An Arabidopsis RNase III-like protein, AtRTL2, cleaves double-stranded RNA in vitro

Abstract

Class 1 ribonuclease III (RNase III), found in bacteria and yeast, is involved in processing functional RNA molecules such as ribosomal RNAs (rRNAs). However, in Arabidopsis thaliana, the lack of an obvious phenotype or quantitative change in mature rRNAs in class 1 RNase III (AtRTL2) mutants and overexpressing plants suggests that AtRTL2 is not involved in rRNA maturation. We characterized the in vitro activity of AtRTL2 to consider its in vivo function. AtRTL2 cleaved double-stranded RNA (dsRNA) specifically in vitro, yielding products of approximately 25 nt or longer in length, in contrast to 10-20 nt long products in bacteria and yeasts. Although dsRNA-binding activity was not detected, the dsRNA-binding domains in AtRTL2 were essential for its dsRNA-cleaving activity. Accumulation of small RNAs derived from transgene dsRNAs was increased when AtRTL2 was transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. These results raise the possibility that AtRTL2 has functions distinct from those of other class 1 RNase IIIs in vivo.

Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation

“…The results presented in Figure 2B and the results by Kiyota et al (2011) suggest that RTL2 ectopic expression stimulates the (A) GUS activity in 15 L1/dcl2 dcl4 untransformed controls and 15 independent L1/dcl2 dcl4/ProUBQ10:RTL2 transformants. GUS activity is expressed as units of fluorescence/min/µg protein.…”

“…Similarly, increased or decreased accumulation of siRNAs was observed when overexpressing RTL2 in N. benthamiana together with artificial or natural dsRNA substrates. Indeed, RTL2 overexpression increased the production of siRNAs from GFP dsRNA (Kiyota et al, 2011), GUS dsRNA, and At3g18145 39UTR hairpin Size distribution and number of reads and DNA methylation visualized by PCR and quantitative PCR in Col and rtl2 bulks of flowers at two selected RTL2-dependent loci (A) and two selected RTL2-sensitive loci (B). Percentage of DNA methylation is calculated as follows: % DNA methylation = (12 (2^2 Dct)) *100 with Dct = ct(McrBC) -ct(mock).…”

“…Whether these molecules could be subsequently processed into 21-, 22-, or 24-nucleotide siRNAs by the DCLs is not known. However, Kiyota et al (2011) showed that coinfiltration of Pro35S:RTL2 and Pro35S:GFP constructs in leaves of the Nicotiana benthamiana 16c line, which carries a Pro35S:GFP transgene that is prone to trigger S-PTGS, enhanced the production of GFP siRNAs. To further analyze the function of RTL2, a Pro35S:IR-GUS construct carrying an inverted repeat of the 59 end of GUS was infiltrated in wild-type N. benthamiana leaves together with Pro35S:GFP or Pro35S:RTL2 constructs.…”

“…Given that RTL2 enhances the production of exogenous siRNAs when transiently overexpressed in plants (Kiyota et al, 2011), we investigated whether RTL2 could have an endogenous function in the processing of siRNAs. Unlike Dcr1, RTL2 is not able to produce siRNAs by itself.…”

Comparison of preribosomal RNA processing pathways in yeast, plant and human cells – focus on coordinated action of endo‐ and exoribonucleases

“…Similarly, specific in vitro activity on structured RNA substrates, dependent on the intact dsRBD, was demonstrated for A. thaliana Rnt1 orthologue, AtRTL2, even though no binding of this protein to dsRNA was directly demonstrated. Importantly, a single mutation within the RNase III signature motif of AtRTL2 entirely abolished its endoribonucleolytic activity towards dsRNA . AtRTL2 was shown to participate in the processing of 3′‐ETS, akin to its yeast counterpart (Fig.…”