Figure 6.
Tissue-specific differences in factors directly involved in leaky termination. Genome map depicting the ORFs of different isoforms of eRF1 (A) and eRF3 (D). (B) RT-qPCR analysis of eRF1 isoforms across different tissue types. For simplicity, isoforms A, B, C, E, F and G are collectively depicted as eRF1A. Note the logarithmic scale for relative abundance. (C) Relative composition of total eRF1 mRNA pool across different tissue types. (E) RT-qPCR analysis of full-length eRF3 (A, B, D), depicted as eRF3A; N-terminally truncated isoform eRF3C, and tj (left panel); and other accessory factors implicated in termination fidelity (right panel): eIF3 subunits S10 and S9, pAbp, pix, Gle1, Dbp80 and Rox8 transcripts using cDNA prepared from adult fly heads and ovaries. CT values for each transcript were normalized against the respective CT values for αTub84B. (F) RT-qPCR analysis of ArgUCG, ArgUCU, CysGCA and TrpCCA isoacceptor tRNAs that are near-cognate to UGA stop codon. PheGGA serves as a control tRNA which is non-cognate to UGA. For cases where individual isodecoders are quantified using separate primer pairs, the isodecoder identity is indicated by the number at the end of the tRNA. CT values for each transcript were normalized against the respective CT values for 18S rRNA. The ΔCT values obtained from each test transcript were then compared between the tissues to derive ΔΔCT. Error bars represent the upper and lower limit of RQ defined by the standard deviation of ΔΔCT from three biological replicates.