RNA elements directing in vivo assembly of the 7SK/MePCE/Larp7 transcriptional regulatory snRNP

Abstract

Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development.

Figure 1.

In vivo association of Larp7 and MePCE with human cellular RNAs. (A) Detection of RNAs associated with Larp7 and MePCE. HeLa RNAs co-immunoprecipitated with endogenous Larp7 and transiently expressed Flag-tagged MePCE were 3′-terminally labelled and separated on a 6% sequencing gel. Control IP reactions performed from non-transfected cells (NT) or without antibody (Cont IP) are shown. Lane M, size markers in nucleotides. (B) Northern blot analysis. HeLa RNAs co-immunoprecipitated with Larp7 and MePCE-FL were separated on a sequencing gel, electroblotted onto a Nylon membrane and probed with oligodeoxynucleotide probes specific for the 7SK, U4 and U6 snRNAs. RNAs isolated from cell extracts (Ext) and pellets of control IPs (Cont IP) were also analysed. (C) The majority of HeLa 7SK snRNA is associated with Larp7. The levels of Larp7, 7SK and U6 were determined by western and northern blot analyses in Larp7-depleted (α-Larp7) and mock-depleted (Cont IP) extracts.

Figure 2.

7SK elements required for in vivo binding of Larp7. (A) Association of transiently expressed truncated 7SK RNAs with HeLa Larp7. Organization of the p7SK expression construct with the relevant sites is shown. Predicted schematic structures of the expressed internally truncated 7SK RNAs are shown. Co-IP of 7SK test RNAs and the endogenous HeLa 7SK snRNA with Larp7 was monitored by northern blot analysis. RNAs from cell extracts (Ext) and control IPs (no antibody) were also analysed. (B) Association of HeLa Larp7 with transiently expressed 7SK RNAs carrying truncated 3′-hairpins. Structure of the 3′-hairpin of 7SK test RNAs is shown. Deleted nucleotides are boxed (d1 and d4) or they are indicated by arrows (d2 and d3). Transiently expressed 7SKd1, 7SKd2, 7SKd3 and 7SK5′h + 3′hd4 RNAs were co-immunoprecipitated with Larp7 and analysed by northern blotting. Endogenous 7SK is indicated. (C) The 3′-terminal hairpin and the following U-rich tail of 7SK work together in Larp7 binding. Schematic structures of the 3′-terminal regions of 7SKext and 7SK2h RNAs are shown. Dashed lines indicate random sequences inserted between the 3′-hairpin and the terminal U-rich tail. Co-IP of transiently expressed 7SKext and 7SK2h RNAs with Larp7 was measured by northern blot analysis.

Figure 3.

In vivo association of Larp7 with mutant 7SK RNAs. (A) Structural requirements of Larp7 binding to the 3′-hairpin of 7SK. Mutant 7SK RNAs carrying sequence alterations (in shaded boxes) in their 3′-hairpin were expressed in HeLa cells, and their co-IP with endogenous Larp7 was measured by RNase A/T1 mapping. For other details, see the legend to Figure 2. (B) Mutational analysis of the terminal loop of the 3′-hairpin of 7SK. Association of HeLa Larp7 with transiently expressed 7SKtag1 RNAs carrying the L1 to L5 nucleotide alterations was investigated by co-IP and RNase A/T1 mapping. (C) The C328 residue in the 3′-terminal U-rich stretch of 7SK is not required for Larp7 binding. Co-IP of transiently expressed 7SKtag1 and 7SK3′U RNAs with Larp7 was assayed by RNase A/T1 mapping.

Figure 4.

7SK elements supporting in vivo MePCE binding. (A) MePCE binds to the basal stem region of the 5′-hairpin of 7SK. Truncated 7SK5′h + 3′h, 7SK5′h and 7SKd5′h RNAs were co-expressed with Flag-tagged MePCE (MePCE-FL) as indicated above the lanes. MePCE-FL was immunoprecipitated, and co-precipitation of mutant and wild-type 7SK RNAs was monitored by northern blotting. (B) Sequence requirements of MePCE binding to the 5′-hairpin of 7SK. Co-IP of transiently expressed MePCE-FL with mutant 7SK5′h RNAs carrying the indicated nucleotide alterations (shaded boxes) was tested by northern blotting.

Figure 5.

Cap independent in vivo association of 7SK snRNA with MePCE. (A) Pol II-mediated expression of 7SK snRNA. Schematic structure of the pU1P-7SKtag2 expression construct with the promoter (U1 PROM) and terminator (U1-3′) of the human U1 snRNA gene is shown. Nucleotide alterations in the 7SK-coding region (tag2) are indicated. HeLa cells transfected with pU1P-7SKtag2 or p7SKtag2 were incubated with (+) or without (−) α-amanitin (α-ama). Accumulation of 7SKtag2 RNAs was measured by RNase A/T1 mapping. RNAs extracted from cells harbouring the pU1P-7SKtag2 plasmid (Ext) were reacted with an anti-trimethyl-G antibody (α-TMG). (B) Pol II-synthesized 7SK RNA associates with both MePCE and Larp7. Extracts (Ext) prepared from HeLa cells transfected with the indicated combination of the pU1P-7SKtag2, pMePCE-FL and pLARP-FL expression plasmids were subjected to IP with anti-Flag antibody. Recovery of 7SKtag2 RNA was monitored by RNase mapping. (C) In vivo association of MePCE with Pol II-synthesized 7SK RNA lacking Larp7-binding capacity. Schematic structure of the 7SK5′h + 3′hm7 RNA with the m7 nucleotide alterations is shown. Co-IP of transiently expressed 7SK5′h + 3′hm7 RNA with MePCE-FL was monitored by northern blot analysis.

Figure 6.

In vivo interaction of Larp7 and MePCE. (A) MePCE can associate with 7SK lacking functional MePCE-binding site. Extracts (Ext) prepared from HeLa cells transfected with the indicated combination of pMePCE-FL and p7SKd5 were reacted with an anti-Flag antibody. Recovery of 7SKd5 RNA was measured by northern blot analysis. (B) Larp7 can tether MePCE to the 7SK snRNP lacking functional MePCE-binding site. Nucleotide alterations (5′hm7 and 3′m7) introduced into the p7SK5′h + 3′h expression construct are indicated in shaded boxes. HeLa cells were transfected with the p7SK5′hm7 + 3′h, p7SK5′hm7 + 3′hm7 and pMePCE-FL expression constructs as indicated. Co-IP of endogenous 7SK snRNA and transiently expressed 7SK5′hm7 + 3′h and 7SK5′hm7 + 3′hm7 RNAs with MePCE-FL and Larp7 was monitored by northern blot analysis.

Figure 7.

Binding of Larp7 to the 7SK snRNA is essential for P-TEFb recruitment. (A) Separation of the 3′-terminal hairpin and the U-rich tail of 7SK by sequence insertion inhibits P-TEFb binding. The 7SKext RNA (see Figure 2C) was transiently expressed in G3H cells expressing HA-CycT1. After extract preparation, HA-CycT1 was immunoprecipitated (α-HA), and co-purification of 7SKext and wild-type 7SK RNAs was tested by northern blotting. (B) The G312 and C313 loop nucleotides are important for P-TEFb binding. 7SKtag1 RNAs lacking or carrying the L1, L2, L3, L4 and L5 nucleotide transversions (see Figure 3B) were expressed in HeLa cells, and their co-IP with endogenous CycT1 was measured by RNase mapping.

Figure 8.

Co-purification of HeLa 7SK snRNP proteins with La protein. HeLa La protein was immunoprecipitated with a La-specific antibody and co-purification of endogenous 7SK snRNA, Larp7, HEXIM1, CycT1, hnRNP A and transiently expressed MePCE-FL was monitored.

Figure 9.

Proposed schematic structure of the 7SK transcriptional regulatory snRNP. Structure of the 7SK snRNA has been adopted from (11). The 7SK elements directing HEXIM1 binding have been reported earlier (20,21).