DNA–RNA hybrid formation mediates RNAi‐directed heterochromatin formation

Abstract

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNA-RNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNA-RNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNA-RNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNA-RNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNA-RNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNA-RNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNA-RNA hybrid formation plays a role in chromatic ncRNA function.

Accumulation of RNA on chromatin disrupts heterochromatic silencing

“…Consistent with a previous observation (Nakama et al 2012), we found low levels of DNA:RNA hybrids at subtelomeres in wild-type cells (Fig. 4F).…”

“…It has been shown that chromatin retention of RNA and DNA:RNA hybrid formation establish RNAi-mediated heterochromatin (Sun et al 2013;SkourtiStathaki et al 2014;Yu et al 2014). Deletion of RNase H, however, has a defect in heterochromatin maintenance, indicating that an excess of DNA:RNA hybrids has a negative impact on heterochromatin assembly (Nakama et al 2012). DNA:RNA hybrids were ′ tlh: second half) or LEU2 were inserted ∼5.5 kb upstream of tlh under the thiamine-inducible nmt1 promoter.…”

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

“…4А). Эти олигонуклеотиды должны формировать нековалентные комплексы c EGPO и блокировать его действие [17]. С помощью ОТ-ПЦР показали, что три олигонуклеотида блокировали АС, что подтвердило правильность гипотезы и позволило определить размер EGPO на уровне 47 нуклеотидов (рис.…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…The first is a siRNA/DNA-binding model [65,136] , during which the RITS complex binds directly to chromatin. This binding seems to be dependent on the interaction between the siRNA and its DNA-target sequence.…”