Cap-binding Protein Complex Links Pre-mRNA Capping to Transcription Elongation and Alternative Splicing through Positive Transcription Elongation Factor b (P-TEFb)

Tina Lenasi,B Matija Peterlin,Matjaz Barboric

doi:10.1074/jbc.m111.235077

Abstract

Promoter-proximal pausing of RNAPII coincides with the formation of the cap structure at the 5' end of pre-mRNA, which is bound by the cap-binding protein complex (CBC). Although the positive transcription elongation factor b (P-TEFb) stimulates the release of RNAPII from pausing and promotes transcription elongation and alternative splicing by phosphorylating the RNAPII C-terminal domain at Ser2 (S2-P RNAPII), it is unknown whether CBC facilitates these events. In this study, we report that CBC interacts with P-TEFb and transcriptionally engaged RNAPII and is globally required for optimal levels of S2-P RNAPII. Quantitative nascent RNA immunoprecipitation and ChIP experiments reveal that depletion of CBC attenuates HIV-1 Tat transactivation and impedes transcription elongation of investigated CBC-dependent endogenous genes by decreasing the levels of P-TEFb and S2-P RNAPII, leading to accumulation of RNAPII in the body of these genes. Finally, CBC is essential for the promotion of alternative splicing through facilitating P-TEFb, S2-P RNAPII, and splicing factor 2/alternative splicing factor occupancy at a splicing minigene. These findings disclose a vital role of CBC in connecting pre-mRNA capping to transcription elongation and alternative splicing via P-TEFb.

Highlights

The mature mRNA is generated by the synthesis and cotranscriptional processing of pre-mRNA
The stoichiometry of the interaction between cap-binding protein complex (CBC) and positive transcription elongation factor b (P-TEFb) is unclear at present, these findings indicate that CBC associates with the active pool of P-TEFb in cells
We provide evidence for a novel mechanism that connects pre-mRNA capping with two downstream transcriptional events, RNA polymerase II (RNAPII) elongation and alternative splicing

Summary

Introduction

The mature mRNA is generated by the synthesis and cotranscriptional processing of pre-mRNA. CBC Facilitates the Gene Occupancy of P-TEFb and S2-P RNAPII for Tat-stimulated HIV-1 Transcription Elongation— To test directly whether depletion of CBC antagonized transcription elongation from the HIV-1 LTR, we developed a qNARIP assay (for details, see “Experimental Procedures,” supplemental Fig. S3, and supplemental Control Experiments) and used it for analyzing the nascent CAT transcripts in HL3T1 cells expressing F.Tat. Briefly, we cross-linked the cells, sonicated nuclear extracts, immunoprecipitated the pre-mRNA with an antibody directed against RNAPII, and measured relative levels of nascent transcripts at their 5Ј region (5Јr; initiated transcripts) and downstream region (Dr; elongated transcripts) by using two different primer pairs (Fig. 2D).

Results

Conclusion