The Drosophila 7SK snRNP and the essential role of dHEXIM in development

Abstract

Regulation of the positive transcription elongation factor, P-TEFb, plays a major role in controlling mammalian transcription and this is accomplished in part by controlled release of P-TEFb from the 7SK snRNP that sequesters the kinase in an inactive state. We demonstrate here that a similar P-TEFb control system exists in Drosophila. We show that an RNA previously suggested to be a 7SK homolog is, in fact, associated with P-TEFb, through the action of a homolog of the human HEXIM1/2 proteins (dHEXIM). In addition, a Drosophila La related protein (now called dLARP7) is shown to be the functional homolog of human LARP7. The Drosophila 7SK snRNP (d7SK snRNP) responded to treatment of cells with P-TEFb inhibitors and to nuclease treatment of cell lysates by releasing P-TEFb. Supporting a critical role for the d7SK snRNP in Drosophila development, dLARP7 and dHEXIM were found to be ubiquitously expressed throughout embryos and tissues at all stages. Importantly, knockdown of dHEXIM was embryonic lethal, and reduction of dHEXIM in specific tissues led to serious developmental defects. Our results suggest that regulation of P-TEFb by the d7SK snRNP is essential for the growth and differentiation of tissues required during Drosophila development.

Figure 1.

Purification of recombinant dHEXIM and detection of endogenous dHEXIM. (A) Diagram showing the location of regions highly conserved between human HEXIM1 and Drosophila HEXIM (dHEXIM). (B) Silver-stained SDS PAGE analysis of wildtype (WT) and RNA-binding domain mutant (ILAA) of HEXIM1 and dHEXIM. (C) western blot analysis of recombinant dHEXIM expressed in E. coli (Ec) and in endogenous dHEXIM in Kc cells (Kc) probed with affinity purified anti-dHEXIM antibody.

Figure 2.

Characterization of dHEXIM. (A) EMSA with dsRNA and recombinant dHEXIM proteins. The indicated amounts of wildtype (WT) and KHRR to ILAA mutant dHEXIM were incubated with labeled dsRNA and analyzed on a native gel as described in ‘Materials and Methods’ section. (B) Glycerol gradient analysis of control and RNase A treated Kc cell extracts. A Kc cell lysate was prepared and analyzed by glycerol-gradient sedimentation as described in ‘Materials and Methods’ section. Half of the lysate was treated with RNase A and the other half served as a control. Gradients were fractionated from the top so that increasing fraction number correlates with increasing particle size. Aliquots of each fraction were subjected to western blotting with the indicated antibodies. (C) Quantification of signals in (B).

Figure 3.

Characterization of dLARP7. (A) Diagram showing the location of regions highly conserved between human LARP7 and Drosophila HEXIM (dLARP7). (B) Western blot analysis of endogenous dLARP7 in Kc cell whole-cell (W) and nuclear (N) extract probed with affinity purified anti-dLARP7 antibody. Arrow indicates dLARP7. (C) Glycerol-gradient analysis of control and RNase A treated Kc cell extracts. A Kc cell lysate was prepared and analyzed by glycerol gradient sedimentation as described in ‘Materials and Methods’ section. Half of the lysate was treated with RNase A and the other half served as a control. Gradients were fractionated from the top so that increasing fraction number correlates with increasing particle size. Aliquots of each fraction were subjected to western blotting with the indicated antibodies.

Figure 4.

Characterization of the Drosophila 7SK snRNP. (A) Drosophila 7SK gene. The diagram shows the Pol III promoter driving 7SK RNA. The track below the diagram shows conservation of the region across all Drosophila species (from UCSC Genome Browser). (B) Sequence of 7SK RNA. Underlined nucleotides denote a region that is highly conserved between human and Drosophila 7SK. (C) Association of 7SK with LARP7. As described in ‘Materials and Methods’ section, the affinity purified LARP7 antibody was used to pull down LARP7 complexes and the RNA found in the bound ‘B’ and unbound ‘UB’ fractions was detected on a denaturing gel by ethidium bromide staining and by northern blot. (D) EMSA of 7SK RNA with dHEXIM on native gel as described in ‘Materials and Methods’ section. (E) Immunoprecipitation analysis of 7SK snRNP. Antibodies against HEXIM ‘H’, LARP7 ‘L’ and Cyclin T ‘C’ were used in immunoprecipitate the proteins from Kc cell extract. Western blots using the indicated antibodies were used to probe the bound and unbound material from each immunoprecipitation. A northern blot of similar fractions from another set of immunoprecipitations (denoted by an asterisk) was used to examine the presence of 7SK. The immunoprecipitations were not as efficient for this set of reactions. (F) Kinase assay with DSIF substrate. P, P-TEFb; C, Control 7SK snRNP; R, RNaseA treated 7SK snRNP; d, 50 µM DRB. The experiment shown in lanes 3–6 was repeated three times giving an average 1.8-fold stimulation with RNaseA.

Figure 5.

Glycerol gradient analysis of lysates from Kc cells treated with P-TEFb inhibitors or HEXIM siRNA. Treatments and generation of lysates is described in ‘Materials and Methods’ section. Antibodies used in westerns are indicated. (A) Control cells. (B) Cells treated with Flavopiridol for 1 h. (C) Cells treated with DRB for 1 h. (D) Effect of knockdown of dHEXIM. Kc cells were treated with RNAi to lacZ ‘L’ as a control or dHEXIM ‘H’ and equal amounts of SDS lysed cells were analyzed by western blot using the dHEXIM antibody. Cell lysates were also prepared and analyzed by glycerol-gradient sedimentation and western analysis using antibodies to Cyclin T.

Figure 6.

Expression profile of dLARP7 during fly development. All panels show immunofluorescence using confocal microscopy and antibodies to dLARP7 (red) individually or merged with DAPI staining (blue). (A) dLARP7 is differentially expressed in the larval eye-antennal and wing discs. AD, antennal disc; NT, notum; WP, wing pouch. The ommatidial cluster of the eye disc is demarcated by a dashed line and zoomed in the lower panel. (B) dLARP7 is nuclear localized in the larval tissues. Nuclei in the indicated tissues are shown. Arrow heads indicate nucleoli. The inset in the merged image from the Gut shows just the DNA staining of the dashed region to highlight the nucleoli (arrow). (C) Nuclear localization of dLARP7 in the adult ovarian egg chamber.

Figure 7.

Expression profile and knockdown of HEXIM during development. (A) Immunostaining of dHEXIM during embryogenesis using confocal microscopy. Developmental stages are indicated on each panel. All embryos are oriented anterior to the left, dorsal uppermost. (B) Immunostaining of dHEXIM during organogenesis (third instar larvae). Top left to bottom right: wing disc, eye-antennal discs, brain and ring gland, leg disc, fat bodies and salivary glands. (C) The expression domain of the rotund-GAL4 driver (rn-GAL4) is revealed by GFP signal in third instar wing and leg discs (left column), and the expression pattern of dHEXIM in wing and leg discs expressing dHEXIM RNAi under the control of the rotund-GAL4 driver (right column). (D) Mutant phenotypes of dHEXIM knockdown with rotund-GAL4 and lozenge-GAL4 (lz-GAL4). In dHEXIM⁻ leg, most of the tarsal segments from 1 to 5 are missing, thus leading to much foreshortened legs (left column). dHEXIM^- wing is strongly atrophied and crumpled (middle column). dHEXIM⁻ pharates (right column) are headless and fail to progress to adult stage.