GOBsmacking tales from the RNA world

Graeme O'Neill speaks with RNAi pioneer Peter Waterhouse and meets Drosha, Dicer, Argonaute and other characters.

RNA interference is an ancient mechanism that probably appeared in ancestral eukaryotic cells some two billion years ago, according to RNAi pioneer Dr Peter Waterhouse, of CSIRO Plant Industry.

The main cast of dicing and slicing RNA endonucleases have quirky names like Dicer, Dicer-like, and Drosha. There is no Slicer - in animal cells, the cutting blade of the RNA-induced silencing complex (RISC), is an RNAase called Argonaute2, which silences genes by slicing up their messenger RNAs.

The Argonaute family of proteins was discovered earlier, and the role of Argonaute2 in RNA interference was unsuspected. Mutation experiments have confirmed its role as the catalytic engine for mammalian RNA.

Synthetic short interfering RNAs (siRNAs) fail to induce RISC-mediated suppression of target genes in mammalian cells lacking functional Argonaute-2. Loss of Argonaute2 is also lethal to embyronic mice, confirming the central role of RNAi in co-ordinating the intricate, time-dependent switching of gene expression during embryogenesis.

Mammalian cells express a single Argonaute 'slicer' gene, and only one Dicer. Where mammals have a single Dicer, Waterhouse says, insects, fungi and ciliates have two, suggesting that the double-Dicer configuration is an ancestral state predating the divergence of plants and animals some 2 billion years ago.

All higher plants have at least four Dicers, poplar has five, but rice trumps all with six. "The fifth Dicer is shared by all monocots, and it looks like it is diverging and taking on another function," Waterhouse says.

Dicer processes micro-RNAs, which occur in all eukaryotes, and are now recognised as central players in regulating gene expression. Dicer cleaves <23-meric microRNAs (miRNAs) from larger hairpin molecules coded within introns or intergenic DNA.

RISCs suppress genes by cleaving their messenger RNAs into small (<24-mer) fragments, which ribosomes cannot process into peptides.

Three prime

miRNAs suppress gene activity by a different mechanism that does not involve destruction of the target RNA.

After being cleaved from a precursor hairpin molecule, multiple miRNAs seek out their cognate mRNAs, and bind by complementary base-pairing to motifs in the 3' untranslated region (3'UTR).

The mechanism tolerates a large degree of base-pair mismatches, potentially allowing a messenger to bind more than one species of miRNA, and vice-versa. The mechanism's promiscuity hints that gene repression is combinatorially regulated.

The bound miRNA somehow prevents ribosomes attaching to the still-intact mRNA and translating its code into protein. The miRNA then acts as a 'tag' that causes the inactivated mRNA molecule to be captured by an intracellular structure called a P-body, where it may remain available for future use.

"Animals may have only one Dicer, but they also have Drosha, and they act in concert," Waterhouse says. "What seems to happen is that the microRNA precursor hairpin molecule is transcribed, and Drosha then chops off the leading and trailing sequences of the stem loop region, which are about 70 bases long.

"The stem loop structure is then exported to the cytoplasm, where Dicer chops cuts it into 21-base micro-RNAs and hands them on to Argonaute, the main component of the RISC complex. The RISC tends to be at the 3' end of the messenger RNA, but it doesn't cleave it - it just stops translation."

GOBs

There is no Drosha in plants, and technically, no Dicer - the multiple Dicer-like proteins perform the role of the single mammalian Dicer, as well as other functions.

"There have been a couple of reports of translational repression involving the same gene in plants, but it's based on a single microRNA, and might involve other mechanisms," Waterhouse says. "It's questionable as to whether translational repression occurs in plants.

"Dicer-like 1 acts a bit like Drosha, cutting away the leader and trailer sequences of the hairpin, and then cleaves the miRNAs. All this takes place in the plant cell nucleus, and the miRNAs are then released into the cytoplasm."

Each miRNA contains a sequence that targets it to Argonaute, in the RISC complex. "One idea, which I favour, is that three proteins form the core of the activity: Dicer, Argonaut and another type of protein that our lab has identified in plants, that we are calling a go-between or 'GOB'.

"There's an homologous protein in Drosophila, R2D2. We think GOBs take the miRNAs from Dicer, and passage them through the nuclear membrane into the cytoplasm, and hand them over to Dicer.

"But it could be that Dicer, Argonaute and GOB work together in a complex inside the nucleus, and that Dicer and GOB are involved in recognising the hairpin precursor, and binding to its double-stranded RNA.

"Dicer may then cleave the hairpin and transfer the miRNAs to Argonaute, which may initially be present in the nucleus. Once Argonaute is charged up with its microRNA, it changes configuration, dissociates from Dicer and GOB, and exits into the cytoplasm [to complex with a RISC]. The RISC then uses the microRNA as a template to find messenger RNAs for cleavage by Argonaute."

Dicer 2

Unlike Dicer-like 1, Dicer-like proteins 2, 3 and 4 cleave messenger RNAs into 24 base pair fragments, which are moved to a nuclear complex that mediates direct, RNA-induced transcriptional silencing of genes.

The nucleoprotein complex may include the Argonaute 4 protein. The gene-silencing mechanism involves methylation, de-acetylation and condensation of chromatin, rendering the gene inaccessible to RNA polymerases and other components of the cell's transcription machinery.

"If we insert one of our [synthetic] hairpin molecules into a plant, as a transgene, it is processed by Dicer 4, which cleaves it into 21-mer sequences and loads it into the RISC, where it serves as a template for identifying and cleaving mRNAs from the target gene," Waterhouse says.

Dicer 4 is thus one of biotechnology's most important new tools - it also appears to be involved protecting plants against infection, by cleaving the double-stranded RNA genomes off from invading viruses.

Two other classes of RNA molecules, the short interfering RNAs (siRNAs) and transacting RNAs, are cleaved from endogenous double-stranded RNA molecules. They are also coded in inter-genic DNA, and their transcription appears to be triggered by the activity of microRNAs released by Dicer 4 from hairpin precursors.

"It's a way of making more microRNAs, but instead of siRNAs being cleaved from hairpin precursors, they're transcribed from DNA by an RNA-dependent polymerase. In plants, they appear to mediate the transition from juvenile to adult forms."