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

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.

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

2003 ◽  
Vol 161 (2) ◽  
pp. 309-319 ◽  
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.

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

2004 ◽  
Vol 279 (50) ◽  
pp. 52447-52455 ◽  
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.

scholarly journals Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

2020 ◽  
Vol 48 (7) ◽  
pp. 3906-3921 ◽  
Author(s):  
Volker Nitschko ◽  
Stefan Kunzelmann ◽  
Thomas Fröhlich ◽  
Georg J Arnold ◽  
Klaus Förstemann
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Convergent Transcription ◽  
Dsrna Binding ◽  
Functional Screens

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.

scholarly journals Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains

2021 ◽  
Author(s):  
Simon H. Stitzinger ◽  
Salma Sohrabi-Jahromi ◽  
Johannes Söding
Keyword(s):  
High Throughput ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Binding Domains

AbstractNumerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA binding domains display low specificity and affinity, to the extent that for most RNA-binding domains, the enrichment of the best binding motif measured by high-throughput RNA SELEX or RNA bind-n-seq is usually below 10-fold, dramatically lower than that of DNA-binding domains. Here, we develop a thermodynamic model to predict the binding affinity for proteins with any number of RNA-binding domains given the affinities of their isolated domains. For the four proteins in which affinities for individual domains have been measured the model predictions are in good agreement with experimental values. The model gives insight into how proteins with multiple RNA-binding domains can reach affinities and specificities orders of magnitude higher than their individual domains. Our results contribute towards resolving the conundrum of missing specificity and affinity of RNA binding proteins and underscore the need for bioinformatic methods that can learn models for multi-domain RNA binding proteins from high-throughput in-vitro and in-vivo experiments.

scholarly journals RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽  
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.

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

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.

scholarly journals The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

1991 ◽  
Vol 11 (7) ◽  
pp. 3419-3424 ◽  
Author(s):  
C G Burd ◽  
E L Matunis ◽  
G Dreyfuss
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Binding Activity ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

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

2009 ◽  
Vol 29 (6) ◽  
pp. 1487-1497 ◽  
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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