A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis (Zhai J, Bischof S, Wang H, Feng S, Lee TF, Teng C, Chen X, Park SY, Liu L, Gallego-Bartolome J, Liu W, Henderson IR, Meyers BC, Ausin I, Jacobsen SE, PMID: 26451488)
In this work the authors demonstrate that the Arabidopsis Pol IV-dependent siRNA precursors, named P4RNAs, are not as long as it was previously assumed: P4RNAs are indeed 30÷40-nt. The characterization of the P4RNAs length and sequence composition give insights to the mechanisms of Pol IV transcription initiation and termination and of DCL processing of the P4RNAs into siRNAs.
P4RNAs are the precursors of Pol IV siRNAs
P4RNAs are 30÷40-nt, as shown by the size distribution of the PATH libraries, and are dependent on both Pol IV and RDR2, suggesting that in vivo the two enzymes work in tight association.
Multiple experiments confirm that these long RNAs are the precursors of siRNAs and not misprocessed siRNAs, for example in the dcl2/3/4 mutant, siRNAs are mainly lost while P4RNAs are increased in abundance but AGO4 still selectively binds to the remaining 22-24-nt siRNAs and not to the longer RNAs. At Pol IV siRNA loci, siRNAs and P4RNAs show positively correlated abundances and interestingly, restricting the analysis on the Pol IV siRNA loci with a strand bias of siRNA accumulation and DNA methylation, the P4RNAs accumulation shows the same strand bias. This result suggests that Pol IV-derived strands, rather than the RDR2-derived strands, are strongly favored to become the final 24-nt siRNAs.
Because of the small length of P4RNAs on average only one 24-nt siRNA is processed by each P4RNA precursor.
P4RNA 5’ end
Pol IV is demonstrated to have retained the same TSS preference from its evolutionary ancestor Pol II (Y/R rule) but the two polymerases are here shown to occupy different genomic territories.
At 5’ end, P4RNAs are enriched in A, as it is known for the siRNAs, and the majority appear to have a 5’ monophosphate: I think this last result was in some way expected because of the cloning technique used to construct the PATH libraries.
P4RNAs that perfectly match to the genome are shown to have an enrichment of ACU in their three last positions, but more than 50% of the total P4RNAs present mismatches at their 3’ ends and these non-templated P4RNAs have a different nucleotide pattern in their 3’ end. 3’ end mismatches are enriched in CG dinucleotides, being C the last matched base and G the first mismatched base, so where a C is found on the template DNA. The level of nucleotide mismatches at 3’ end is strongly decreased in ddm1/dcl3 compared to dcl3, proving the DNA methylation is influencing the misincorporation of nucleotides by Pol IV. In this model, the DNA cytosine methylation causes the termination of Pol IV transcription to give rise to the short siRNA precursors. What still remains unclear to me is: why exactly after 30÷40-nt? It would be interesting to know what is the frequency of finding a methylated C after a Pol II-like TSS in the genome.
By contrast to the P4RNAs, only 1% of the total siRNAs have mismatches at their 3’ end. This result, together with the shared 5’ A enrichment and strand bias between siRNAs and P4RNAs, suggest that siRNAs are preferentially cleaved from the 5’ portion of their P4RNA precursors.
Another recent work “Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis” (Blevins T, Podicheti R, Mishra V, Marasco M, Wang J, Rusch D, Tang H, Pikaard CS, PMID: 26430765) confirms the short nature of the siRNA precursors but with a main difference: here, the precursors of siRNAs are found to have a strong preference for a 5’ purine but with similar frequencies for A and G. Compared to precursors with 5’ A, those with 5’ G have 3’ end pattern more similar to that of siRNAs, suggesting that these 5’ G precursors might be processed from their 3’ portion to give rise to siRNAs. It would be interesting to understand why these 5’ G siRNA precursors were not observed in the previous described work.