binding Domains ◽

Dsrna Binding ◽

Localization Signal ◽

Editing Enzyme

ABSTRACT Double-stranded RNA (dsRNA)-binding proteins interact with substrate RNAs via dsRNA-binding domains (dsRBDs). Several proteins harboring these domains exhibit nucleocytoplasmic shuttling and possibly remain associated with their substrate RNAs bound in the nucleus during nuclear export. In the human RNA-editing enzyme ADAR1-c, the nuclear localization signal overlaps the third dsRBD, while the corresponding import factor is unknown. The protein also lacks a clear nuclear export signal but shuttles between the nucleus and the cytoplasm. Here we identify transportin-1 as the import receptor for ADAR1. Interestingly, dsRNA binding interferes with transportin-1 binding. At the same time, each of the dsRBDs in ADAR1 interacts with the export factor exportin-5. RNA binding stimulates this interaction but is not a prerequisite. Thus, our data demonstrate a role for some dsRBDs as RNA-sensitive nucleocytoplasmic transport signals. dsRBD3 in ADAR1 can mediate nuclear import, while interaction of all dsRBDs might control nuclear export. This finding may have implications for other proteins containing dsRBDs and suggests a selective nuclear export mechanism for substrates interacting with these proteins.

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Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting

The Journal of Cell Biology ◽

10.1083/jcb.200301034 ◽

2003 ◽

Vol 161 (2) ◽

pp. 309-319 ◽

Cited By ~ 18

Author(s):

Michael Doyle ◽

Michael F. Jantsch

Keyword(s):

Rna Editing ◽

Rna Binding ◽

Double Stranded Rna ◽

Binding Domains ◽

Initial Substrate ◽

Rna Binding Domains ◽

Editing Enzyme ◽

Deaminase Domain

The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA–binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

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Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122duringXenopusDevelopment

Journal of Biological Chemistry ◽

10.1074/jbc.m406107200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52447-52455 ◽

Cited By ~ 12

Author(s):

Garry P. Scarlett ◽

Stuart J. Elgar ◽

Peter D. Cary ◽

Anna M. Noble ◽

Robert L. Orford ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Rna Binding ◽

Transcription Activator ◽

Double Stranded Rna ◽

Binding Domains ◽

Rna Binding Domains ◽

Ccaat Box

CBTF122is a subunit of theXenopusCCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (anin vivotarget of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA bindingin vitro. In addition, these mutations alter the ability of CBTF122fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 genein vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding bothin vitroandin vivo.

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Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

Journal of Virology ◽

10.1128/jvi.00009-07 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5449-5459 ◽

Cited By ~ 27

Author(s):

Beau J. Fenner ◽

Winnie Goh ◽

Jimmy Kwang

Keyword(s):

Rna Silencing ◽

Rna Binding ◽

Amino Acid Residues ◽

Alanine Scanning Mutagenesis ◽

Marine Aquaculture ◽

Double Stranded Rna ◽

Dsrna Binding ◽

Fish Cells ◽

Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

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A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2212 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2212-2219 ◽

Cited By ~ 115

Author(s):

Rosa María Marión ◽

Puri Fortes ◽

Ana Beloso ◽

Carlos Dotti ◽

Juan Ortín

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Rna Binding ◽

Intracellular Localization ◽

Binding Activity ◽

Ns1 Protein ◽

Binding Domains ◽

Human Sequence ◽

Dsrna Binding

ABSTRACT In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a λ cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparentKd of 10−9 M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.

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How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

Acta Biochimica Polonica ◽

10.18388/abp.2016_1339 ◽

2017 ◽

Vol 63 (4) ◽

Cited By ~ 2

Author(s):

Natalia Koralewska ◽

Weronika Hoffmann ◽

Maria Pokornowska ◽

Marek Cezary Milewski ◽

Andrea Lipinska ◽

...

Keyword(s):

Rna Binding ◽

Feedback Loops ◽

Small Interfering Rnas ◽

Binding Studies ◽

Hairpin Rna ◽

Rna Molecules ◽

Short Rnas ◽

Microrna Processing

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by variety of proteins are being elucidated, less is known about non-protein factors; e.g. RNA molecules, that may influence the enzyme activity. Therefore, we decided to investigate the problem of whether the RNA molecules can function not only as Dicer substrates but also as its regulators.Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding with the enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

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