Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE179616 | GEO | 2021/10/14
REPOSITORIES: GEO
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Project description:In plants, the biogenesis of 24 nt and 23 nt small interfering RNAs (siRNAs) requires NUCLEAR RNA POLYMERASE IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). We show that single-stranded M13 bacteriophage DNA can be used as a template for siRNA synthesis in vitro. Deep sequencing of RNAs produced from the in vitro reactions of Pol IV, RDR2 and DCL3 shows that Pol IV transcribes the DNA into first-strand RNAs which RDR2 then uses as templates to synthesize complementary second strands. These siRNA precursor transcripts made by Pol IV and RDR2 are mostly 30-50 nt. An untemplated 3' terminal nucleotide is a characteristic of RDR2 transcripts. DCL3 dicing of double-stranded precursor RNAs synthesized by Pol IV and RDR2 generates siRNAs that are mostly 24 nt, with a smaller population of 23 nt also produced.
2019-07-01 | GSE126086 | GEO
Project description:Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Keywords: siRNA, transfection, chemical modification, off-targets
2008-04-02 | GSE10960 | GEO
Project description:RNaseIII enzymes catalyze the cleavage of double stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis RNASE THREE LIKE2 (RTL2) carries one RNaseIII and two dsRNA-binding (DRB) domains, and is the unique Arabidopsis RNAse III enzyme resembling the budding yeast siRNA-producing Dcr1 enzyme. Here we show that RTL2 modulates the production of siRNAs, and that this activity depends on both RNaseIII and DRB domains. However, RTL2 mode of action differs from that of Dcr1. Indeed, whereas Dcr1 directly cleaves dsRNAs into 23-nt siRNAs, RTL2 likely cleaves dsRNAs into molecules longer than siRNAs, which are subsequently processed by the DCL enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2- dependent loci) or reduces the efficiency of DCL processing (RTL2-sensitive loci). Because the vast majority of plant small RNAs are 24-nt siRNAs that guide DNA methylation at transposons and intergenic regions, RTL2 depletion mostly modifies DNA methylation in these regions. Nevertheless, 13% of the siRNA-producing loci affected by RTL2 depletion correspond to protein coding genes. We show that changes in 24-nt siRNA levels also affect DNA methylation levels at such loci, and inversely correlate with mRNA steady-state levels, thus implicating RTL2 in the control of protein-coding gene expression.
2015-10-01 | GSE71782 | GEO
Project description:Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Experiment Overall Design: For details please see: Chen PY, Weinmann L, Gaidatzis D, Pei Y, Zavolan M, Tuschl T, Meister G. RNA (2008) 14(2):263-74.
2008-06-11 | E-GEOD-10960 | biostudies-arrayexpress
Project description:DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide siRNA and long miRNA biogenesis and mutants have been characterized from Arabidopsis and rice. Here, a tomato DCL3 mutant was generated through the use of trans-activated artificial microRNA and characterized. Global tomato DCL3 (SlDCL3) silencing was induced by crossing the generated responder line (OP:amiRDCL3) with constitutive driver line. Constitutive trans-activation knocked down SlDCL3 levels by ~77%, resulting in dramatically decreased 24-nucleotide small RNA levels, but a significant increase in 21- and 22- nucleotide small RNAs, which was correlated with specific upregulation of SlDCL4 and SlDCL2b. Moreover, in the majority of small RNA-generating loci an almost complete overlap between the control and 35S>>amiRSlDCL3 siRNA signatures, was observed, strongly suggesting that the reduction in 24-nucleotide siRNAs was compensated by increased biogenesis of 21-22 nt siRNAs from the same genomic sequences. Collectively, these results suggest the requirement of SlDCL3 for the biogenesis of 23-24 nt sRNAs and its substitution by SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt small RNAs. In addition, differential expression between control and SlDCL3-silenced small RNAs indicated a significant reduction in the abundance of 24-nt putative long miRNAs, which was validated by northern blots, implicating SlDCL3 in their biogenesis.
2014-03-31 | GSE52265 | GEO
Project description:DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide siRNA and long miRNA biogenesis and mutants have been characterized from Arabidopsis and rice. Here, a tomato DCL3 mutant was generated through the use of trans-activated artificial microRNA and characterized. Global tomato DCL3 (SlDCL3) silencing was induced by crossing the generated responder line (OP:amiRDCL3) with constitutive driver line. Constitutive trans-activation knocked down SlDCL3 levels by ~77%, resulting in dramatically decreased 24-nucleotide small RNA levels, but a significant increase in 21- and 22- nucleotide small RNAs, which was correlated with specific upregulation of SlDCL4 and SlDCL2b. Moreover, in the majority of small RNA-generating loci an almost complete overlap between the control and 35S>>amiRSlDCL3 siRNA signatures, was observed, strongly suggesting that the reduction in 24-nucleotide siRNAs was compensated by increased biogenesis of 21-22 nt siRNAs from the same genomic sequences. Collectively, these results suggest the requirement of SlDCL3 for the biogenesis of 23-24 nt sRNAs and its substitution by SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt small RNAs. In addition, differential expression between control and SlDCL3-silenced small RNAs indicated a significant reduction in the abundance of 24-nt putative long miRNAs, which was validated by northern blots, implicating SlDCL3 in their biogenesis. Examination of small RNA populations in control (35S:LhG4) and SlDCL3 silenced seedlings (35S>>amiRSlDCL3) 2 biological replicates each
2014-03-31 | E-GEOD-52265 | biostudies-arrayexpress
Project description:Small RNAs play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode DICER-LIKE (DCL) RNaseIII enzymes, including DCL1, which produces miRNAs, and DCL2, DCL3 and DCL4, which produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Small RNA sequencing in plants over-expressing RTL1 or RTL2 or lacking RTL2 revealed that RTL1 over-expression inhibits the accumulation of all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. By contrast, RTL2 plays minor, if any, role in the small RNA repertoire. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by degrading dsRNA before they are processed by DCL2, DCL3 and DCL4. The substrate of RTL1 cleavage is likely long perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from short imperfect dsRNA. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. These results reveal another level of complexity in the evolutionary battle between viruses and plant defenses. Flower sRNA profiles in diverse conditions involving RTL1 and RTL2
2013-09-10 | E-GEOD-49866 | biostudies-arrayexpress
Project description:RNA-directed DNA methylation (RdDM) is a prominent pathway in plants to defend against invasive nucleic acids. In the canonical RdDM, 24-nt small interfering RNAs (siRNAs) are produced through DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system in which transcriptional gene silencing (TGS) is independent of DLC3. In a forward genetics screen performed with this system, already known components of RdDM and RNA-binding protein RBP45D were identified. RBP45D promotes DNA methylation, and a lack of RBP45D causes late flowering especially under heat, presumably mediated by elevated FLC levels. RBP45D is localized to the nucleus where it is associated with snRNAs and snoRNAs. RBP45D maintains siRNA production originating from the transgene, but does not promote mRNA levels or influence pre-mRNA splicing of known RdDM genes. We hypothesize that RBPD45 facilitates DCL3-independent siRNA production through stabilisation of either the precursor RNA or the – not yet identified – slicer protein.
2022-05-13 | GSE178678 | GEO
Project description:In plants, transcriptional silencing by RNA-directed DNA methylation (RdDM) is mediated by ARGONAUTE 4 (AGO4) and 24 nt short-interfering RNAs (siRNAs) that are generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs serve as the passenger strands of 23/24 nt duplexes loaded into AGO4. The 24 nt siRNAs then guide AGO4 slicing of the passenger strands, generating 11 and 12 nt cleavage products. Unexpectedly, we find that the 12 nt products remain associated with the guide strand-AGO4 complexes. Long noncoding RNAs generated at RdDM loci are similarly sliced and retained by AGO4. These results suggest a model in which RNA POLYMERASE V transcripts at target loci are sliced repeatedly as transcription elongation proceeds, sequentially releasing AGO4-siRNA-scaffold RNA complexes that independently recruit the RdDM machinery. Consistent with this hypothesis, plant lines expressing wild-type versus slicing-defective AGO4 show quantitative variation in cytosine methylation and siRNA levels within RdDM loci.
2023-02-17 | GSE201234 | GEO
Project description:In plants, transcriptional silencing by RNA-directed DNA methylation (RdDM) is mediated by ARGONAUTE 4 (AGO4) and 24 nt short-interfering RNAs (siRNAs) that are generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs serve as the passenger strands of 23/24 nt duplexes loaded into AGO4. The 24 nt siRNAs then guide AGO4 slicing of the passenger strands, generating 11 and 12 nt cleavage products. Unexpectedly, we find that the 12 nt products remain associated with the guide strand-AGO4 complexes. Long noncoding RNAs generated at RdDM loci are similarly sliced and retained by AGO4. These results suggest a model in which RNA POLYMERASE V transcripts at target loci are sliced repeatedly as transcription elongation proceeds, sequentially releasing AGO4-siRNA-scaffold RNA complexes that independently recruit the RdDM machinery. Consistent with this hypothesis, plant lines expressing wild-type versus slicing-defective AGO4 show quantitative variation in cytosine methylation and siRNA levels within RdDM loci.
2023-02-17 | GSE201233 | GEO