6 research outputs found
Exogenous and endogenous dsRNAs perceived by plant Dicer-like 4 protein in the RNAi-depleted cellular context
Abstract Background In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles. Methods In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism, in which we could highlight the role of dicing as neither Argonautes nor RNA-dependent RNA polymerase is present. We have therefore tested the DCL4 functionality in processing exogenous dsRNA-like substrates, such as a replicase-assisted viral replicon defective-interfering RNA and RNA hairpin substrates, or endogenous antisense transcripts. Results DCL4 was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci. Conclusions We provide new keys to interpret what was studied so far on antiviral DCL4 in the host system. The results all together confirm the role of sense/antisense RNA-based regulation of gene expression, expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications
RNA and Protein Determinants Mediate Differential Binding of miRNAs by a Viral Suppressor of RNA Silencing Thus Modulating Antiviral Immune Responses in Plants
Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and āGāU wobblesā on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response
Coordinated Action of Two Double-Stranded RNA Binding Motifs and an RGG Motif Enables Nuclear Factor 90 To Flexibly Target Different RNA Substrates
The mechanisms of how RNA binding
proteins (RBP) bind to and distinguish
different RNA molecules are yet uncertain. Here, we performed a comprehensive
analysis of the RNA binding properties of multidomain RBP nuclear
factor 90 (NF90) by investigating specifically the functional activities
of two double-stranded RNA binding motifs (dsRBM) and an RGG motif
in the proteinās unstructured C-terminus. By comparison of
the RNA binding affinities of several NF90 variants and their modes
of binding to a set of defined RNA molecules, the activities of the
motifs turned out to be very different. While dsRBM1 contributes little
to RNA binding, dsRBM2 is essential for effective binding of double-stranded
RNA. The proteinās immediate C-terminus, including the RGG
motif, is indispensable for interactions of the protein with single-stranded
RNA, and the RGG motif decisively contributes to NF90ās overall
RNA binding properties. Conformational studies, which compared wild-type
NF90 with a variant that contains a pseudophosphorylated residue in
the RGG motif, suggest that the NF90 C-terminus is involved in conformational
changes in the protein after RNA binding, with the RGG motif acting
as a central regulatory element. In summary, our data propose a concerted
action of all RNA binding motifs within the frame of the full-length
protein, which may be controlled by regulation of the activity of
the RGG motif, e.g., by phosphorylation. This multidomain interplay
enables the RBP NF90 to discriminate RNA features by dynamic and adaptable
interactions
A Viral Suppressor Modulates the Plant Immune Response Early in Infection by Regulating MicroRNA Activity
Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plantās immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSRās miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro. Most importantly, in contrast to controls with a Īp19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation
Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template
The
hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is
a central enzyme of the intracellular replication of the viral (+)ĀRNA
genome. Here, we studied the individual steps of NS5B-catalyzed RNA
synthesis by a combination of biophysical methods, including real-time
1D <sup>1</sup>H NMR spectroscopy. NS5B was found to bind to a nonstructured
and a structured RNA template in different modes. Following NTP binding
and conversion to the catalysis-competent ternary complex, the polymerase
revealed an improved affinity for the template. By monitoring the
folding/unfolding of 3ā²(ā)ĀSL by <sup>1</sup>H NMR, the
base pair at the stemās edge was identified as the most stable
component of the structure. <sup>1</sup>H NMR real-time analysis of
NS5B-catalyzed RNA synthesis on 3ā²(ā)ĀSL showed that
a pronounced lag phase preceded the processive polymerization reaction.
The presence of the double-stranded stem with the edge base pair acting
as the main energy barrier impaired RNA synthesis catalyzed by NS5B.
Our observations suggest a crucial role of RNA-modulating factors
in the HCV replication process