LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated

Abstract

Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.

Figure 1.

LARP7 is associated with P-TEFb, HEXIM, MEPCE, 7SK snRNA and U6 snRNA. (A) Schematic comparison of La with LARP7 with information obtained by querying NCBI's Conserved Domain Database. La—La domain, RRM1—RNA recognition motif 1, RRM3— RNA recognition motif 3. (B) SDS gel showing the composition of an affinity-purified LARP7-TAP eluate. The tagged protein is indicated by an asterisk. (C) Northern blots were performed on total RNA extracted from HEK 293 cells (T; 900 ng) or RNA extracted from the LARP7-TAP eluate (L; 60 ng) and probed with RNA oligos specific for U2, U6 or 7SK snRNAs. (D) Network highlighting the interactions of LARP7 with various RNA processing factors, P-TEFb (CCNT1/cyclin T1 and Cdk9 subunits), its negative regulatory factors (HEXIM1 and HEXIM2) and the 7SK snRNA capping enzyme MEPCE. (E) Western blot of HeLa whole cell extract (WCE) or HeLa nuclear extract (NE) probed with the affinity purified LARP7 antibody generated in sheep. Arrowhead—LARP7.

Figure 2.

LARP7 co-sediments with the 7SK snRNP before and after P-TEFb and HEXIM release. (A) Glycerol gradient sedimentation analysis of untreated HeLa cells. Western blotting was performed with the indicated antibodies. (B) Gradient analysis of cells treated with 500 nM Flavopiridol for 1 h. (C) Northern blot of 7SK from gradient samples used in A and B. (D) Ethidium bromide stain of the RNA gels in C prior to northern transfer.

Figure 3.

Immunoprecipitation of LARP7 confirms that it is a stable component of the 7SK snRNP. (A) Glycerol gradient fractions were pooled and subjected to LARP7 immunoprecipitation as described in the text. Nucleic acid and protein bound to the beads were either Trizol extracted or released in SDS loading buffer and analyzed by northern blotting, ethidium bromide staining and western blotting with the indicated antibodies. (B) Autoradiograph from the EMSA of E. coli expressed recombinant LARP7 bound to radioactively labeled 7SK. (C) Co-immunoprecipitation of the 7SK snRNP with HEXIM1 (HEX), hnRNP A1 (A1) and LARP7 antibodies before and after DRB treatment. Samples were then subjected to northern blotting for 7SK and western blotting with the indicated antibodies. Two exposures of the hnRNP A1 western blot are shown.

Figure 4.

Knockdown of LARP7 decreases 7SK levels, increases free P-TEFb, and enhances Tat transactivation. (A) LARP7 knockdown (72 h post-transfection) was monitored by western blotting in extracts from HEK 293 cells mock-transfected or transfected with control or LARP7 siRNA (tubulin was used as a loading control). (B) 7SK steady-state levels were assessed by RNA blotting using total RNA extracted from HEK 293 cells mock-transfected or transfected with control or LARP7 siRNA (48 and 72 h post-transfection) and probed with RNA oligonucleotides that specifically detect 7SK, U6 or U2 snRNAs. (C) Knockdown of LARP7 in HeLa cell glycerol gradient input or the leftover nuclear pellet 72 h post-transfection was determined by western blot and comparison with an actin loading control. (D) Same conditions as C, except the siRNA used targeted MEPCE. (E) Glycerol gradient analysis of the large form of P-TEFb after siRNA knockdown of LARP7 and MEPCE using the Cdk9 antibody. (F) HEK 293 cells were mock-transfected or transfected twice with control, MEPCE, LARP7 or double (MEPCE and LARP7) siRNAs and an HIV-1 LTR-luciferase reporter construct. A Tat-expressing plasmid was co-transfected where indicated. Luciferase activity was measured 24 h after transfection. Results are presented as fold activation relative to transfections in the absence of the Tat expression plasmid. N = 3.

Figure 5.

Direct knockdown of 7SK disrupts the large form of P-TEFb and decreases total P-TEFb. (A) HeLa cells were mock-treated or treated with 7SK siRNA for the indicated periods of time. One control and two independent siRNA treatments are shown for each time point. C-Control, R-siRNA-treated. (B) HeLa cells were mock-treated or treated with 7SK siRNA for 48 h, subjected to glycerol gradient sedimentation, and analyzed by western blot using the indicated antibodies. (C) HeLa cells were mock-treated or treated with 7SK siRNA for 64 h. Quantitative northern blotting for 7SK and western blotting for cyclin T1 and Cdk9 were performed. C-Control, R-siRNA-treated. (D) HeLa cells were treated with the indicated amount of LARP7 siRNA for 48 h, lysed in SDS loading buffer and western blotted with the indicated antibodies. Non-specific bands (ns) are provided as a loading control.

Figure 6.

Model of the 7SK snRNP before and after release of P-TEFb. Prior to treatment with P-TEFb inhibitors or cellular stress, the 7SK snRNP contains P-TEFb, a HEXIM dimer, MEPCE, LARP7 and 7SK. Following release, the 7SK snRNP is composed of, LARP7, 7SK and hnRNP proteins.