Argonaute-dependent sRNA biogenesis are found in many organisms. Here, by using the Neurospora
milR-1 milRNA as an example, we established a biochemical mechanism for an Argonaute-dependent sRNA production process and uncovered the collaborative and distinct roles of the Argonaute QDE-2, exosome and QIP in the milR-1 maturation process. Our biochemical and genetic results suggest that the maturation of milR-1 is a five-step process (). First, the 170-nt pri-milR-1 is directly cleaved by Dicer to generate the ~33-nt double-stranded pre-milRNA. Although we showed that the recombinant DCL-2 is sufficient to convert pri-milRNA into the functional pre-milR-1, pre-milR-1’ was not produced by Dicer cleavage in vitro
, and it is likely that additional factors are involved in this step in vivo
. We previously showed that the down-regulation of MRPL3, a putative RNAse III domain-containing protein, resulted in a decrease in the levels of pre-milR-1 (Lee et al., 2010
), suggesting that MRPL-3 may also participate in this process.
The second step is Argonaute QDE-2 binding of the duplex pre-milRNA. Third, QIP, which is recruited by QDE-2, separates the pre-milRNA duplex into single-stranded RNAs in collaboration with QDE-2; the pre-milRNA strand remains QDE-2-associated, and the pre-milRNA* strand is released from the complex and degraded way. We demonstrated that QIP is indeed a 3’ to 5’exoribonuclease () and importantly, it also posses an activity that can trigger the stand-separation of QDE-2 associated duplex RNA in vitro
( and Figure S4C
). The role of QIP in strand-separation of the pre-milR-1 duplex in vivo
was further supported by the increase of pre-milR-1 duplex levels in qip
mutants ( and ) and by its role in converting the QDE-2-associated siRNA duplex into single strands (Maiti et al., 2007
). Because both pre-milR-1 and siRNA are maintained in duplex forms in qde-2
mutants () (Maiti et al., 2007
), QDE-2 should also contributes to the duplex seapation process in vivo
. This notion is consistent with the existence of low levels of single-stranded pre-milRNA in the qip
mutants () and with previous results that the Drosophila
Ago1 can passively separate miRNA duplexes in vitro
(Kawamata et al., 2009
). The ability of QIP to separate QDE-2-bound duplex pre-milRNA and its role in siRNA RISC activation suggest that strand-separation of Argonaute-bound duplex siRNA and miRNA is an important step in the activation of siRISC and miRISC. Our results and the fact that the budding yeast homolog of QIP, Gfd2p, was identified as a high copy suppressor of an RNA helicase mutant (Estruch and Cole, 2003
) suggest that similar exoribonucleases may also posses dsRNA strand-separation activity.
The selective dissociation of the milRNA* strand of the pre-milR-1 from QDE-2 is likely pre-determined by the asymmetric loading of the duplex pre-milRNA onto QDE-2, so that the strand separation process preferentially dissociates milRNA* strand from the complex. The asymmetric loading of siRNAs, duplex-miRNAs, and pre-piRNAs into Argonaute proteins was recently suggested to be due to the selective binding of Argonaute proteins to small RNAs with a 5’ U (Kawaoka et al., 2011
; Mi et al., 2008
; Seitz et al., 2011
). The pre-milR-1 milRNA strand also has a strong preference for 5’ U, thus, the asymmetric loading of the pre-milRNA duplex may be due to the selective binding of QDE-2 to the pre-milRNA strand.
In the fourth step, the exosome trims the QDE-2 bound pre-milRNAs from 3’ to 5’ end into sRNAs of intermediate sizes. Our conclusion that the exosome carries out this trimming is supported by several lines of evidence. Recombinant QIP cannot efficiently process the single-stranded full-length pre-milR-1and prefers shorter pre-milR-1 substrates (). In addition, there is the accumulation of a ladder of single-stranded pre-milRNAs with sizes between the mature milR-1 and pre-milR-1 in the qip mutants (), indicating that QIP can only efficiently process pre-processed pre-milR-1. Importantly, the silencing of the essential exosome components in Neurospora abolishes the QDE-2-dependent maturation of milR-1 and the production of the processed pre-milRNA ladder, indicating that the exosome is required for milR-1 maturation and is responsible for the generation of the prem-milR-1 substrates for QIP. Furthermore, even in the absence of QIP, a low level of the processed pre-miRNA with sizes similar to the mature milR-1 can be observed (), suggesting that exosome alone is able to convert pre-milRNA into mature milRNA, albeit very inefficiently.
In the fifth and final step, the exosome-processed pre-milRNAs are further processed into mature milRNAs in a process involving both QIP and exosome. The accumulation of the intermediate-sized partially processed pre-milRNAs in the qip mutants indicates that they are substrates of QIP. Supporting this notion, QIP can efficiently convert this ladder of processed pre-milRNAs into mature milRNA in vitro (). In addition, we found that the addition of exosome strongly promoted the QIP-mediated maturation of QDE-2-bound pre-milRNA (), indicating that QIP and exosome collaborate in the 3’ to 5’ progressive trimming of pre-milR-1 into the mature milRNAs.
Our results also shed important insights in the role of Argonauate protein in small RNA maturation. Based on our results, the Argonaute QDE-2 has three essential roles in the milR-1 maturation process: 1) it binds to pre-milR-1 and determines which milR-1 strand will be matured, 2) it recruits exoribonucleases to process pre-milR-1, and 3) it determines the sizes of milR-1 by protecting the mature milR-1 from further processing. We showed that the duplex pre-milR-1 was first become single-stranded before being proceed, however, pre-milR-1 and pre-milR-1* have complete different outcome after processing: while pre-milR-1 was matured into milR-1, the pre-milR-1* was degraded away. In addition, we showed that although the single-stranded pre-milR-1 is associated with QDE-2, the single-stranded pre-milR-1* is not (Figure S4D
). This result is also consistent with the much slower degradation kinetics of pre-milR-1* than that of pre-milR-1 () and the fact that QIP can degrade the synthetic unprotected pre-milR-1 into very small RNA fragments (). Thus, the different processing outcomes of pre-milR-1 and pre-milR-1* is due to the binding of pre-milR-1 by QDE-2.
Crystal structure of an archaeal Argonaute suggested both the 5’ and 3’ ends of the guide RNA are anchored on the protein and the 5’ end of the guide RNA is anchored within a highly conserved basic pocket, suggesting that when in a complex, ~21nt from the 5’end of the small RNA are in complex with and protected by Argonaute (Ma et al., 2005
; Parker et al., 2004
; Wang et al., 2008
). Although the association between QDE-2 and pre-milR-1 protect the region containing the mature milR-1, the 3’ ends of the large pre-milR-1 should be accessible by the nucleases. Thus, the sizes of mature milR-1 are determined by the region of pre-milR-1 that is protected by QDE-2 from further processing.
Recently, a cell-free system derived from a silkworm ovary-derived cell line was established and was shown to recapitulate key steps of animal piRNA biogenesis (Kawaoka et al., 2011
). The proposed 3’ end trimming model for piRNA maturation is very similar to that we proposed for biogenesis of the Neurospora
milR-1. However, the enzyme or enzymes that trim the piRNA-precursor in a PIWI-dependent manner have not been identified. Our study suggests that the highly conserved RNA exosome and DEDDh superfamily of exonucleases may function as the trimmer enzymes in piRNA and other small RNA processing. Since the submission of our paper, the putative exoribonuclease Nibbler has been shown to modify the 3’ ends of mature miR-34 miRNAs in Drosophila
(Han et al., 2011
; Liu et al., 2011
). Interestingly, even though QIP and Nibbler are not sequence homologs, they both belong to the DEDDh superfamily of the ribonuclease. In addition, miR-451 in vertebrates is also generated by an Argonaute-dependent mechanism that is similar to that of the milR-2 in Neurospora
(Cheloufi et al., 2010
; Cifuentes et al., 2010
; Lee et al., 2010
; Yang et al., 2010
). For these two miRNAs, however, the slicer activity of the Argonaute proteins is required to generate miRNA precursors before they are trimmed into maturation by un-identified nuclease.
Exosome components were previously suggested to be involved in small RNA decay (Bail et al., 2010
; Halic and Moazed, 2010
; Ibrahim et al., 2010
), and exosome has also been shown to process the 3’ ends of some Drosophila
mirtron-derived miRNAs (Flynt et al., 2010
). In the fission yeast, the Argonaute-dependent priRNA levels and size distributions were found to be modestly effected in a dis3
mutant (Halic and Moazed, 2010
), but a clear role for exosome in priRNA production is still unclear. In Neurospora
, exosome is clearly essential for Neurospora
milR-1 maturation. On the other hand, we found that the silencing of the exosome components resulted in the significant accumulation of siRNA and other milRNAs examined (), indicating that exosome also functions in sRNA decay pathways in Neurospora
. Therefore, exosome has at least two opposing roles in regulating sRNA levels.