scholarly journals Binding of dsRNA by D. melanogaster Dicer-2 is substrate-dependent and regulated by Loquacious-PD

2020 ◽  
Author(s):  
M. Jonely ◽  
R. K. Singh ◽  
B. L. Bass ◽  
R. Noriega
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Molecular Recognition ◽  
Rna Binding ◽  
Regulatory Proteins ◽  
Dependent Manner ◽  
Biochemical Activity ◽  
Double Stranded Rna ◽  
Molecular Scale ◽  
Local Binding

ABSTRACTDrosophila melanogaster Dicer-2 is a large, multidomain protein that cleaves double-stranded RNA (dsRNA) into small interfering RNAs in a terminus-dependent manner as part of the RNA interference pathway. We characterize the local binding environment involved in this substrate-selective molecular recognition event by monitoring the time-resolved photophysics of a cyanine dye linked to the dsRNA terminus. We observe substantial changes in the molecular rigidity and local freedom of motion of the probe as a function of distinct conformations of the biomolecular complex between Dicer-2 and dsRNA as a function of dsRNA termini, the presence of regulatory proteins, and the addition of a biochemical energy source (ATP) or a non-hydrolysable equivalent (ATP-γS). With a clustering analysis based solely on these molecular-scale measures of the local binding environment at the dsRNA terminus, we identify sub-populations of similar conformations that define distinct modes of molecular recognition which are correlated with biochemical activity. These observations reveal the important role of substrate-selective molecular recognition properties for proteins with multiple domains that can bind RNA, regulatory proteins, and cofactors.STATEMENT OF SIGNIFICANCEThe molecular-scale determinants of protein-RNA binding remain elusive, particularly when different subunits of a single protein confer specificity toward small structural differences of their RNA partners. An important case is that of Drosophila melanogaster Dicer-2, a critical component of the antiviral RNA interference response. Dicer-2 discriminates between double stranded RNA with blunt or 3’ overhang termini, a feature suggested to mediate recognition of “self” vs. “non-self” substrates. We study these interactions at the binding site with a fluorescent label at the RNA terminus, monitoring intramolecular and collective measures of flexibility to report on the local environment. Dicer-2 has distinct modes of molecular recognition which are regulated by accessory proteins and ATP, leading to different conformations and tuning biochemical activity.

Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4147-4156 ◽  
Author(s):  
P. Svoboda ◽  
P. Stein ◽  
H. Hayashi ◽  
R.M. Schultz
Keyword(s):  
Rna Interference ◽  
Map Kinase ◽  
Selective Reduction ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Double Stranded Rna ◽  
Mouse Oocytes ◽  
Tissue Plasminogen ◽  
Maternal Mrnas ◽  
Specific Mrna

Specific mRNA degradation mediated by double-stranded RNA (dsRNA), which is termed RNA interference (RNAi), is a useful tool with which to study gene function in several systems. We report here that in mouse oocytes, RNAi provides a suitable and robust approach to study the function of dormant maternal mRNAs. Mos (originally known as c-mos) and tissue plasminogen activator (tPA, Plat) mRNAs are dormant maternal mRNAs that are recruited during oocyte maturation; translation of Mos mRNA results in the activation of MAP kinase. dsRNA directed towards Mos or Plat mRNAs in mouse oocytes effectively results in the specific reduction of the targeted mRNA in both a time- and concentration-dependent manner. Moreover, dsRNA is more potent than either sense or antisense RNAs. Targeting the Mos mRNA results in inhibiting the appearance of MAP kinase activity and can result in parthenogenetic activation. Mos dsRNA, therefore, faithfully phenocopies the Mos null mutant. Targeting the Plat mRNA with Plat dsRNA results in inhibiting production of tPA activity. Finally, effective reduction of the Mos and Plat mRNA is observed with stoichiometric amounts of Mos and Plat dsRNA, respectively.

A staufen-like RNA-binding protein in translocation channels linking nurse cells to oocytes in Notonecta shows nucleotide-dependent attachment to microtubules

1999 ◽  
Vol 112 (17) ◽  
pp. 2947-2955
Author(s):  
S. Hurst ◽  
N.J. Talbot ◽  
H. Stebbings
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Mrna Localization ◽  
Dependent Manner ◽  
Nurse Cells ◽  
Double Stranded Rna ◽  
Standard Procedures ◽  
Rna Probes ◽  
Peptide Antibody

In Drosophila melanogaster the staufen gene encodes an RNA-binding protein that is essential for the correct localization of certain nurse cell-derived transcripts in oocytes. Although the mechanism underlying mRNA localization is unknown, mRNA-staufen complexes have been shown to move in a microtubule-dependent manner, and it has been suggested that staufen associates with a motor protein which generates the movement. We have investigated this possibility using Notonecta glauca in which nurse cells also supply the oocytes with mRNA, but via greatly extended nutritive tubes comprised of large aggregates of parallel microtubules. Using a staufen peptide antibody and RNA probes we have identified a staufen-like protein, which specifically binds double-stranded RNA, in the nutritive tubes of Notonecta. We show that while the staufen-like protein does not co-purify with microtubules from ovaries using standard procedures it does so under conditions of motor-entrapment, specifically in the presence of AMP-PNP. We also show that the staufen-like protein is subsequently removed by ATP and GTP, but not ADP. Nucleotide-dependent binding to microtubules is typical of a motor-mediated interaction and the pattern of attachment and detachment of the staufen-like protein correlates with that of a kinesin protein within the ovaries. Our findings indicate that the staufen-like RNA-binding protein attaches to, and is transported along, Notonecta ovarian microtubules by a kinesin motor.

scholarly journals Substrate-dependent Contribution of Double-stranded RNA-binding Motifs to ADAR2 Function

2006 ◽  
Vol 17 (7) ◽  
pp. 3211-3220 ◽  
Author(s):  
Ming Xu ◽  
K. Sam Wells ◽  
Ronald B. Emeson
Keyword(s):  
Rna Binding ◽  
Structural Similarity ◽  
Dependent Manner ◽  
Structural Determinants ◽  
Binding Motifs ◽  
Double Stranded Rna ◽  
Subnuclear Localization ◽  
Rna Targets ◽  
Editing Activity ◽  
Site Selective

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine (A-to-I). ADAR2 contains two tandem double-stranded RNA-binding motifs (dsRBMs) that are not only important for efficient editing of RNA substrates but also necessary for localizing ADAR2 to nucleoli. The sequence and structural similarity of these motifs have raised questions regarding the role(s) that each dsRBM plays in ADAR2 function. Here, we demonstrate that the dsRBMs of ADAR2 differ in both their ability to modulate subnuclear localization as well as to promote site-selective A-to-I conversion. Surprisingly, dsRBM1 contributes to editing activity in a substrate-dependent manner, indicating that dsRBMs recognize distinct structural determinants in each RNA substrate. Although dsRBM2 is essential for the editing of all substrates examined, a point mutation in this motif affects editing for only a subset of RNAs, suggesting that dsRBM2 uses unique sets of amino acid(s) for functional interactions with different RNA targets. The dsRBMs of ADAR2 are interchangeable for subnuclear targeting, yet such motif alterations do not support site-selective editing, indicating that the unique binding preferences of each dsRBM differentially contribute to their pleiotropic function.

scholarly journals Double-Stranded-RNA-Binding Protein 2 Participates in Antiviral Defense

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Károly Fátyol ◽  
Katalin Anna Fekete ◽  
Márta Ludman
Keyword(s):  
Rna Interference ◽  
Virus Infection ◽  
Nicotiana Benthamiana ◽  
Rna Binding ◽  
Binding Protein ◽  
Systemic Necrosis ◽  
Double Stranded Rna ◽  
Content Type ◽  
Dsrna Binding ◽  
Dsrna Binding Protein

ABSTRACT Double-stranded RNA (dsRNA) is a common pattern formed during the replication of both RNA and DNA viruses. Perception of virus-derived dsRNAs by specialized receptor molecules leads to the activation of various antiviral measures. In plants, these defensive processes include the adaptive RNA interference (RNAi) pathway and innate pattern-triggered immune (PTI) responses. While details of the former process have been well established in recent years, the latter are still only partially understood at the molecular level. Nonetheless, emerging data suggest extensive cross talk between the different antiviral mechanisms. Here, we demonstrate that dsRNA-binding protein 2 (DRB2) of Nicotiana benthamiana plays a direct role in potato virus X (PVX)-elicited systemic necrosis. These results establish that DRB2, a known component of RNAi, is also involved in a virus-induced PTI response. In addition, our findings suggest that RNA-dependent polymerase 6 (RDR6)-dependent dsRNAs play an important role in the triggering of PVX-induced systemic necrosis. Based on our data, a model is formulated whereby competition between different DRB proteins for virus-derived dsRNAs helps establish the dominant antiviral pathways that are activated in response to virus infection. IMPORTANCE Plants employ multiple defense mechanisms to restrict viral infections, among which RNA interference is the best understood. The activation of innate immunity often leads to both local and systemic necrotic responses, which confine the virus to the infected cells and can also provide resistance to distal, noninfected parts of the organism. Systemic necrosis, which is regarded as a special form of the local hypersensitive response, results in necrosis of the apical stem region, usually causing the death of the plant. Here, we provide evidence that the dsRNA-binding protein 2 of Nicotiana benthamiana plays an important role in virus-induced systemic necrosis. Our findings are not only compatible with the recent hypothesis that DRB proteins act as viral invasion sensors but also extends it by proposing that DRBs play a critical role in establishing the dominant antiviral measures that are triggered during virus infection.

scholarly journals Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins

2002 ◽  
Vol 156 (1) ◽  
pp. 53-64 ◽  
Author(s):  
Amy M. Brownawell ◽  
Ian G. Macara
Keyword(s):  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Dependent Manner ◽  
Intact Cells ◽  
Double Stranded Rna ◽  
Permeabilized Cells ◽  
Dsrna Binding ◽  
Rna Export

We have identified a novel human karyopherin (Kap)β family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

scholarly journals RNA-binding protein Syncrip regulates Starvation-Induced Hyperactivity in adult Drosophila

2020 ◽  
Author(s):  
Wanhao Chi ◽  
Wei Liu ◽  
Wenqin Fu ◽  
Shengqian Xia ◽  
Ellie S. Heckscher ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Behavioral Response ◽  
Rna Binding ◽  
Target System ◽  
Rna Seq ◽  
Behavioral Trait ◽  
Alternatively Spliced ◽  
Genetic Contributions

AbstractHow to respond to starvation determines fitness. One prominent behavioral response is increased locomotor activities upon starvation, also known as Starvation-Induced Hyperactivity (SIH). SIH is paradoxical as it promotes food seeking but also increases energy expenditure. Despite its importance in regulating fitness, the genetic contributions to SIH as a behavioral trait remains unexplored. Here, we examined SIH in the Drosophila melanogaster Genetic Reference Panel (DGRP) and performed genome-wide association studies. We identified 23 significant loci, corresponding to 14 genes, significantly associated with SIH in adult Drosophila. Gene enrichment analyses indicated that genes encoding ion channels and mRNA binding proteins (RBPs) were most enriched in SIH. We are especially interested in RBPs because they provide a potential mechanism to quickly change protein expression in response to environmental challenges. Using RNA interference, we validated the role of Syp in regulating SIH. Syp encodes Syncrip, an RBP. While ubiquitous knockdown of Syp led to lethality during development, adult flies with neuron specific Syp knockdown were viable and exhibited decreased SIH. Using the Temporal and Regional Gene Expression Targeting (TARGET) system, we further confirmed the role of Syp in adult neurons in regulating SIH. Lastly, RNA-seq analyses revealed that Syp was alternatively spliced under starvation while its expression level was unchanged. Together, this study not only demonstrates genetic contributions to SIH as an important behavioral trait but also highlights the significance of RBPs and post-transcriptional processes in the brain in regulating behavioral responses to starvation.Author summaryAnimals living in the wild often face periods of starvation. How to physiologically and behaviorally respond to starvation is essential for survival. One behavioral response is Starvation-Induced Hyperactivity (SIH). We used the Drosophila melanogaster Genetic Reference Panel, derived from a wild population, to study the genetic basis of SIH. Our results show that there is a significant genetic contribution to SIH in this natural population, and that RNA binding proteins (RBPs) are especially important. Using RNA interference and the TARGET system, we confirmed the role of an RBP Syp in adult neurons in SIH. Further studies using RNA-seq and Western blotting showed that Syp was alternatively spliced under starvation while its expression level was unchanged, highlighting an essential role of post-transcriptional modification.

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