Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference
Thirdly, we tested whether inactivation of lin-35 increases gene silencing resulting from expression of an endogenously transcribed dsRNA. To do this, we took advantage of a system in which worms express GFP exclusively in the hypodermal seam cells, along with a dsRNA targeting the green fluorescent protein (GFP) mRNA [9]. In wild-type animals, where RNAi works with normal efficiency, there is a low level of GFP fluorescence in the seam cells due to targeting by the co-expressed dsRNA (54% of worms have GFP expression visible in their midbody seam cells; Figure 1b, Table 2). If RNAi is used to target genes required for RNAi, however, this reduces GFP knock-down, and there is an observed increase in GFP levels (for example, for rde-4, 67% of worms have GFP expression visible in their midbody seam cells; Figure 1c, Table 2); conversely, targeting genes whose loss increases RNAi efficiency results in a further reduction of GFP expression (for example, for eri-1, 13% of worms have GFP expression visible in their midbody seam cells; Figure 1d, Table 2; p < 0.001, Chi squared test). We found that targeting lin-35 causes a strong enhancement of GFP silencing in the seam cells (1% of worms have GFP expression visible in their midbody seam cells; Figure 1e, Table 2; p < 0.001). When combined with the enhanced RNAi phenotypes described above, this result is consistent with a model in which inactivation of lin-35 enhances the efficiency of RNAi. In addition, since in this system the dsRNA is expressed in the same cells in which the targeting occurs, we conclude that inactivation of lin-35 must enhance the cellular process of RNAi-induced gene silencing, rather than just altering the uptake or systemic transport of dsRNA. Taken together, these results indicate that mutations in lin-35 cause an increase in the effectiveness of RNAi and that this results in stronger and more penetrant RNAi phenotypes for many genes, making lin-35(n745) an invaluable research tool. We note that similar findings were reported by the Ruvkun lab while this manuscript was in preparation [10].
Finally, although inactivation of LIN-35 results in RNAi hypersensitivity, it is possible that some of the genes with an enhanced phenotype in lin-35(n745) animals could represent genetic interactions between lin-35 and a target gene via a mechanism that is independent of the RNAi hypersensitivity of this strain. To directly identify these genes, we took advantage of a strain carrying a mutation in a lin-35 pathway gene that does not show an increased sensitivity to RNAi. In both mammals and worms, LIN-35/Rb proteins are proposed to function by directly binding E2F family proteins [11,12]. The strain efl-1(se1) [13] carries a weakloss-of-function mutation in the worm E2F family gene efl-1, which is known to function with lin-35 in regulating cell-cycle progression [14], as well as development of the vulva [12] and pharynx [15]. efl-1(se1) animals do not show an increased sensitivity to RNAi, as judged by testing genes with an enhanced RNAi phenotype in rrf-3(pk1426) animals, or by inhibiting expression of efl-1 in the RNAi reporter strain GR1401. Thus, to identify genes that interact genetically with the lin-35 pathway, we tested whether genes that have an enhanced RNAi phenotype in lin-35(n745) animals, but not in rrf-3(pk1426) animals, also had enhanced RNAi phenotypes in efl-1(se1) animals (Additional data file 2). We found three genes that fulfilled these criteria (Table 3). The first of these genes is pha-1, which has previously been identified as genetically interacting with lin-35 and efl-1 [15], so validating the success of our approach. The other two genes represent novel lin-35 pathway genetic interaction partners: dpy-22 is predicted to encode a component of the mediator complex that, like LIN-35 and EFL-1, probably also functions in chromatin remodelling [16], and Y106G6E.6 encodes a Casein Kinase I family member. Intriguingly, targeting Y106G6E.6 by RNAi results in abnormalities in early embryonic polarity (C Panbianco and J Ahringer, personal communication); strong reduction of efl-1 function has previously been shown to affect embryonic polarity [13]. EFL-1 affects embryonic polarity at least in part through regulation of MAP kinase activity in the oocyte [13] and our data thus suggest that LIN-35, EFL-1, and Y106G6E.6 cooperate in some way to regulate MAPK activity in the C. elegans oocyte. There is no previously published functional association between p105Rb, E2F and a CKI family member and this underlies the strength of genetic interaction mapping as a way to reveal gene function.
lin-35 animals are more sensitive to RNAi than previously described RNAi hypersensitive strains
We compared the RNAi sensitivity of animals carrying strong loss-of-function mutations in the two previously described genes that are known to negatively regulate RNAi in C. elegans, rrf-3 or eri-1, to that of lin-35(n745) animals. rrf-3(pk1426)[5] and eri-1(mg366)[6] enhanced the RNAi phenotypes of 70 and 69 of 1,749 genes tested, respectively, compared to 113 genes enhanced by lin-35(n745) (Figure 1a; Additional data file 2). Every gene displaying an increased phenotype with rrf-3(pk1426) or eri-1(mg366) also has an increased RNAi phenotype with lin-35(n745). In addition, many genes that have enhanced RNAi phenotypes in rrf-3(pk1426) or eri-1(mg366) have even stronger phenotypes in lin-35(n745).
Although the RNAi clones that we tested in each of the four strains represented a functionally biased set of genes, we also found very similar results when using random RNAi clones targeting genes with many diverse functions. In addition to the approximately 1,800 RNAi clones originally screened, we also screened the first 682 RNAi clones targeting genes on C. elegans chromosome III. These genes have very diverse molecular functions (Additional data file 4) and we found that 42 of these clones also had RNAi phenotypes that were stronger in lin-35(n745) than in rrf-3(pk1426) worms (Additional data file 5). In addition, it is not just the number of genes with enhanced RNAi phenotypes that is greater in lin-35 than in the other strains; the strengths of the RNAi phenotypes are also enhanced. For example, 11 of the genes we tested from chromosome III had an RNAi phenotype in rrf-3 worms that was further enhanced in lin-35 worms (Additional data file 5).
These results show that lin-35(n745) worms are more sensitive to RNAi than any previously described single mutant strain and are an ideal strain for new RNAi-based screens. This is a key finding - merely finding another hypersensitive strain is not a particularly useful research tool unless it is an improvement on the previously identified strains. Our ranking of the three strains is based on the use of a large set of test genes, and thus our conclusion is robust and not a curiosity of a few atypical RNAi phenotypes. We note, however, that Wang et al. [10] also provide evidence that a lin-35(n745); eri-1(mg366) double mutant strain may display a further enhancement in RNAi sensitivity to lin-35(n745), suggesting that these two genes may partially function in parallel.
lin-35(n745) animals display increased sensitivity to RNAi in the nervous system
For unknown reasons, many neuronally expressed genes appear largely refractory to RNAi in wild-type worms, precluding reverse genetic analyses [4]. We generated strong phenotypes for several neuronally expressed genes in lin-35(n745) animals (Table 1), suggesting RNAi-based screens for neuronal functions might be feasible in this strain. To test further for enhanced RNAi sensitivity in the nervous system of lin-35(n745) animals, we focused on genes expressed in the six touch receptor neurons of C. elegans. These neurons sense gentle touch to the body, and several mechanosensory abnormal (mec) genes have been identified that are needed for their development or function [17,18]. Although RNAi has been detected in these neurons when dsRNA is injected into animals [19], it is not seen when dsRNA is delivered by feeding in wild-type animals (AC, C Keller, and MC, unpublished data), rendering high-throughput RNAi screens impractical.
We tested the touch sensitivity of wild-type and lin-35(n745) animals fed on bacteria targeting eight mec genes (mec-2, mec-3, mec-4, mec-8, mec-9, mec-10, mec-12 and mec-18) and two unrelated genes (gfp and sym-1). In wild-type worms, none of the bacterial strains caused touch insensitivity - that is, the Mec phenotype - either in adults that had fed on the bacteria throughout their entire larval development or in their progeny (n > 30 for each). Thus, if bacterial-mediated RNAi is having an effect in the touch neurons of wild-type animals, the effect is too small to generate a detectable phenotype. In contrast, in parallel experiments, lin-35 adults that had been fed with bacteria targeting mec-2, mec-3, mec-4, mec-9 and mec-18 throughout their larval development were touch insensitive, although the animals displayed the Mec phenotype with differences in penetrance and expressivity. Penetrance ranged from 47% (mec-9) to 83% (mec-2). Bacteria expressing mec-2, mec-3, and mec-4 dsRNA consistently gave a highly penetrant phenotype with strong expressivity (that is, the animals had a touch insensitivity similar to animals with null alleles). Bacteria making dsRNA for mec-12 produced a highly penetrant phenotype (63%) with intermediate strength (the animals responded to a few touches). mec-18 bacteria produced less consistent but easily detectable results; in some experiments the penetrance was high (60%) and expressivity strong, whereas in others the penetrance was lower (45%) and the expressivity intermediate. Bacteria producing mec-9 dsRNA gave the weakest positive results with penetrance of 47% and intermediate expressivity. These weaker effects seen with mec-9, mec-12 and mec-18 may be a consequence of the high expression of these genes in the to
Comments: 0
Votes:8