Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway.

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.

Analysis of siRNA Precursors Generated by RNA Polymerase IV and RNA-Dependent RNA Polymerase 2 in Arabidopsis.

Noncoding RNAs perform diverse regulatory functions in living cells. In plants, two RNA polymerase II-related enzymes, RNA polymerases IV and V (Pol IV and V), specialize in the synthesis of noncoding RNAs that silence a subset of transposable elements and genes via RNA-directed DNA methylation (RdDM). In this process, Pol IV partners with RNA-dependent RNA polymerase 2 (RDR2) to produce double-stranded RNAs that are then cut by an RNase III enzyme, Dicer-like 3 (DCL3), into 24 nt small interfering RNAs (siRNAs). The siRNAs are loaded into an Argonaute family protein, primarily AGO4, and guide the complex to complementary DNA target sequences where RdDM and repressive chromatin modifications ensue. The dependence of 24 nt siRNA biogenesis on Pol IV and RDR2 has been known for more than a decade, but the elusive pre-siRNA transcripts synthesized by Pol IV and RDR2 have only recently been identified. This chapter describes the approaches that enabled our identification of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in Arabidopsis thaliana. These included the use of a triple Dicer mutant (dcl2 dcl3 dcl4) to cause P4R2 RNAs to accumulate, genome-wide identification and mapping of P4R2 RNAs using a modified Illumina small RNA-Seq protocol, and multiple bioinformatic pipelines for data analysis and displaying results.

Transcriptome-wide identification and functional investigation of the RDR2- and DCL3-dependent small RNAs encoded by long non-coding RNAs in Arabidopsis thaliana.

The involvement of the long non-coding RNAs (lncRNAs) in small RNA (sRNA)-related pathways remains elusive. Taking advantage of the public sRNA sequencing data, we searched for RNA-dependent RNA polymerase 2 (RDR2)- and Dicer-like 3 (DCL3)-dependent sRNAs generated from the lncRNAs of Arabidopsis thaliana. First, 55,162 sRNAs were identified to be RDR2- and DCL3-dependent. These sRNAs were then mapped onto the lncRNAs. As a result, a total of 26,643 sRNAs found their loci on 3,834 lncRNAs, and 29,388 sRNAs found their loci on 4,174 reverse complementary (RC) sequences of the lncRNAs. To support the formation of the double-stranded precursors for sRNA generation, double-stranded RNA sequencing (dsRNA-seq) reads were mapped onto the sense and antisense strands of the lncRNAs with RDR2- and DCL3-dependent sRNA loci. As a result, 1,075 regions longer than 100 nt were identified to be covered by dsRNA-seq reads on 390 sense strands of the lncRNAs, and 1,352 regions were identified on 544 RC strands. Besides, 2,238 out of 3,211 dsRNA-seq read-covered sRNA loci were supported by degradome sequencing data on the sense strands of the lncRNAs. Interestingly, dozens of dsRNA-seq read-covered regions with AGO4-associated sRNA loci showed site-specific chromatin modification patterns. Thus, some of the lncRNAs were integrated into the RDR2- and DCL3-dependent sRNA biogenesis pathway. Moreover, our results indicated that the site-specific chromatin modifications mediated by the AGO4-associated sRNAs might play a regulatory role on the transcription activity of the lncRNA genes.

Chromatin structure and gene expression changes associated with loss of MOP1 activity in Zea mays.

Though the mechanisms governing nuclear organization are not well understood, it is apparent that epigenetic modifications coordinately modulate chromatin organization as well as transcription. In maize, MEDIATOR OF PARAMUTATION1 (MOP1) is required for 24 nt siRNA-mediated epigenetic regulation and transcriptional gene silencing via a putative Pol IV- RdDM pathway. To elucidate the mechanisms of nuclear chromatin organization, we investigated the relationship between chromatin structure and transcription in response to loss of MOP1 function. We used a microarray based micrococcal nuclease sensitivity assay to identify genome-wide changes in chromatin structure in mop1-1 immature ears and observed an increase in chromatin accessibility at chromosome arms associated with loss of MOP1 function. Within the many genes misregulated in mop1 mutants, we identified one subset likely to be direct targets of epigenetic transcriptional silencing via Pol-IV RdDM. We found that target specificity for MOP1-mediated RdDM activity is governed by multiple signals that include accumulation of 24 nt siRNAs and the presence of specific classes of gene-proximal transposons, but neither of these attributes alone is sufficient to predict transcriptional misregulation in mop1-1 homozygous mutants. Our results suggest a role for MOP1 in regulation of higher-order chromatin organization where loss of MOP1 activity at a subset of loci triggers a broader cascade of transcriptional consequences and genome-wide changes in chromatin structure.