A capping-independent function of MePCE in stabilizing 7SK snRNA and facilitating the assembly of 7SK snRNP

Abstract

The 7SK snRNP represents a major reservoir of activity where P-TEFb, a general transcription factor key for RNA polymerase II elongation, can be withdrawn to promote gene expression, cell growth and development. Within this complex, 7SK snRNA is a central scaffold that coordinates key protein-protein interactions and maintains P-TEFb in an inactive state. Although the stability of 7SK directly affects the amount of active P-TEFb in vivo, relatively little is known about how it is maintained and how the 7SK methylphosphate capping enzyme MePCE and LARP7, a La-related protein associated with the 3'-poly(U) of 7SK, contribute to this process. Here, we show that 7SK is capped by the LARP7-free MePCE and in probably a co-transcriptional manner prior to its sequestration into 7SK snRNP. However, upon interacting with LARP7 within 7SK snRNP, MePCE loses its capping activity, probably due to the occlusion of its catalytic center by LARP7. Despite its lack of capping activity in 7SK snRNP, MePCE displays a capping-independent function to promote the LARP7-7SK interaction, which in turn stabilizes 7SK and facilitates the assembly of a stable MePCE-LARP7-7SK subcomplex. Our data indicate that MePCE and LARP7 act cooperatively to stabilize 7SK and maintain the integrity of 7SK snRNP.

Figure 1.

7SK RNA in 7SK snRNP is likely already capped by MePCE, which is the highly predominant, if not the only, source of 7SK capping activity in vivo. (A) NE prepared from either normal HeLa cells (control) or HeLa cells stably expressing a specific shRNA targeting MePCE (shMePCE) was incubated with in vitro transcribed 7SK (r7SK) in capping reactions that also contained [³H]SAM. After the reaction, the RNA products, which were isolated and resolved in a polyacrylamide–urea gel, were subsequently analyzed by autoradiography (top panel) and ethidium bromide (EB) staining (middle panel). The levels of MePCE in NEs of control and MePCE KD cells were examined by anti-MePCE WB in the bottom panel. (B) r7SK or e7SK, which was isolated from purified 7SK snRNP, was incubated in capping reactions with affinity purified F-MePCE and [³H]SAM. The reaction products were analyzed by EB staining (bottom) and autoradiography (top) as in A.

Figure 2.

The 7SK-independent interaction between MePCE and LARP7 nucleates the formation of a stable MePCE–LARP7–7SK subcomplex within 7SK snRNP. (A) NEs prepared from W10, a HeLa-based cell line stably expressing F-MePCE, and the parental HeLa cells were analyzed by WB for the indicated proteins. (B) W10 cells were treated with the indicated agents. NEs prepared from the treated cells were analyzed by WB with the indicated antibodies (right panel) and subjected to anti-Flag immunoprecipitation. Upon elution with the Flag peptide, the compositions of the immunoprecipitates (αFlag IP) were analyzed by WB and NB as indicated (left panel). (C) Prior to the elution with the Flag peptide, the immobilized αFlag IP were washed with a buffer containing the indicated KCl concentrations, and their compositions were subsequently analyzed as in B. (D) NEs of W10 (left panel) and FPS86 cells, a HeLa-based cell line stably expressing F-LARP7 (right panel), were incubated with (+) or without (−) RNase A prior to anti-Flag immunoprecipitation. αFlag IP were analyzed as in B. (E) NEs of HeLa cells were pretreated with (+) or without (−) RNase A prior to immunoprecipitations with the indicated antibodies. The immunoprecipitates were analyzed as in B.

Figure 3.

LARP7 prevents MePCE from capping 7SK in vivo and in vitro. (A) NE from either normal (control) or HeLa cells stably expressing shLARP7 was incubated with r7SK and [³H]SAM in capping reactions. The RNA products were isolated, resolved in a polyacrylamide–urea gel, and analyzed by autoradiography (top left) and EB staining (bottom left). The levels of the indicated proteins in NEs of control and LARP7 KD cells were examined by WB in the right panel. (B) Left panels: Purified F-LARP7 (1.2 pmols) and/or MePCE (1.3 pmols) proteins were incubated with r7SK and [³H]SAM in capping reactions. The RNA products were analyzed as in A. (Right panel) F-LAPR7 and MePCE used in the capping assay were examined on a silver-stained SDS–gel. An asterisk indicates a minor contaminating band in the F-LARP7 preparation. (C) The affinity purified F-MePCE free of any associated 7SK snRNP components and the F-LARP7-bound MePCE, which was isolated through anti-Flag immunoprecipitation and then either untreated (−) or treated (+) with MCN were analyzed by WB and NB for the presence of the indicated factors (left panel). These MePCE samples were subsequently analyzed in 7SK capping reactions as in A.

Figure 4.

LARP7 may occupy the catalytic center of MePCE to inhibit the latter’s capping activity. (A) Wild-type F-MePCE and the VLD-AAA mutant were affinity purified with anti-Flag beads from NEs of transfected HeLa cells under highly stringent conditions (see ‘Materials and Methods’ section) to remove any associated 7SK snRNP components. Upon their normalization to approximately the same level by anti-Flag WB (bottom panel), these two proteins were tested in 7SK capping reactions. The RNA products were isolated and analyzed by autoradiography (top panel) and EB staining (middle panel). (B) Wild-type and VLD-AAA mutant F-MePCE were affinity purified from NEs of transfected HeLa cells under relatively mild conditions to retain their associated factors, which were subsequently detected by WB and NB as indicated.

Figure 5.

MePCE not bound to LARP7 are responsible for all the 7SK capping activity in the nucleus and preferentially associated with the 7SK gene promoter. (A) HeLa NEs were subjected to immunodepletion with the indicated antibodies and then analyzed by WB for the presence of the indicated proteins (right panel). The depleted NEs were then tested in 7SK capping reactions, with the RNA products analyzed by autoradiography (top left) and EB staining (bottom left). (B) The levels of input chromatin derived from the HeLa-based W10 cells stably expressing F-MePCE were carefully adjusted (in 2-fold increments) and then PCR amplified with primer sets corresponding to the indicated genes (top panel). Once the input chromatin corresponding to the five genes were normalized to approximately the same level, ChIP with anti-Flag mAb was performed. After DNA purification, PCR reactions containing α-[³²P]dCTP were carried out and the products analyzed by gel electrophoresis and autoradiography (bottom panel).

Figure 6.

Capping-independent stimulation of the LARP7–7SK binding by MePCE, which leads to the stabilization of 7SK by the cooperative actions of MePCE and LARP7. (A) Gel mobility shift assay was performed with ³²P-labeled wild-type 7SK or 7SK (Δ4U’s) and in the presence (+) or absence (−) of the indicated proteins or SAM. The positions of free 7SK as well as the various 7SK-containing complexes are indicated on the left. (B) 7SK capping reactions containing the same concentration (1×) as or twice (2×) or thrice (3×) more MePCE than that used in gel shift reactions in A were performed in the presence of SAM and absence of LARP7. The RNA products were isolated and analyzed by autoradiography (top panel) and EB staining (bottom panel). (C) HeLa cells were transfected with constructs expressing the indicated shRNAs to KD the expression of MePCE or/and LARP7. The levels of 7SK and the other indicated proteins in NEs of transfected cells were analyzed by WB and NB.