Induction of qde-2 expression by dsRNA.
Since QDE-2, the Argonaute-like protein, is a core component of the Neurospora RNAi pathway, we hypothesized that its expression should be highly regulated. To test this possibility, we examined QDE-2 protein levels using a QDE-2-specific antibody in Neurospora RNAi mutants in which one or more of the RNAi genes were disrupted. As shown in Fig. , levels of QDE-2 were reduced in the dcl-1ko dcl-2rip double mutant and in the qde-1ko and qde-3ko single mutants compared to those in the wild-type strain. No QDE-2 protein was detected in the qde-2rip strain, indicating the specificity of our QDE-2 antibody. These data indicate that the expression of QDE-2 is regulated by other components of the RNAi pathway. Since QDE-1 and QDE-3 are involved in the synthesis of endogenous dsRNA from aberrant RNAs, and DCLs are responsible for the production of siRNA, this suggests that the production of dsRNA or siRNA promotes the expression of QDE-2 proteins.
FIG. 1. Induction of qde-2 expression by dsRNA expression. (A) Western blot analysis showing that the levels of QDE-2 are low in the RNAi mutants. The asterisk indicates a nonspecific cross-reacting protein band recognized by our QDE-2 antibody. Equal protein (more ...)
We then examined the QDE-2 levels in albino-1 (al-1, involved in carotenoid biosynthesis)-quelled strains. In these strains, al-1 was silenced by the introduction of multiple copies of an al-1 DNA fragment, resulting in yellow (partially quelled) or white (fully quelled) conidia and hyphae (data not shown). Since quelling involves the production of dsRNA, the degree of al-1 silencing may reflect the amounts of dsRNA produced. As shown in Fig. , compared with levels observed in the wild-type strain (orange conidia), QDE-2 levels were increased in two partially quelled strains and further increased in two fully quelled strains, suggesting that the production of dsRNA in these strains leads to the increase in QDE-2 expression.
To directly investigate the effect of dsRNA on QDE-2, we examined whether the induction of dsRNA alone was sufficient for the increase in QDE-2 expression. Previously, we and others developed a method to inducibly express dsRNA from inverted repeat sequences to silence gene expression in Neurospora
). The expression of the inverted repeats of the gene of interest are controlled by the QA-inducible (qa-2
) promoter (21
), so that the addition of QA to the medium will lead to the production of dsRNA and initiate gene silencing. The expression of dsRNA leads to the production of homologous siRNA in the wild-type strain but not in the dcl
double mutant (10
). QDE-2 levels were examined in wild-type and qde-2rip
strains and in three wild-type transgenic strains, dsal-1
, and dsfrh
, which carry constructs to induce dsRNA specific for al-1
, a circadian clock gene), and frh
(an essential RNA helicase gene required for circadian clock function), respectively. As shown in Fig. , the presence of QA led to a significant increase in QDE-2 levels in all three strains with the dsRNA constructs. In contrast, QA had no effect on QDE-2 in the wild-type strain. Since these strains produce different dsRNAs, the effect on QDE-2 is dependent on the production of dsRNA rather than gene-specific dsRNA. In addition, we found that the QDE-2 levels in strains with a mutated al-1
gene were comparable to that of the wild-type strain, indicating that carotenoid biosynthesis is not involved in the regulation of QDE-2 expression (data not shown).
To determine whether the increase of QDE-2 expression by dsRNA is regulated at the transcriptional level, Northern blot analysis was performed. As shown in Fig. , the addition of QA led to a significant increase in qde-2
mRNA levels in strains containing the dsRNA constructs, indicating that dsRNA increases QDE-2 expression at the transcriptional level. Although the qa-2
promoter is tightly regulated by the presence of QA, it is not a very strong promoter (13
). The dramatic inductions of qde-2
mRNA and QDE-2 protein in the presence of QA in the dsRNA construct-containing strains suggest that the amount of dsRNA produced endogenously is low.
dsRNA, not siRNA, induces QDE-2 expression, and DCLs are required for the maintenance of QDE-2 levels posttranscriptionally.
The low levels of QDE-2 in the dcl-1ko dcl-2rip
double mutant (Fig. ) suggest that DCLs are required for the maintenance of steady-state levels of QDE-2. Further supporting this notion, we found that the induction of dsRNA in the dcl-1ko dcl-2rip
double mutant failed to increase the level of QDE-2 (Fig. ). In contrast, QDE-1 was not required for the induction of QDE-2 by dsRNA. It has been previously shown that QDE-1 is not required for gene silencing when dsRNA is produced from an exogenous hairpin construct (10
FIG. 2. DCLs posttranscriptionally regulate the steady-state levels of QDE-2, and dsRNA, but not siRNA, is responsible for the transcriptional activation of qde-2. (A) Western blot analysis showing that the level of QDE-2 could not be induced by dsRNA in the (more ...)
The data presented above suggest that the DCLs and siRNA either regulate QDE-2 posttranscriptionally or are required for the transcriptional activation of qde-2
. To distinguish between these two possibilities, Northern blot analyses were performed to examine the expression of qde-2
mRNA after the induction of dsRNA in the dcl-1ko dcl-2rip
double mutant (Fig. ). We found that the transcriptional activation of qde-2
by dsRNA was maintained in the dcl-1ko dcl-2rip
double mutant. In the wild-type strain, qde-2
mRNA levels were induced after 1 h, peaked 4 to 6 h after the addition of QA, and decreased afterwards. In comparison, qde-2
was activated to a higher level in the mutant, and high levels of qde-2
mRNA were maintained 12 h after the addition of QA. In addition, the basal level of qde-2
was higher in the mutant than in the wild-type strain. Despite the increase in qde-2
mRNA in the dcl-1ko dcl-2rip
double mutant, QDE-2 levels failed to increase significantly. In contrast, in the wild-type strain, the increase of the QDE-2 protein was seen 1 to 2 h after the QA treatment; QDE-2 levels were maintained at high levels (~10-fold of the basal level) after 8 h. Since DCL-1 and DCL-2 are responsible for all Dicer activity in Neurospora
), these data indicated that dsRNA, but not siRNA, is responsible for the transcriptional activation of qde-2
expression, and DCLs are not required for dsRNA sensing in the qde-2
transcriptional activation pathway. However, DCLs are required for QDE-2 accumulation posttranscriptionally, suggesting that siRNA, the cleavage product of dsRNA by DCL, plays a role in the accumulation of the QDE-2 protein.
Induction of DCL-2 by dsRNA.
The induction of qde-2
expression by dsRNA prompted us to examine whether the other key component of the RNAi pathway, DCL-2, is also regulated by dsRNA. Although the functions of DCL-1 and DCL-2 are partially redundant, DCL-2 is responsible for more than 90% of the Neurospora
Dicer activity (10
). As shown in Fig. , dcl-2
mRNA was strongly induced by the production of dsRNA in two strains with different dsRNA constructs, indicating that dsRNA also transcriptionally activates dcl-2
expression. Examination of the DCL-2 protein showed that the production of dsRNA also led to a significant increase in DCL-2 levels (Fig. ). In contrast, the presence of QA had no effect on dcl-2
or DCL-2 levels in the wild-type strain. Interestingly, the kinetics of DCL-2 induction by dsRNA were significantly delayed compared to those of QDE-2; DCL-2 levels did not peak until 24 h after the addition of QA. These data suggest that the induction of dcl-2
by dsRNA is a secondary response rather than an immediate response as presumed for qde-2
FIG. 3. Induction of dcl-2 expression by dsRNA. (A) Northern blot analysis showing the induction of dcl-2 mRNA in strains with dsRNA constructs. (B) Western blot analysis showing the induction of DCL-2 protein by dsRNA. The number of hours indicates the time (more ...)
To understand whether QDE-2 participates in the signaling pathway that mediates the induction of gene expression by dsRNA, we examined the induction of DCL-2 by dsRNA in a qde-2 null strain (qde-2rip). As shown in Fig. , the induction of DCL-2 by dsRNA was normal in the qde-2rip strain. Together with the data presented in the above-described figures, these data demonstrate that the components of the RNAi pathway are induced by dsRNA but are not required for dsRNA sensing and transcriptional activation in Neurospora.
Induction of QDE-2 by dsRNA is required for optimal gene silencing.
Since QDE-2 and DCL-2 are two central players in the RNAi pathway, the induction of QDE-2 and DCL-2 by dsRNA suggests that their regulation is important for the efficiency of the pathway. To test this hypothesis, we examined whether the high QDE-2 level induced by dsRNA is required for the efficiency of gene silencing. A construct in which the qde-2 is under the control of the qa-2 promoter (qaQDE-2) and a construct containing the wild-type qde-2 gene with its own promoter were created and transformed into the qde-2rip dsal-1 strain. As shown in Fig. , the wild-type qde-2 construct complemented the function of the endogenous qde-2, and the transformant exhibited dsRNA-induced QDE-2 expression (Fig. ) and silencing of the al-1 gene, as indicated by the white aerial hyphae and conidia in the presence of QA (Fig. ). On the other hand, the expression levels of QDE-2 in qaQDE-2 transformants in the presence of QA were comparable to the uninduced level of QDE-2 in the qde-2rip dsal-1 qde-2 strain. In addition, the qde-2rip dsal-1 qaQDE-2 strains showed only very weak silencing of al-1 in the presence of QA, indicating a severe compromise of RNAi efficiency. These results suggest that the high levels of QDE-2 induced by dsRNA are required for efficient RNAi and that the induction of QDE-2 expression by dsRNA is mediated by the qde-2 promoter.
FIG. 4. Induction of QDE-2 by dsRNA is required for efficient RNAi. (A) Western blot analysis showing the expression of QDE-2 in the indicated strains. (B) Photograph of the corresponding strains growing in slants. (C) Graphic depiction of the indicated qde-2 (more ...)
To further confirm this result, we created constructs that contain both the qde-2 gene with segments of its upstream sequence of different lengths (Fig. ) and the dsal-1 cassette. These constructs were transformed into the qde-2rip strain at the his-3 locus. As shown in Fig. , Pqde-2A, which contains 1.9 kb of qde-2 upstream sequence, fully complemented the function of the endogenous qde-2, as indicated by the induction of QDE-2 and silencing of al-1 in the presence of QA in the transformants. In contrast, in the Pqde-2B transformants, dsRNA-induced QDE-2 expression was abolished; the QDE-2 levels remained at the basal level in the presence of QA. These data indicate that a cis element within the 0.4-kb qde-2 promoter is required for dsRNA-induced expression. Also, as expected, the aerial hyphae and conidia of the Pqde-2B strains remained orange in the presence of QA, indicating a severe compromise of the RNAi pathway. Together, these data indicate that QDE-2 is a limiting factor in the RNAi pathway and that the induction of QDE-2 by dsRNA at the transcriptional level is critical for RNAi function.
Genome-wide search revealed that additional RNAi components and genes homologous to host defense responses are DRAGs.
The observation that qde-2 and dcl-2 were induced by dsRNA indicates the existence of a transcription-based dsRNA response program in Neurospora. To understand the function of this response, we carried out a genome-wide study to identify other DRAGs in Neurospora by microarray and qRT-PCR analyses. To identify genes that are immediately activated by dsRNA, cultures of the dsal-1 strain were treated with QA for 6 h. The wild-type cultures treated with QA were used as the controls. As shown in Fig. , as expected, qde-2 mRNA was dramatically induced in the dsal-1 cultures. In contrast, the qRT-PCR analysis showed that the levels of alpha-actin and qa-2 (a QA-inducible gene) mRNAs were comparable in both strains (Fig. ), indicating that the QA responses were similar in both strains. Thus, activation of genes in the dsal-1 strain should be due to the production of dsRNA. In addition to the microarray experiments, genes known to be involved in the RNAi machinery and some genes with low signal levels in the microarray experiments were analyzed by qRT-PCR.
FIG. 5. Genome-wide identification of DRAGs. QA was added to wild-type (WT) and dsal-1 triplicate cultures for 6 h before their harvest. (A) Northern blot analysis of qde-2 for samples used in the microarray experiments. (B and C) qRT-PCR analysis of gene expression. (more ...)
The microarray experiments showed that the expression of the vast majority of Neurospora genes is not activated by dsRNA. To generate the list of genes induced by dsRNA, only genes that exhibited an average of more than a 1.5-fold increase in the dsal-1 groups and also showed a >1.3-fold increase in all triplicate samples were included. In addition, genes with a weak signal (the signal values are less than 1.5-fold of the background value) were excluded.
To confirm the microarray results, more than half of the identified DRAGs were examined by qRT-PCR analysis, which is a more sensitive and quantitative method than the microarray assay (29
). In addition to the genes identified by microarray experiments, genes known to be involved in the RNAi machinery and some genes with low signal levels in the microarray experiments were also analyzed by qRT-PCR. qRT-PCR results showed that more than 90% of the genes that we identified by the microarray analysis were up-regulated. However, it also revealed that the change (n
-fold) in the microarray experiments was, in most case, underestimated. The smaller change (n
-fold) observed in microarray experiments is due mostly to the higher background signal generated in the microarray experiments than in qRT-PCR. In addition, we observed similar induction of most DRAGs by dsRNA in independent microarray experiments using the dsfrh
strain (data not shown), indicating that induction was not due to the silencing of al-1
By combining the results of microarray and qRT-PCR analyses, we identified 60 DRAGs with inductions that ranged from half to several hundredfold after dsRNA production (Table and Fig. ). This number is certainly an underestimate due to the lack of sensitivity of the microarray analysis. In addition, since dsRNA was induced for only 6 h in these experiments, the genes identified should mostly be the ones that are immediately activated by dsRNA, and genes induced secondarily, such as dcl-2, were likely missed. It is also possible that the induction of some of the DRAGs is specific for the strain and protocol used in our experiments. In these experiments, no genes were found to be consistently down-regulated after the induction of dsRNA.
dsRNA-activated gene expression after 6 h of induction of dsal-1a
We then asked whether the induction of these DRAGs by dsRNA, like the induction of qde-2, was also independent of the DCLs. To address this, we examined the induction of eight DRAGs by dsRNA in the dcl double mutants by qRT-PCR. As shown in Fig. , their robust induction by dsRNA was maintained in the mutants. These data suggest that the induction of most, if not all, DRAGs by dsRNA is mediated by a signaling pathway that is independent of DCLs and siRNA.
qRT-PCR analysis showing the induction of DRAGs by dsRNA in the dcl double mutants. For cultures containing QA, QA was added for 6 h before harvesting.
The classification of the DRAGs based on their known or putative functions revealed that they belong to the major functional groups described below.
(i) RNAi machinery.
As expected, qde-2 was identified as a DRAG by microarray experiments. Although dcl-2 was not found to be significantly induced in the microarray analysis, qRT-PCR revealed that its level was doubled after the induction of dsRNA (Fig. ), suggesting that dcl-2 is not a gene that is immediately activated by dsRNA. In addition, qde-1 and dcl-1, but not qde-3, were found to be modestly induced by dsRNA in the qRT-PCR analysis. qip, a recently identified exonuclease that interacts with QDE-2 and facilitates singled-stranded siRNA production in the RISC complex, was also up-regulated by dsRNA. Thus, most known components of the Neurospora RNAi pathway are induced by dsRNA.
, which encodes one of the three RNA-dependent RNA polymerases in Neurospora
), is the most highly induced DRAG (~30-fold induction) in this group of genes. This result suggests that RRP-3, a homolog of QDE-1, may play a role in the formation of viral and retrotransposon dsRNA (39
(ii) IFN-stimulated and antiviral genes.
Genes with homology to mammalian ISGs form a major class of DRAGs. Neurospora
has four genes that are homologous to the mammalian myxovirus (influenza virus) resistance (Mx) proteins, and three are strongly induced by dsRNA; one (NCU04935.1) was induced >200-fold. Mx proteins are conserved large GTPases with homology to dynamin. They inhibit viral growth by interfering with virus replication, and their induction by IFNs in mammals is an important part of the antiviral response (23
). The induction of Mx genes by dsRNA in Neurospora
and mammals suggests a conserved dsRNA response from fungi to mammals and indicates the potential importance of Mx proteins in antiviral defense systems.
IFN-induced 6-16 family proteins are small proteins containing the IGS12 domain with unknown functions in mammals (37
has five genes encoding 6-16 family proteins (NCU04486.1, NCU04488.1, NCU04489.1, NCY04490.1, and NCU04491.1) that are clustered in a single chromosomal locus. Interestingly, all six genes in this locus (the five 6-16 family protein genes and NCU04487.1) are significantly induced by dsRNA, whereas genes flanking this region (NCU04492.1 and NCU04485.1) are not (data not shown).
(iii) RNA/DNA binding and regulation.
The RNA/DNA binding and regulation DRAGs include a 3′-5′ exonuclease (NCU07036.1), an RNA helicase (NCU04472.1), and set-6
(NCU09495.1); all were dramatically induced by dsRNA (from 40- to 200-fold). The exonuclease belongs to the RNase D family. Mut-7, an RNase D-like protein, is involved in transposon silencing in Caenorhabditis elegans
). The RNA helicase induced by dsRNA belongs to the superkiller-2
) subfamily of helicases. In yeast, ski-2
is part of the host defense system that represses the propagation of dsRNA viruses by working with the exosome complex to degrade viral RNA (55
). In addition, several RNA helicases, including RIG-I, are involved in the antiviral response and the RNAi pathway in animals (18
, which encodes one of the SET-domain-containing proteins in Neurospora
), was induced ~80-fold by dsRNA. SET domains are a signature of lysine protein methyltransferases and are found in histone methyltransferases. In Neurospora
, DIM-5, a SET-domain-containing protein, is a histone H3 Lys9 histone methyltransferase (50
). Although the function of set-6
is not known, its strong induction by dsRNA suggests that it may have a role in chromatin remodeling in response to dsRNA expression.
(iv) Stress response and protein degradation.
Several heat shock proteins (HSPs) and one DNAJ-like cochaperone are up-regulated upon dsRNA induction in Neurospora
. DRAGs involved in stress responses include a Cu/Zn superoxide dismutase, a multidrug resistance protein, and proteins involved in peroxisome function. In addition, two genes involved in regulating proteasome function were induced by dsRNA. In mammals, HSPs and proteasomal subunits are also known to be induced after viral infections or by IFNs (19
). The induction of these genes in Neurospora
suggests a stress response for dsRNA production.
DRAGs involved in metabolism include genes involved in fatty acid and carbohydrate metabolism and transport, such as phosphoenolpyruvate carboxykinase, acetyl coenzyme A (acetyl-CoA) synthetase, two carnitine acetyltransferase, and carnitine/acylcarnitine carrier. Interestingly, phosphoenolpyruvate carboxykinase is an ISG in mammals (19
). Fatty acid metabolism has been shown to play a role in hepatitis C virus replication, and acetyl-CoA synthetase, an enzyme involved in fatty acid metabolism, was found to be up-regulated upon hepatitis C virus infection in mammals (26
). The his-3
gene, which encodes the histidinol dehydrogenase, a key enzyme involved in the histidine biosynthesis pathway, was also significantly induced by dsRNA.
(vi) Genes with unknown functions.
There are 17 DRAGs that are genes with unknown functions. Among these genes, NCU05628.1 (induced ~188-fold) has similarity to RNase H, which is structurally similar to the PIWI domain of the Argonaute proteins (43
). Interestingly, two of its neighboring genes (NCU05629.1 and NCU05631.1) are also significantly induced by dsRNA. Several DRAGs that encode small proteins of unknown function are some of the most highly induced genes identified in our experiments. One of them, NCU08351.1 (81 amino acids), was the most highly induced DRAG in the microarray analysis.