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In Letters to Nature: Researchers Uncover Enzymes Involved in miRNA Biogenesis, RISC

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Researchers’ understanding of the mechanics of RNAi and microRNAs just got a little clearer, based on findings detailed in two letters to this week’s Nature.

In the first, Greg Hannon of Cold Spring Harbor Laboratory, Ronald Plasterk of the Hubrecht Laboratory, and colleagues reported that they have identified a heretofore unknown component of RISC in C. elegans, Drosophila, and mammals.

The letter’s authors said that they had already identified four RISC components — short interfering RNAs, Argonaute 2, VIG, and FXR. Now, they have found a fifth, called Tudor-SN (tudor staphylococcal nuclease), which is a protein containing five repeats of a staphylococcal/micrococcal nuclease domain. Four of the repeats are intact, while the fifth is fused at its amino terminus to a tudor domain.

As it turns out, they wrote, purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases, despite containing non-canonical active-site sequences.

“Notably, both Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease,” they wrote. “Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.”

Hannon, Plasterk, and colleagues began by biochemically purifying a RISC complex from Drosophila that degrades its mRNA target. In multiple independent experiments, they identified the previously characterized proteins, as well as Tudor-SN. Further experimentation indicated that all four proteins are present in a single complex, and that a “similar association of C. elegans orthologues of Drosophila RISC proteins” exists.

The letter’s authors noted that they had a hard time detecting interactions between orthologues of Drosophila RISC components in naïve mammalian cells, but that “formation of a complex containing these proteins was induced if we first triggered an RNAi response by transfection with siRNAs.”

“Specifically, we showed interactions between an Argonaute family protein, AGO2, the fragile X mental retardation protein, and the mammalian Tudor-SN homologue, p100,” they wrote. “Notably, complex formation occurred without changes in the expression levels of individual RISC components, indicating that association of pre-existing proteins is nucleated when a[n] siRNA becomes available in the cell.

“Our results point to a common architecture for RISC in animals as a complex that contains small RNA (miRNA or siRNA) and protein components that include an Argonaute family member, VIG, Tudor-SN, and, at least in Drosophila and mammals, a fragile X family member.”

The researchers wrote that they also sought to assess the possibility that Tudor-SN might contribute to the catalysis of RISC, examining its intrinsic nuclease activity.

They reported that, like micrococcal nucleases, Tudor-SN can cleave both RNA and DNA substrates. Additionally, pdTp, a known specific competitive inhibitor of staphylococcal nucleases, appears to inhibit Tudor-SN at certain concentrations.

“Importantly, RISC activity is also inhibited by pdTp,” they noted. “These data are consistent with the possibility that Tudor-SN contributes at least some of the nuclease activity observed in RNAi effector complexes.”

They wrote that their experiments, which include testing the role of Tudor-SN in dsRNA-mediated silencing in vivo, indicate that the protein is a “bona fide RISC component. This is reflected by the co-purification of Tudor-SN and RISC in Drosophila, C. elegans, and mammalian cells.”

But the letter’s authors caution that it remains uncertain whether Tudor-SN is a “catalytic engine of RNAi” in light of several possible inconsistencies in the data: firstly, purified recombinant Tudor-SN is non-sequence-specific, in contrast to RISC, which is highly specific for its mRNA targets; secondly, Tudor-SN will cleave both RNA and DNA, while RISC shows no DNase activity; thirdly, specific cleavage of mRNAs within the siRNA-mRNA hybrid has been observed, making it difficult to “rationalize with the known activities of Tudor-SN and related enzymes.”

The researchers offered two possible solutions to the aforementioned issues. They suggested that it is possible that RISC contains multiple nucleases, only one of which can catalyze site-specific mRNA cleavage, or that Tudor-SN simply does not catalyze RISC.

“Answers to these questions will come only from understanding RISC in sufficient detail to allow reconstitution of its native activity from purified components such that we can study in detail the individual contributions of each to the varied roles of the RNAi effector machinery,” they concluded.

Enter Drosha

In the second letter, V. Narry Kim, of the Institute of Molecular Biology and Genetics and School of Biological Sciences at Seoul National University in South Korea, and colleagues report that they have identified Drosha as the processing enzyme involved in the first step of the creation of mature miRNAs.

According to the researchers, miRNAs are transcribed as long primary transcripts (so-called pri-miRNAs) that are processed into stem-loop precursors that are roughly 70 nucleotides long (known as pre-miRNAs). These pre-miRNAs end up moving to the cytoplasm, where they are chopped up by Dicer into mature miRNAs of about 22 nucleotides in length.

“Although the Dicer-mediated step has been studied intensively, the initiation step remains uncharacterized,” they wrote.

To resolve this matter, Kim and colleagues determined the sites of cleavage, mapping the 5’ and 3’ ends of pre-miRNA. Pre-miR-30a was cloned and found to be a stem-loop of 63 nucleotides with a 2-nucleotide overhang at its 3’ end, which the researchers wrote is “characteristic of the products of the RNase-III-mediated cleavage reaction.”

To further understand the nuclease activity toward pri-miRNA, they also examined the cis-acting requirements for processing. They determined that the cis-acting elements reside in the immediate vicinity and/or inside the stem-loop pre-miRNA. The internal loops and bulges were then removed at or near the cleavage sites through site-directed mutagenesis.

“These mutants were processed efficiently,” the researchers wrote, “indicating that the internal loops and the bulge are not essential for processing. On the contrary, mutations that disturbed the stem structure markedly reduced the efficiency of the initial processing … These results demonstrate the requirement for dsRNA structure around the cleavage sites, supporting the notion that the nuclease of interest may belong to the RNase III family.”

According to Kim et al., human database searches turned up three RNase-III-type proteins: L44, a component of the large subunit of the mitochondrial ribosome; Dicer, of course; and Drosha, which has previously been implicated in pre-ribosomal RNA processing, “making it a good candidate for the nuclear processing factor for miRNA.”

To confirm their suspicions about Drosha, they prepared Dicer and Drosha for in vitro processing. Drosha produced fragments about 70 nucleotides long, while Dicer cleaved the input RNA into fragments roughly 22 nucleotides in length.

Among the key findings from this experiment was that the final RNA products from the Drosha/Dicer combination were more homogenous than RNA fragments produced with Dicer alone. Additionally, Kim and colleagues found that a combination of Drosha and Dicer yielded more 22-nucleotide-long fragments than when twice the amount of Dicer alone was applied. Taken together, these results indicate a synergistic relationship between the two proteins.

The researchers also carried out RNA interference to deplete Drosha in cultured cells and examined the effect on miRNA biogenesis. “All the tested miRNAs significantly decreased after Drosha RNAi, suggesting that Drosha may be widely used for the maturation of most, if not all, miRNAs,” they wrote in the Nature letter.

The researchers noted that, given that Drisha has been implicated in rRNA processing, they do not exclude the possibility that the protein may have dual roles in rRNA and miRNA processing.

They also state that additional work needs to be conducted to understand how miRNA-processing enzymes are regulated, and how Drosha and Dicer specifically recognize their targets when the primary sequences of diverse miRNAs show no conserved elements.

“It will be important to determine structures of miRNA precursors and RNase III proteins,” they wrote. “Further studies of Drosha will help to elucidate the action mechanism of RNase III, and to unravel the mechanism of miRNA biogenesis.”

— DM

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