Figure 3

Co-knockdown of Zn72D and bel partially restores productively spliced mle mRNA levels. (A) Depicted is the mle gene, which contains 5 exons (white boxes) and 4 introns (black horizontal lines). Poly(A)-1 and -2 are depicted as gray vertical lines within the fifth exon. (Bottom) Four mle transcript isoforms differ in retention of part of intron 2 (Isoforms I1 and I2) and usage of the upstream or downstream poly(A) sites (P1 and P2). The partially retained intron is shown as a thicker horizontal black line. (B) Northern analysis with RNA collected from S2 cells untreated or treated with dsRNAs to Zn72D, bel, or Zn72D+bel. The blot was probed with a probe antisense to mle cDNA. The top band corresponds to isoform I2, P2; the middle band corresponds to both isoforms I1, P2 and I2, P1; and lower band corresponds to isoform I1, P1. In the absence of Zn72D, only I2 isoforms remain. Upon Zn72D+bel knockdown, there are predominantly I2 isoforms but a subtle increase in I1 isoforms compared to Zn72D knockdown. (C) The blot on the left was stripped and reprobed with a probe located between the two poly(A) sites. This probe only recognizes the P2 isoforms. There are similar slight increases in the level of correctly spliced I1 isoforms when comparing the left and right blots, indicating no difference in recovery of the two isoforms of mle mRNA that use alternative poly(A) sites. (D) S2 cells were untreated or treated with mle, Zn72D, bel, or Zn72D+bel dsRNAs and qRT-PCR was performed with primers to the mle transcript that specifically amplify isoform 1. Samples were normalized first to rp49 and then to the untreated sample, setting untreated to 1. The error bars represent the average of four independent experiments, with qPCR performed in triplicate, and error was determined using standard error propagation methods.