dPNUTS complexes with and regulates RNAPII phosphorylation.

A) dPNUTS complexes contain RNAPII and PP1; inhibition of PP1 activity in dPNUTS complexes leads to hyperphosphorylation of RNAPII. dPNUTS-S and dPNUTS were immunoprecipitated (IP) from embryonic nuclear extracts and precipitates were probed with ARNA-3 anti-RNAPII antibody. Lane 1, neither hypo- or hyper-phosphorylated RNAPII (RNAPIIa and RNAPIIo respectively) precipitate with pre-immune serum; Lane 2, both RNAPIIa and RNAPIIo precipitate with dPNUTS-S; Lane 3, RNAPIIa, but almost no RNAPIIo, is detected in dPNUTS precipitates. Lane 4, pre-immune serum does not precipitate RNAPII; Lane 5, Inhibitor 2 does not affect the ability of RNAPIIa and RNAPIIo to associate with dPNUTS-S (compare Lane 2); Lane 6, inhibition of PP1 results in conversion of RNAPIIa to RNAPIIo in dPNUTS precipitates (compare Lane 3). Ratios of RNAPIIa and RNAPIIo levels, as derived from densitometry measurements of the respective bands, are shown above the blots. B) Western Blot showing levels of RNAPII CTD Ser5-P (4H8) in extracts from either 1st (L1) or 2nd (L2) instar larvae of the indicated genotypes: homozygous revertant dPNUTSexKG/dPNUTSexKG (exKG/exKG); homozygous null mutant dPNUTS9B/dPNUTS9B (9B/9B) or dPNUTS13B/dPNUTS13B (13B/13B); isogenic control strain w1118/w1118; homozygous hypomorphic mutant dPNUTSKG572/dPNUTSKG572 (KG/KG). 1st instar larval samples from dPNUTS9B/9B and dPNUTS13B/13B were independent extracts run in parallel on the same gel. Blot with anti-Actin antibody shows relative loading. C) Precipitation of RNAPII Ser5-P with dPNUTSW726A but not dPNUTSWT. dPNUTS complexes from Drosophila embryonic nuclear extracts expressing Myc-tagged dPNUTSWT or dPNUTSW726A under the control of da-GAL4 were isolated by immunoprecipitation with anti-Myc antibody. Control precipitations were performed on w1118 extracts (−). This was followed by immunoblotting with anti-RNAPII CTD Ser5-P (4H8) antibody to test for co-immunoprecipitation. Lower panels show immunoblot analyses of total lysates, confirming the levels of total RNAPII and Myc-dPNUTS. D) Levels of RNAPII CTD Ser5-P (H14) on polytene chromosome squashes from salivary glands expressing either histone-H2B YFP or Myc-dPNUTSW726A prepared on the same slide to ensure identical staining conditions (H14 staining in green; DNA staining in magenta). Insets are enlarged views of the distal end of the X chromosome. Arrows indicate approximate lines along which quantitation of fluorescence (in E) was performed. E) Representative line scans through the regions illustrated in D, showing levels of RNAPII CTD Ser5-P staining in the two genotypes. Bar graphs represent the average fluorescence in this region from 6 independent images/genotype. Genotypes are indicated by the colour key.