The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: almost identical yet different.

Since the discovery of the RNA interference pathway, several other small RNA pathways have been identified. These make use of the same basic machinery to generate small RNA molecules that can direct different types of (post)transcriptional silencing. The specificity for the different silencing pathways (which type of silencing a small RNA initiates) is likely accomplished by the effector molecules that bind the small RNAs: the Argonaute proteins. Two Argonaute proteins, ALG-1 and ALG-2, have been implicated in one of the silencing pathways, the microRNA (miRNA) pathway, in Caenorhabditis elegans. The two proteins are highly similar, and previous work suggested redundancy of the two proteins. Here, we present genetic and biochemical data that hint at individual nonredundant functions for ALG-1 and ALG-2 in the processing of precursor miRNAs to mature miRNAs.

Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.

Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains approximately 22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. Here we characterize the Caenorhabditis elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41).

The silence of the genes.

About two years ago, it was recognized that introduction of double-stranded RNA (dsRNA) had a potent effect on gene expression, in particular on mRNA stability. Since then, this process has been found to occur in many different organisms, and to bear a strong resemblance to a previously recognized process in plants, called cosuppression. Both genetic and biochemical studies have started to unravel the mysteries of RNA interference: genes involved in this process are being identified and in vitro studies are giving the first hints of what is happening to both the dsRNA and the affected mRNA molecules after the introduction of the dsRNA.

A genetic link between co-suppression and RNA interference in C. elegans.

Originally discovered in plants, the phenomenon of co-suppression by transgenic DNA has since been observed in many organisms from fungi to animals: introduction of transgenic copies of a gene results in reduced expression of the transgene as well as the endogenous gene. The effect depends on sequence identity between transgene and endogenous gene. Some cases of co-suppression resemble RNA interference (the experimental silencing of genes by the introduction of double-stranded RNA), as RNA seems to be both an important initiator and a target in these processes. Here we show that co-suppression in Caenorhabditis elegans is also probably mediated by RNA molecules. Both RNA interference and co-suppression have been implicated in the silencing of transposons. We now report that mutants of C. elegans that are defective in transposon silencing and RNA interference (mut-2, mut-7, mut-8 and mut-9) are in addition resistant to co-suppression. This indicates that RNA interference and co-suppression in C. elegans may be mediated at least in part by the same molecular machinery, possibly through RNA-guided degradation of messenger RNA molecules.

Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.

While all known natural isolates of C. elegans contain multiple copies of the Tc1 transposon, which are active in the soma, Tc1 transposition is fully silenced in the germline of many strains. We mutagenized one such silenced strain and isolated mutants in which Tc1 had been activated in the germline ("mutators"). Interestingly, many other transposons of unrelated sequence had also become active. Most of these mutants are resistant to RNA interference (RNAi). We found one of the mutated genes, mut-7, to encode a protein with homology to RNaseD. This provides support for the notion that RNAi works by dsRNA-directed, enzymatic RNA degradation. We propose a model in which MUT-7, guided by transposon-derived dsRNA, represses transposition by degrading transposon-specific messengers, thus preventing transposase production and transposition.

Target choice determinants of the Tc1 transposon of Caenorhabditis elegans.

The Tc1 transposon of Caenorhabditis elegans always integrates into the sequence TA, but some TA sites are preferred to others. We investigated a TA target site from the gpa-2 gene of C.elegans that was previously found to be preferred (hot) for Tc1 integration in vivo . This site with its immediate flanks was cloned into a plasmid, and remained hot in vitro , showing that sequences immediately adjacent to the TA dinucleotide determine this target choice. Further deletion mapping and mutagenesis showed that a 4 bp sequence on one side of the TA is sufficient to make a site hot; this sequence nicely fits the previously identified Tc1 consensus sequence for integration. In addition, we found a second type of hot site: this site is only preferred for integration when the target DNA is supercoiled, not when it is relaxed. Excision frequencies were relatively independent of the flanking sequences. The distribution of Tc1 insertions into a plasmid was similar when we used nuclear extracts or purified Tc1 transposase in vitro , showing that the Tc1 transposase is the protein responsible for the target choice.

Crystal structure of the specific DNA-binding domain of Tc3 transposase of C.elegans in complex with transposon DNA.

The crystal structure of the complex between the N-terminal DNA-binding domain of Tc3 transposase and an oligomer of transposon DNA has been determined. The specific DNA-binding domain contains three alpha-helices, of which two form a helix-turn-helix (HTH) motif. The recognition of transposon DNA by the transposase is mediated through base-specific contacts and complementarity between protein and sequence-dependent deformations of the DNA. The HTH motif makes four base-specific contacts with the major groove, and the N-terminus makes three base-specific contacts with the minor groove. The DNA oligomer adopts a non-linear B-DNA conformation, made possible by a stretch of seven G:C base pairs at one end and a TATA sequence towards the other end. Extensive contacts (seven salt bridges and 16 hydrogen bonds) of the protein with the DNA backbone allow the protein to probe and recognize the sequence-dependent DNA deformation. The DNA-binding domain forms a dimer in the crystals. Each monomer binds a separate transposon end, implying that the dimer plays a role in synapsis, necessary for the simultaneous cleavage of both transposon termini.