Co-knockdown of Zn72D and bel restores MSL localization and X-linked gene expression. (A) GFP-MSL2 expressing S2 cells were either untreated or treated with Zn72D, bel, or Zn72D+bel dsRNAs. The left panels show GFP fluorescence (green), the middle panels show DAPI staining to highlight the nucleus (blue), and right panels show the merged image. (B) Western blots with anti-Zn72D (top), anti-Bel (middle), or anti-γ-tubulin (bottom) as a loading control, in knockdowns indicated above. (C) qRT-PCR for the X-linked genes arm, CG14804, and mRpL16 and the autosomal gene RpII140 was done after S2 cells were untreated (gray bars), or treated with dsRNAs to knockdown mle (white), Zn72D (black), bel (blue), or Zn72D+bel (red). Samples were normalized first to rp49 and then to the untreated sample, setting untreated to 1. The error bars represent the average of three independent experiments, with qPCR performed in triplicate, and error was determined using standard error propagation methods. (D) Zn72D and Bel are both present in the cytoplasm. Cells were stained with antibodies to HA (red), Bel (green), and DAPI (blue) to delineate the nucleus. The nucleolar staining in the anti-Bel image was the result of antibody cross-reactivity, as when bel is knocked down, the nucleolar staining remained while the Bel staining decreased (data not shown). (E) Zn72D and Bel are present in both nuclear and cytoplasmic extracts. The cytoplasmic extract is enriched for β-tubulin and depleted for H3K4me3, whereas the nuclear extract is depleted for β-tubulin and enriched for H3K4me3.

Co-knockdown of Zn72D and bel restored MLE protein levels to 70% of normal levels. (A) One representative quantitative western blot for MLE protein upon knockdown of mle, zn72d, bel, or zn72d+bel. (B) MLE levels were quantitated by first normalizing to the amount of γ-tubulin and then averaging the numbers obtained from the 3 experiments with 2 technical replicates per experiment. Standard deviation was found for the average of each experiment.

Identification of proteins that co-immunoprecipitate with Zn72D. (A) IPs were performed with an anti-HA antibody in wild-type (wt) S2 cells and in S2 cells expressing HA-Zn72D. Proteins that co-IP with HA-Zn72D were identified by mass spectrometry. (B) (Top) HA-Zn72D co-IPs with Myc-CG5641 when cells expressing HA-Zn72D were transiently transfected with a vector expressing Myc-CG5641 (left 2 lanes). IPs were performed using an anti-Myc antibody, and immunoblotting was performed with an HA antibody. Right 2 lanes are controls in which no Myc-CG5641 was transfected. (Bottom left) HA-Zn72D co-IPs with FMR1. IPs were performed using an anti-FMR1 antibody and immunoblotting was performed to detect HA. IPs were performed in cells that did (2 left lanes) and did not (2 right lanes) express HA-Zn72D. (Bottom right) The FMR1 antibody specifically recognizes FMR1 protein. S2 cell extracts from untreated and FMR1 knockdown cells were probed with anti-FMR1 and anti-γ-tubulin. (C) The interaction between Zn72D and Bel is RNA-independent. IPs using anti-HA antibody were performed on S2 cells expressing HA-Zn72D, in the absence (lane 3) or presence (lane 6) of RNase A. Lane 9 is a control IP performed with an anti-HA antibody using S2 cells not expressing HA-Zn72D. HA and Bel were detected using immunoblotting. The ethidium bromide stained gel to the right shows equivalent amounts of input extract loaded per lane, either untreated or treated with RNase A. For parts B and C, "in" indicates 2% of the input into each IP and "FT" indicates 2% of flow-through from each IP.

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.