mRNA levels of act-5 and act-3 in WT and mutant alleles. qPCR analysis of act-5 (A) and act-3 (B) mRNA levels in WT and act-5(ptc), act-5(?1), and act-5(?2) mutants. act-3 mRNA levels are upregulated when act-5 mutant mRNA levels are reduced (i.e., only in the act-5(ptc) allele). WT expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 1?source data 1. Two-tailed Student?s t-test was used to calculate P values

(A) Schematic of all known act-5 isoforms and alleles used in this study. Black boxes indicate the deletion and nonsense alleles used in this study; black arrows point to the position of the qPCR primers. The act-5(ok1397) isoform was only identified in the act-5(Δ2) allele. (B) Partial sequence of act-5 (WT, ptc and Δ1 alleles). The PTC in the act-5(ptc) allele is 264 nucleotides from the next exon-intron junction and 888 nucleotides from the stop codon. Red indicates the mutation; PTC is underlined; ‘nnn’ and ‘---’ indicate deleted nucleotides in the act-5(Δ1) allele. The dt2017 deletion leads to a PTC which is located in the last exon (153 bases from the stop codon) and is thus predicted not to trigger NMD (Kashima et al., 2010; Lindeboom et al., 2016). act-5(dt2019) = act-5(ptc); act-5(dt2017) = act-5(Δ1); act-5(ok1397) = act-5(Δ2).

WT expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 1—source data 2. Two-tailed Student’s t-test was used to calculate P values.

(A) qPCR analysis of act-3 pre-mRNA levels in WT and act-5(ptc) mutants. (B) Average dCt values from qPCR analysis of act-3 pre-mRNA levels in WT and act-5(ptc) mutants. WT expression levels are set at 1. Data are mean ± S.E.M.. Two-tailed Student’s t-test was used to calculate P values.

act-5p::rfp extrachromosomal reporter expression was observed in the intestine in 153 of 300 WT animals. (B) act-3p::rfp extrachromosomal reporter expression was observed in the pharynx in 182 of 400 WT animals. (C) act-3p::rfp extrachromosomal reporter expression was observed in the pharynx and intestine in 138 of 320 act-5(ptc) mutants.

act-5p::rfp extrachromosomal reporter expression was observed in the pharynx and intestine in 148 of 300 act-5(ptc) mutants.

mRNA levels of unc-89 and sax-3 in WT and mutant alleles. qPCR analysis of unc-89 (C) and sax-3 (D) mRNA levels in WT and unc-89(ptc1), unc-89(ptc2), unc-89(ptc3), and unc-89(?) mutants. sax-3 mRNA levels in unc-89 alleles are upregulated when unc-89 mutant mRNA levels are reduced, except in the deletion allele. WT expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 3?source data 2. Two-tailed Student?s t-test was used to calculate P values.

Schematic of a subset of the 16 known unc-89 isoforms as well as the deletion and nonsense alleles used in this study (black boxes). Black arrows point to the position of the qPCR primers. (B) Partial sequence of the longest unc-89 isoform with the single nucleotide change causing PTCs. Red indicates the mutations; PTCs are underlined. The distance from each PTC to the next exon-intron junction and to the stop codon is shown in Figure 3—source data 3. unc-89 (gk469156) = unc-89(ptc1); unc-89 (gk506355) = unc-89(ptc2); unc-89 (gk509355) = unc-89(ptc3); unc-89(bns7000) = unc-89(Δ).

(A) qPCR analysis of sax-3 pre-mRNA levels in WT and unc-89(ptc1), unc-89(ptc2), unc-89(ptc3) mutants. (B) Average dCt values from qPCR analysis of sax-3 pre-mRNA levels in WT and unc-89(ptc1), unc-89(ptc2), unc-89(ptc3) mutants. WT expression levels are set at 1. Data are mean ± S.E.M.. Two-tailed Student’s t-test was used to calculate P values.

qPCR analysis of unc-89 (A) and sax-3 (B) mRNA levels in WT and act-5(ptc) mutants as well as of act-5 (C) and act-3 (D) in WT and unc-89(ptc1), unc-89(ptc2) and unc-89(ptc3) mutants. WT expression levels are set at 1. Data are mean ± S.E.M.; Two-tailed Student’s t-test was used to calculate P values.

List of genes and RNAi clones tested in the screen; average dCt values of qPCR analyses of act-5 and act-3 mRNA levels in WT and act-5 mutants as well as of unc-89 and sax-3 mRNA levels in WT and unc-89 mutants.

WT expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 4—source data 1. Two-tailed Student’s t-test was used to calculate P values.

WT expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 4—source data 1. Two-tailed Student’s t-test was used to calculate P values.

WT expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 4—source data 1. Two-tailed Student’s t-test was used to calculate P values.

Factors regulating transcriptional adaptation analyzed in double mutants.

qPCR analysis of act-5 (A) and act-3 (B) mRNA levels in nrde-3(gg66) mutants and act-5(ptc); nrde-3(gg66) double mutants as well as of unc-89 (C) and sax-3 (D) mRNA levels in nrde-3(gg66) and unc-89(ptc); nrde-3(gg66) double mutants. Single mutant nrde-3(gg66) expression levels are set at 1. Data are mean ± S.E.M.; average dCt values are shown in Figure 5—source data 1. Two-tailed Student’s t-test was used to calculate P values.