Reconstitution of a functional 7SK snRNP

Abstract

The 7SK small nuclear ribonucleoprotein (snRNP) plays a central role in RNA polymerase II elongation control by regulating the availability of active P-TEFb. We optimized conditions for analyzing 7SK RNA by SHAPE and demonstrated a hysteretic effect of magnesium on 7SK folding dynamics including a 7SK GAUC motif switch. We also found evidence that the 5΄ end pairs alternatively with two different regions of 7SK giving rise to open and closed forms that dictate the state of the 7SK motif. We then used recombinant P-TEFb, HEXIM1, LARP7 and MEPCE to reconstruct a functional 7SK snRNP in vitro. Stably associated P-TEFb was highly inhibited, but could still be released and activated by HIV-1 Tat. Notably, P-TEFb association with both in vitro-reconstituted and cellular snRNPs led to similar changes in SHAPE reactivities, confirming that 7SK undergoes a P-TEFb-dependent structural change. We determined that the xRRM of LARP7 binds to the 3΄ stem loop of 7SK and inhibits the methyltransferase activity of MEPCE through a C-terminal MEPCE interaction domain (MID). Inhibition of MEPCE is dependent on the structure of the 3΄ stem loop and the closed form of 7SK RNA. This study provides important insights into intramolecular interactions within the 7SK snRNP.

Figure 1.

Magnesium induced structure of in vitro 7SK RNA. SHAPE reactivity heat maps showing flexibility of nucleotides 4–272. The color of each cell corresponds to the reactivity of that base from black (non-reactive), through goldenrod (reactive), to yellow (highly reactive). Bases with high background are colored blue. (Left) Magnesium acetate concentrations during the initial folding of 7SK RNA increase as indicated. Regions that change in reactivity are represented by up arrowheads for increases and down arrowheads for decreases. (Right) 7SK RNA was initially folded at 3 mM magnesium and diluted to the indicated concentrations for analysis. Regions that retain 3 mM reactivities are represented by filled arrowheads and open arrowheads represent regions that change after removal of magnesium.

Figure 2.

SHAPE analysis of GAUC mutants. (A) SHAPE results for wild type (WT, from Figure 1) and the three GAUC to CUAG mutations (green numbered boxes) folded under the indicated magnesium folding conditions. (B) Secondary structure predictions of low and high magnesium folded 7SK using RNAstructure and corresponding SHAPE constraints and modeled with forna. The GAUC regions are labeled and SHAPE value colors are mapped on the predicted structures. (C) Secondary structure predictions of GAUC mutants (red boxes) at high magnesium.

Figure 3.

Stepwise in vitro assembly of the P-TEFb containing 7SK snRNP. Each reaction contained 1 pmol of 7SK folded at 3 mM magnesium acetate and diluted to 100 μM and the indicated amounts of proteins added, unless stated otherwise. EMSAs used 4% native PAGE and kinases assays 9% SDS PAGE. (A) Stoichiometric EMSA of HEXIM1 and P-TEFb binding to labeled 7SK RNA folded under the indicated magnesium conditions. (B) Kinase assay of P-TEFb activity using titrations of HEXIM1 and 7SK RNA using silver stain (above) and phosphorimaging (below). Amounts of proteins and RNA are in molar equivalents with 1 signifying 0.02 pmol. (C) Parallel EMSA and SHAPE analysis of individual HEXIM1, LARP7, and MEPCE proteins binding to 7SK RNA compared to low magnesium folded 7SK (from Figure 1). Triangles indicate increasing amounts of proteins (0.1, 0.3, 1 pmol). (D) Parallel EMSA and SHAPE of the indicated combination of proteins binding to 7SK. Proteins are at 1 pmol except for P-TEFb increasing from 0.5 to 1 to 2 pmol. 7SK–protein shifts are labeled with the first letter of the proteins where possible, asterisk denote complexes that did not enter the gel. (E) Stoichiometric EMSA of the assembly of snRNP complex in vitro. (F) Kinase assay as in B. Wild type (W) and GAUC1 mutant (M) RNA.

Figure 4.

Functional analysis of the in vitro 7SK snRNP. Complete 7SK snRNP complexes were assembled under the indicated conditions and sedimented through a glycerol gradient as described in Materials and Methods. All gels are 9% SDS PAGE. (A) The indicated fractions were analyzed using a kinase assay with and without RNase A treatment. (B) Western blot analysis of the glycerol gradients for the indicated proteins. In vitro P-TEFb release assay by kinase assay using (C) HIV-1 Tat and (D) Brd4 P-TEFb interaction domain (PID).

Figure 5.

SHAPE analysis of cellular and in vitro 7SK snRNPs. (A) SHAPE results for (Left) 7SK folded under the indicated magnesium conditions (from Figure 1), (Center) in vitro 7SK snRNP complexes formed under the indicated magnesium conditions and purified by glycerol gradient, and (Right) the indicated cellular 7SK snRNP populations. (B) Secondary structure predications using RNAstructure and SHAPE constraints of cellular and in vitro P-TEFb containing snRNPs colored as in Figure 2.

Figure 6.

The role of the C-terminus of LARP7 and 7SK RNA in inhibition of MEPCE. Unless indicated, 6% TBE urea gels were used. (A) Structural domains of LARP7 and mutants indicating the two RNA binding domains of LARP7 and a predicted helical C-terminal region. (B) Stoichiometric EMSA of full length and truncated LARP7 with MEPCE on 7SK folded as indicated analyzed on a 4% native gel. Amounts of proteins and RNA are in pmol. (C) Methyltransferase assay using 5 pmol MEPCE to methylate recombinant 7SK with ¹⁴C-S-adenosylmethionine and the indicated molar equivalents of LARP7 or LARP7 mutants. (D and E) Methyltransferase assays with the indicated wild type or mutant 7SK RNAs and molar equivalents of LARP7. Gel was analyzed by ethidium bromide staining (EtBr) and phosphorimaging of ¹⁴C-labeled 7SK (7SK). 7SK RNAs are represented as cartoons with the mutated regions denoted by red arrows. (F) SHAPE analysis of the indicated mutant 7SK RNAs folded under indicated Mg conditions. GAUC motifs are shown as numbered green boxes and sites of mutations as ***.

Figure 7.

Model of 7SK snRNP cycle. 7SK in the P-TEFb-containing snRNP is in an ‘open’ conformation with a standard 7SK motif and an extended 5΄ stem loop, which allows HEXIM1 to bind and inhibit P-TEFb. MEPCE is associated with the 5΄ end and LARP7 is associated with the 3΄ stem loop. Upon release of P-TEFb, HEXIM1 is also released leading to an altered 7SK motif structure that is stabilized by hnRNPs. This structure has the 5΄ end of 7SK pairing near the 3΄ end resulting in a ‘closed’ conformation. In this conformation, LARP7 and MEPCE interact resulting in the inhibition of MEPCE. Re-sequestration of P-TEFb may be achieved through a chromatin bound 7SK intermediate containing the alternative 7SK motif. A 7SK motif switch allows HEXIM1 binding and subsequent recruitment of P-TEFb.