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. 2024 Aug 20;13:RP97487. doi: 10.7554/eLife.97487

Figure 3. Gene-specific requirements for NRDE-3 can be bypassed in two ways.

(A) Gene schematics (as in Figure 1) of rde-4 and eri-1. (B, C) Loss of ERI-1 can bypass the NRDE-3 requirements for silencing bli-1 but not the other requirements for silencing bli-1 or unc-22. Feeding RNA interference (RNAi) targeting bli-1 (B) or unc-22 (C) with fractions silenced, numbers scored, comparisons, asterisks, and error bars as in Figure 1. (B) Loss of ERI-1 (mg366, jam263, and jam264 alleles) can compensate for the role of NRDE-3 (tm1116 and jam205 alleles) but not of RDE-10 (jam206 allele) or MUT-16 (jam148, jam265, jam266, jam267, and jam268 alleles) in bli-1 silencing. See Table 3 for additional information. (C) Silencing of unc-22 is not restored by loss of ERI-1 (mg366, jam260, jam261, and jam262 alleles) in mutants that also lack any two of mut-16 (jam148 and jam240 alleles), rde-10 (jam196 and jam206 alleles), or nrde-3 (jam205 allele). See Table 3 for additional information. (D, E) Overexpression of RDE-4 in the hypodermis can bypass the requirement for NRDE-3 in bli-1 silencing. (D) Minimal amounts of RDE-4 are sufficient for somatic silencing. (Top) Schematic depicting generation of male progeny with paternal inheritance of a single-copy transgene (Si[…]) that expresses rde-4(+) or rde-1(+) under the control of the mex-5 promoter (mex-5p) in the germline (green) of rde-4(−) or rde-1(−) animals, respectively (germline- and intestine-enriched RDE, based on rescue of RNAi in rde-1(−) animals (39)). (Bottom) Male cross progeny with the transgene were scored after feeding only F1 animals, showing that unlike animals with germline- and intestine-enriched RDE-1, animals with similarly enriched RDE-4 can rescue both unc-22 and bli-1 silencing. Thus, small amounts of RDE-4 potentially mis-expressed in the hypodermis or a non-autonomous effect of RDE-4 from the germline or intestine is sufficient for silencing in the muscle and hypodermis. † indicates p < 0.05 when compared to either wild type or the rde-4(−) mutant and other symbols are as in (B). (E) Silencing of bli-1 is restored in nrde-3(tm1116); rde-4(ne301) double mutants when rde-4(+) is overexpressed in the hypodermis (Si[nas-9p::rde-4(+)]). (F) Silencing of bli-1 is not restored in animals lacking MUT-16 or RDE-10, despite the overexpression of RDE-4 in the hypodermis and/or the loss of ERI-1. (G) Summary depicting the bypass of NRDE-3 when ERI-1 is eliminated and/or RDE-4 is overexpressed. The increase in double-stranded RNA (dsRNA) processing increases the contributions of NRDE-3-independent paths to silencing.

Figure 3—source data 1. Excel sheet containing raw data from RNA interference (RNAi) feed depicted in Figure 3B.
Figure 3—source data 2. Excel sheet containing raw data from RNA interference (RNAi) feed depicted in Figure 3C.
Figure 3—source data 3. Excel sheet containing raw data from RNA interference (RNAi) feed depicted in Figure 3D.
Figure 3—source data 4. Excel sheet containing raw data from RNA interference (RNAi) feed depicted in Figure 3E.
Figure 3—source data 5. Excel sheet containing raw data from RNA interference (RNAi) feed depicted in Figure 3F.

Figure 3.

Figure 3—figure supplement 1. Overexpressing RDE-4 in the hypodermis.

Figure 3—figure supplement 1.

Semiquantitative RT-PCR of rde-4 mRNA and tbb-2 mRNA (control) in wild-type animals, rde-4(−) animals, or rde-4(−) animals expressing rde-4(+) in the hypodermis using a single-copy transgene (Si(nas-9p)).+RT and −RT indicate whether reverse transcriptase was used. The normalized mRNA abundance in rde-4(−) animals was subtracted from all lanes. With the observation in Figure 3E and abundance of rde-4 transcripts in the germline in wild-type animals (in situ data from NEXTDB), expression at ~30% of wild type is expected to be an overexpression in the hypodermis.
Figure 3—figure supplement 1—source data 1. Unlabeled gel image of semi-quantitative polymerase chain reaction (PCR) for rde-4 with the addition of reverse transcriptase – depicted in the first row of Figure 3—figure supplement 1.
Figure 3—figure supplement 1—source data 2. Unlabeled gel image of semi-quantitative polymerase chain reaction (PCR) for rde-4 without the addition of reverse transcriptase – depicted in the second row of Figure 3—figure supplement 1.
Figure 3—figure supplement 1—source data 3. Unlabeled gel image of semi-quantitative polymerase chain reaction (PCR) for tbb-2 with the addition of reverse transcriptase – depicted in the third row of Figure 3—figure supplement 1.
Figure 3—figure supplement 1—source data 4. Unlabeled gel image of semi-quantitative polymerase chain reaction (PCR) for tbb-2 without the addition of reverse transcriptase – depicted in the fourth row of Figure 3—figure supplement 1.
Figure 3—figure supplement 1—source data 5. Labeled images of all gels used in Figure 3—figure supplement 1.
Figure 3—figure supplement 1—source data 6. Excel sheet with quantifications of bands depicted in Figure 3—figure supplement 1 to obtain the numbers depicted below the cropped gel.