Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora

Abstract

Small RNA molecules have been found to be specifically associated with posttranscriptional gene silencing (PTGS) in both plants and animals. Here, we find that small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 genes previously shown to be essential for gene silencing, but does not depend on a functional qde-2, indicating that this gene is involved in a downstream step of the gene silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules, suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation.

Figure 1

Small 25-nt sense and antisense RNAs derived from transgenic transcripts are associated with PTGS. RNA blot hybridization: 50 μg (each lane) of enriched low-molecular-weight RNAs prepared from either untransformed wild-type (unsilenced) or al-1 silenced strain (6xw). Identical RNA blots were hybridized with the following RNA probes: (A) Probes 1 and 2 corresponding to the duplicated al-1 region that are able to recognize small RNAs derived from both the transgenic and endogenous al-1 gene. The presence of the upper bands in wild-type and 6xw lanes were due to nonspecific hybridization of the RNA probe. (B) Probes 3 and 4 were, instead, specific for the transgenic chimeric RNA. 30 pmoles per lane of 25-nt DNA oligonucleotides in sense (S) and antisense (AS) orientation were used to verify the hybridization specificity and also as size markers in both A and B. (C) Schematic representation of plasmid pal-1.6 and RNA probes.

Figure 2

Analysis of small RNA accumulation in qde mutants. (A) RNA blot hybridization of low-molecular-weight RNAs prepared from the 107 strain (qde-1⁻), the 820 strain (qde-2⁻), the 627 strain (qde-3⁻), the untransformed wild-type strain (unsilenced), and an al-1 silenced strain (6xw). Sense and antisense probes are the same as in Figure 1. (B) Northern blot on total RNA extracts hybridized with a probe specific for al-1 mRNA.

Figure 3

QDE2 copurifies with small RNAs. (A) Characterization of antibodies. α-Flag monoclonal and α-QDE2 antibodies were used in Western analysis of total proteins extracted from two deletion qde-2 mutants that express Flag–QDE2 protein (lines 1f-qde-2 and 2f-qde-2), a wild-type (unsilenced) strain, and a qde-2 deletion mutant strain 820 (qde-2⁻). The estimated protein size is shown on the left. (B) QDE2 protein was immunopurified with anti-FLAG affinity gel beads from protein extracts of two Flag–QDE2 strains and of the al-1 silenced strain 6xw (silenced). Western analysis, using as probe α-QDE2 antibody, was performed on the flowthrough fraction (FT) and on the immunopurified fraction (Flag-IP). Three bands were specifically detected only in immunopurified fractions of Flag–QDE2-expressing strains (lines 1f-qde-2 and 2f-qde-2). The slower-migrating band comigrates with the wild-type version of QDE2 (unsilenced), total extract, indicating that the two additional faster-migrating bands could be proteolyzed forms produced in the IP elution step. A band corresponding to QDE2 is present in the flowthrough (FT) fraction of the silenced (6xw) strain only, indicating that the wild-type QDE2 is not bound by the anti-Flag affinity gel beads. (C) Small RNA molecules were extracted from the Flag-IP protein fractions and analyzed by Northern blotting using a riboprobe specific for the al-1 sequence. Small RNAs of ∼25 nt were detected in the RNA preparations from the immunopurified fraction of the Flag–QDE2 strain (line Flag-IP/1f-qde-2) but not in the 6xw strain (Flag-IP/6xw). In contrast, small RNAs are detected in the flowthrough of 6xw (FT/Silenced) but not in the Flag–QDE2 strain (FT/1f-qde-2). The weakness of the signal in the flowthrough fraction recovered from the 6xw strain is explained by the high dilution of this fraction. A small-RNA-enriched preparation from the total RNA extracted from the 6xw strain is used as a positive control. Sense (S) and antisense (AS) 25-nt oligonucleotides are used as size markers.