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

Abstract

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).

Figure 1

(a) Double-stranded RNA (ds) and single-stranded RNA (ss), both 500 bp long, were incubated with extract from C. elegans embryos. siRNAs are produced in a time-dependent fashion from the dsRNA but not from the ssRNA (time points: 0, 30, and 60 min). (b) siRNA-producing activity can be immunoprecipitated with antisera (I) raised against DCR-1. The preimmune serum (P) is inactive. The left lane contains an RNA marker. (c) siRNA produced by C. elegans extract (C) is ∼2 nt bigger than siRNA produced by Drosophila extract (D). In both reactions a 500-bp dsRNA was used as substrate. (d) Time courses (0, 30, 60 min) with dsRNA of different lengths, using C. elegans extract. siRNA is produced from dsRNA of 35 bp and longer, but the larger dsRNA molecules are clearly better substrates (equal counts and equal mass of RNA were added to each reaction). (e) siRNA production is ATP-dependent. (Lane 1) The substrate (500 bp) was not incubated with extract; (lane 2) a reaction with ATP-depleted extract; (lane 3) ATP is added back to the depleted extract; and (lanes 4,5) nonhydrolyzable ATP analogs are added (ATPγS and ADPNP). (f) Incubation of 500-bp dsRNA with C. elegans embryo extract results in the formation of a ladder, with a spacing characteristic of siRNAs. (Lane 1) Untreated RNA; (lane 2) the same RNA after treatment with extract; (lanes 3,4) identical reactions to lanes 1 and 2, but with a 100-bp dsRNA substrate with a 400-bp single-stranded tail. The laddering stops at the position where the RNA substrate is no longer double stranded.

Figure 2

(a) A wild-type animal next to a dcr-1 homozygous deletion animal. Fertilization of oocytes does not occur, or occurs sporadically. Lack of fertilization is not caused by a sperm defect as mating to wild-type males does not rescue this phenotype (data not shown). This phenotype can be rescued by introducing a wild-type copy of dcr-1. However, the fertilized eggs do not hatch, and the mother displays an egg-laying-defective phenotype (data not shown). (b) Oocytes are abnormal. Oocytes normally do not divide in the gonad, but in dcr-1(pk1531) mutant animals this is frequently observed (arrows indicate division planes). (c–e) Animals carrying a GFP transgene expressing in the germ line. Wild-type animals (n = 25) display clear fluorescence in the gonad (c), which is silenced by dsRNA against GFP (d) (n = 29). Animals homozygous for the dcr-1 deletion displayed the fluorescence both before (n = 18) and after RNAi (e) (n = 22), indicating that the RNAi response is defective.

Figure 3

(a) The wild-type pattern of jam-1::gfp staining in seam cells in adult animals. The seam cells have fused, and no individual cells can be recognized. (b) In the dcr-1 deletion mutant, the seam cells of the adult animal have failed to fuse and have undergone an additional round of cell division. (c) An animal is presented that contains a high copy number hsp::dcr-1 transgene (see Materials and Methods). The animal is sterile owing to the absence of oocytes, similar to a lin-41 knockout phenotype. The region where the oocytes should be is located between the two black bars. (d) Dicer converts the let-7 double-stranded precursor into 21-nt RNA molecules in vitro. Radiolabeled let-7 precursor RNA (Drosophila sequence; Pasquinelli et al. 2000; shown in lane 5) was incubated with Drosophila embryo extract (lane 2), dicer preimmune precipitate (lane 3), and dicer immune precipitate (lane 4). (Lane 1) A control reaction with 500-bp dsRNA. (e) A Northern blot on which RNA from wild-type and mutant animals is probed for let-7; U6 was used for a loading control. (Lanes 1,2) RNA from L4-stage wild-type and dcr-1(pk1351) homozygous mutant animals, respectively. The processed let-7 signal drops 10-fold (corrected for loading) in the dcr-1 mutant population, and the longer let-7 precursor RNA accumulates. We find a similar result (twofold reduction of the processed let-7) when we inhibit dcr-1 by RNAi, as shown in lanes 3 and 4; the RNA in these two lanes was isolated from adult animals.

Figure 4

Dicer may be involved in two distinct processes, both of which require the processing of dsRNA into small RNA (stRNA, small temporal, or regulatory; or siRNA, short interfering). One of these pathways (dsRNA-induced) leads to RNA degradation via the RISC complex and RNAi. The second pathway (let-7-dependent) leads to translational suppression via interaction between endogenous, small temporal RNAs and their mRNA targets. In the stRNA branch the double-stranded precursor and the match between stRNA and target RNA are not perfect (at least for let-7 and lin-4). We indicate this by including a bulge in the dsRNA molecules. This is not intended to specify size or number of mismatched regions.