qiRNA is a new type of small interfering RNA induced by DNA damage

Abstract

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing. The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20-21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5' end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.

Figure 1

DNA damage induces QDE-2 expression. (a) Northern and Western blot analyses showing the induction of qde-2 mRNA and QDE-2 protein expression by histidine. (b) Western blot analysis showing that the induction of QDE-2 by EMS requires QDE-1, QDE-3 and DCLs. The labels above the lanes indicate the genotypes of the strains. (c) Western blot analyses showing high QDE-2 levels in DNA repair mutants in the absence of a DNA damaging agent.

Figure 2

DNA damage results in the production of qiRNAs and aRNAs. (a) Enrichment of the Myc-QDE-2 associated small RNAs by immunoprecipitation. A wild-type strain that lacks the Myc-QDE-2 construct was used as a negative control. Immunoprecipitated RNA was 3′ end labeled and separated on a 16% acrylamide gel. (b) Mapping of qiRNAs to an rDNA repeat. Regions encoding for the mature rRNAs are shaded. (c) Northern blot analysis of qiRNAs in the indicated strains. An RNA probe specific for the antisense 26S RNA was used. (d) qRT-PCR showing that EMS treatment results in the induction of aRNAs from the rDNA regions. The upper panel shows a schematic diagram of an rDNA repeat and the intergenic rDNA regions (U1, U2 and D1) analyzed by qRT-PCR analysis. A dcl double mutant was used. n= 3; *, P < 0.001. Error bars, s. d. (e) qRT-PCR analysis showing the loss of EMS-induced aRNAs from the rDNA locus in the qde-3 mutant. Results of two independent experiments are shown.

Figure 3

QDE-1 is required for the synthesis of DNA damage-induced rDNA-specific aRNA and exhibits DdRP activity using a ssDNA template. (a) qRT-PCR results showing the levels of the rDNA-specific aRNA and several pol II transcribed genes in the wild-type strain. n= 3; Error bars, s. d. (b) Northern blot analysis showing the levels of aRNA in the wild-type and qde-1^ko strains. Total RNA was used. (c) In vitro RNA polymerase assay using a 175nt ssDNA or ssRNA template and purified Myc-QDE-1. CTL indicates a reaction using purification products from a strain without the construct. (d) The ssDNA-templated reaction products from (c) were treated with RNase H.

Figure 4

The role of qiRNA in DNA damage response. (a) Northern blot analysis showing that the rDNA-derived qiRNAs are loaded onto an active RISC. Myc-QDE-2 or the catalytically inactive Myc-QDE-2(D664A) were immunoprecipitated and the associated RNAs were extracted. An rDNA-specific probe was used. (b) DNA damage results in the decrease of protein synthesis rate, a response that was partially blocked in the qde-1 and qde-3 strains. The radioactivity of ³⁵S labeled protein in various strains were used to compare the rate of total protein synthesis. n = 3; *, P < 0.0001. Error bars, s.d. (c) The qde-1 and the dcl double mutants exhibit increased sensitivity to DNA damaging agents. The numbers of conidia used in the spot test are indicated.