Several helicases have been implicated in the RNAi pathways in virtually all organisms in which RNAi is studied;42, 46, 47, 49–63
however, a specific role for any one helicase in RNAi has yet to be assigned. The most likely role for an RNA helicase would be RNA duplex unwinding, a common and traditional role assigned to RNA helicases. This would place RNA helicases at two positions in the RNAi pathway: unwinding the guide strand from the passenger strand of the Dicer-processed siRNA or miRNA; and unwinding the guide strand RNA from its target mRNA.
Generally, RNA helicases have been implicated in RNAi by two means: (1) in their absence, gene silencing by RNAi is reduced and (2) known protein components of the RNAi pathways are found in complexes with these RNA helicases. Despite these two observations, and the apparent need for dsRNA to be unwound in the RNAi pathway, it is possible that RNA helicases play different or additional roles, beyond acting purely as helicases. Moreover, RNA helicases have been implicated in transcription, RNA degradation, gene regulation, RNA export, and nucleocytoplasmic transport, some or all of which may not require RNA helicase activity. For a review see refs. 64–66
. Additionally, several reports have demonstrated helicase activity to be dispensable for the function of the RNA helicase proteins.67–69
Moreover, data from more recent reports characterizing RNA helicases in RNAi, proposes helicase-independent functions for RNA helicases.
RNA helicase A (RHA), a human DEAH-box protein (also known as DDX9, Dhx9, LKP, and NDHII) has been previously known for its roles in transcription, splicing, nuclear export, and translation. For a review see ref. 70
. More recently, RHA was isolated from an affinity purification-based screen to identify RISC components.58
The data suggests that RHA is able promote active RISC formation by facilitating the association of siRNA or processed Dicer substrate short hairpin RNA (shRNA) with Ago2, and the rest of RISC. Gene silencing, and recruitment of siRNA to Ago2 were reduced in RHA-depleted cells. Notably, both guide strand and passenger strand levels bound to Ago2 were reduced. If RHA operated to unwind the guide strand from the passenger strand, then in the absence of RHA, the amount of passenger strand associated with Ago2 should accumulate to a higher degree compared to the amount of guide strand.58
Also, the RNA helicase activity of RHA was found to be inefficient for 3’ overhang RNA, like siRNA.71
Furthermore, it seems as though RHA is not involved in the recycling of RISC since in the absence of RHA, RISC cleavage activity did not diminish over successive rounds of cleavage. Together, this favors a model in which RHA functions to remodel RISC to allow the dsRNA to load onto the complex.58
DEAD-box RNA helicase homolog of human DDX3, Belle (Bel), was previously uncovered as an RNAi pathway candidate in two screens.55, 62
In a more recent study, Bel was uncovered as a bonafide RNAi component in a screen designed to identify genes that either positively or negatively affect miRNA or siRNA pathways in Drosophila
The requirement of Bel for RNAi was also validated in the Drosophila
eye where bel
mutant tissue was shown to be defective in RNAi. Interestingly, in S2 cells, Bel cofractionated with known RISC members: Ago1, Ago2, Fragile X Mental Retardation 1 (FMR1), and Vasa Intronic Gene (VIG). Since Ago1 and Ago2 function in the miRNA and siRNA respectively,72
Bel seems to be important to both pathways. In addition, an artificial siRNA and an abundant endogenous small interfering RNA (endo-siRNA) could be found in Bel immunoprecipitates.63
Thus, Bel may be part of a complex containing RISC members as well as small RNAs. Although it remains unclear where Bel specifically operates, Bel acts downstream of small dsRNA loading onto the RISC, since steady-state and Ago2-bound levels of a sensor for an endo-siRNA were unaffected in Bel depleted S2 cells.63
It seems possible that at least two types of RNA helicases could function in RNAi: those that unwind RNA duplexes, and those that are needed for proper RNA loading within the RISC (). It should be noted that Ago2 alone is not sufficient to bind dsRNA.73
Although unlikely due to the number of RNA helicases implicated in RNAi, it is also possible that a single helicase could potentially act in both roles and/or at various steps in the pathway. To what extent do the roles of the various helicases implicated in RNAi overlap? Initial studies addressing whether or not helicase activity is important for each RNA helicase involved with RNAi to produce a fully functional RISC would be informative. It would also be interesting to investigate whether or not helicases that unwind the guide strand RNA from the passenger strand RNA are the same helicases as those that unwind guide strand RNA from target mRNA.
Figure 1 The Potential Roles of RNA Helicases in RNAi. Pri-miRNAs are processed to pre-miRNAs by a Drosha-containing complex that may also harbor an RNA helicase (1), such as p68,59 which is necessary for its function. Pre-miRNAs and other sources of long dsRNAs (more ...)
One cannot assume that because Bel, or any other RNA helicase functions downstream of dsRNA loading onto the RISC, that it is necessarily involved in one of the two presumed dsRNA unwinding roles in RNAi. In a study on the role of Armitage (Armi) in Drosophila
RNAi, two groups found this putative DEA(H/D) RNA helicase to be required for RNAi.42, 50
Specifically, the data of Tomari et al. suggests that Armi is necessary for single-stranded siRNA incorporation onto the RISC and thus, facilitates the assembly of functionally active RISC. The composition of RISC intermediate complexes were analyzed to determine at which step in RISC assembly Armi functions. While a Dicer-2 containing complex carrying double-stranded siRNA formed in armi
ovary lysates, formation of a mature RISC consisting of single-stranded siRNA was blocked. The most likely explanation for such an event would be that Armi is required to unwind small RNA duplexes. This does not appear to be the case since RNAi was not reconstituted in armi
mutants when cells were supplied with single-stranded siRNA. These results suggested that perhaps Armi functions in an additional step in the RNAi pathway that follows dsRNA unwinding, but precedes formation of single-stranded siRNA-bound RISC. Additional experiments demonstrated that ATP is required for single-stranded siRNA to load onto the RISC, but not for target mRNA recognition or cleavage.42
This again suggests that a helicase with ATPase activity like Armi may be required for ssRNA loading onto the RISC, however, it is possible that Armi could participate in ssRNA loading without the use of ATP. The requirement for the presence of ATP and Armi for ssRNA loading could be indirect.
Clearly new evidence is emerging which suggests RNA helicases may have functions in RNAi in addition to unwinding dsRNA. However, all of the proposed functions described so far occur downstream of dsRNA loading onto the pre-RISC. Recently however, the DEAD-box RNA helicase p68 (also known as DDX5) was found to associate with a complex containing Drosha, and the DNA damage respondent p53 tumor suppressor in a human colon cancer cell line (HCT116), and human diploid fibroblasts.59
Previously, Dmp68, the Drosophila
homologue of p68, was found to co-purify with Ago2, and to be required for efficient RNAi in S2 cells.53
Moreover, p68 along with another DEAD-box RNA helicase, p72, was found to be a subunit of the Drosha complex, and was also required for processing of miRNAs in mice.74
Additionally, p68 had been shown to physically interact with p53, and act as a coactivator to stimulate p53-dependent transcription.75
Evidence suggests that p53 interacts with the p68-containing Drosha complex by binding p68, and that this p53/p68/Drosha complex is important for the generation of pre-miRNAs from pri-miRNAs. Binding experiments demonstrated that following DNA-damage, p53 recruits the p68/Drosha complex to target pri-miRNAs, thus elevating the level of miRNA processing.59
The identification of p68 interacting at the level of pri-miRNA processing is interesting in that it implies a role for RNA helicases upstream of any of the traditional roles presumed or attributed to RNA helicases in RNAi. Additionally, this finding suggests another potential function for an RNA helicase in RNAi, which does not involve the unwinding of dsRNA since dsRNA is not separated during pri-miRNA processing. While it is unclear what the specific function of p68 could be in regards to pri-miRNA processing, it may involve the rearrangement of RNA-protein interactions. For example, a specific protein arrangement may be important for Drosha to gain access to the pri-miRNAs in order for cleavage to occur. Furthermore, it is possible that in addition to functioning at the level of Drosha-mediated miRNA processing, p68 may have additional roles downstream since the Drosophila homolog, Dmp68, was found in a complex containing the RISC component Ago2.53