Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis.
Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production.
Pontier
Dominique
D
LGDP, UMR 5096, Université de Perpignan, 66860 Perpignan Cedex, France.
Yahubyan
Galina
G
Vega
Danielle
D
Bulski
Agnès
A
Saez-Vasquez
Julio
J
Hakimi
Mohamed-Ali
MA
Lerbs-Mache
Silva
S
Colot
Vincent
V
Lagrange
Thierry
T
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D019175:DNA Methylation; D004251:DNA Transposable Elements / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020868:Gene Silencing; D017343:Genes, Plant; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D021122:Protein Subunits; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
D004251:DNA Transposable Elements; D018744:DNA, Plant; D007527:Isoenzymes; D021122:Protein Subunits; D018749:RNA, Plant; D034741:RNA, Small Interfering; C499181:RNA polymerase IV, Arabidopsis; D012321:DNA-Directed RNA Polymerases