Author Archives: aul24

Arabidopsis RNASE THREE LIKE2 modulates the expression of protein-coding genes via 24-nucleotide small interfering RNA-directed DNA methylation

Elvira-Matelot E, Hachet M, Shamandi N, Comella P, Saez-Vasquez J, Zytnicki M, Vaucheret H

The Arabidopsis RTL2 is an RNASE THREE LIKE protein with one RNAseIII domain and two dsRNA-binding domains. Its transient over-expression in plants is known to enhance the production of exogenous siRNAs. Here the authors investigate its role in the production of endogenous siRNAs.

The ectopic expression of RTL2 stimulates the production of siRNAs from artificial and natural Inverted Repeats constructs. Both its domains are necessary for the RTL2-dependent production of siRNAs from dsRNAs. Contrarily, in other cases the over-expression of RTL2 reduces the production of siRNAs from dsRNAs. The opposite effect of RTL2 on dsRNA substrates likely depends on the structure and/or sequence of the dsRNAs.

Interestingly, the over-expression of RTL2 also stimulates the production of RNA molecules larger than 24 nts from artificial and natural Inverted Repeats constructs. The authors also demonstrate that RTL2 cannot substitute for the function of DCL2, DCL3 and DCL4. The suggested hypothesis is that RTL2 could process dsRNAs into RNA molecules longer than 24 nts, which could subsequently be processed by DCL proteins into siRNAs. In some cases the cleavage of RTL2 results in a better processing by DCLs, in some other cases it results in a worse processing by DCLs.

By a sliding window approach combined with the analysis of reads at each position of the genome, a total of 481 sRNA loci are found to be differentially expressed in the rtl2 mutant compared to wt: 183 are RTL2-dependent loci, 298 are RTL2-sensitive loci. These RTL2-targeted sRNA loci produce siRNAs that are mostly dependent on DCL2DCL3DCL4, Pol IV, Pol V and DRM2, indicating the involvement of these sRNA loci in the RdDM process.

Recent works reported that siRNA precursors, named P4R2 RNAs (Pol IV- and RDR2-dependent), preferentially start with an A or a G, and preferentially end with a U (Blevins et al. 2015 and Zhai et al., 2015). The authors found that the RTL2 loci show different 5’ and 3’ nucleobase preferences compared to the total Pol IV loci previously identified. This tendency is reverted to the same behavior observed for the total Pol IV loci in the rtl2 mutant, suggesting that RLT2 modifies the 5’ and 3’ end compositions of the target loci, making them similar to those of the total P4R2 RNAs.

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RTL2 targets mainly TEs and intergenic regions but also protein-coding genes, influencing the DNA methylation and mRNA expression level of the target loci.

The biogenesis of siRNAs is not yet completely understood and these findings suggest that the siRNA precursors might undergo multiple successive processings operated by different proteins. The one precursor one siRNA model might not be valid for all sRNA genes, leaving the question: what are the genetic/epigenetic features of the sRNA genes that differentiate their sRNA precursors production and processing?


A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis

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.

P4RNA 3’end

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.