Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis

Abstract

Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5′ monophosphate, lack a poly(A) tail at the 3′ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants.

Dicer-independent RNA-directed DNA methylation in Arabidopsis

“…Since our study did not include a RNA fragmentation step, our sequencing reads should represent the native state of Pol IV-dependent transcripts, and no further assembly of the reads is needed. On the other hand, our results are consistent with the finding of Li et al that P4 RNAs contain a monophosphate in their 5′ end and no poly(A) tail in the 3′ end [32]. The T4 RNA ligase used for our RNAseq library construction requires 5′ monophosphate and 3′ hydroxyl groups of the RNAs.…”

“…incorporation of NTPs facilitates release of the transcript, which is then transcribed by RDR2. Of the 4 464 > 24 nt P4 RNA clusters identified in dcl1/2/3/4 mutant plants, we found that 1 751 overlap with DRM2-dependent (D2) loci and 651 overlap with CMT2-dependent (C2) loci, and 73 overlap with both D2 and C2 loci, consistent with the findings that both D2 and C2 loci produce 24-nt siRNAs [13,32]. During the preparation of this manuscript, two independent groups reported short, Pol IV-dependent precursors of 24-nt siRNAs [58,59], which corroborate our results.…”

“…P4 RNAs are mostly 25-35 nt in length and are more abundant at RdDM loci whose methylation is independent of, or partially dependent on, DCL proteins. A previous study that characterized Pol IV-and RDR2-dependent RNAs suggested that the majority of Pol IV-dependent RNAs are 100-500 nt in length [32]. However, we did not observe > 100 nt Pol IV-dependent transcripts by northern blot analyses (data not shown).…”

“…Importantly, many of the P4 RNAs overlap with each other. The RNA-seq library preparation procedure in the Li et al study contained a transcript fragmentation step and accordingly neighboring reads no more than 60 nt apart were joined together computationally [32]. Since our study did not include a RNA fragmentation step, our sequencing reads should represent the native state of Pol IV-dependent transcripts, and no further assembly of the reads is needed.…”

“…It has been thought that other DCL proteins could also process RDR products, compensating for DCL3 when it is absent, and that the 21/22-nt siRNAs, though less efficient, could also be fed into Argonaute (AGO) proteins [20]. A recent study utilized a dcl2/3/4 mutant background to characterize Pol IV-dependent transcripts and found that they were mostly 100-500 nt in length, had 5′ monophosphates and lacked 3′ polyA tails [32].…”