Promoter-Proximal Transcription Termination by the Integrator and Rixosome Complexes

Abstract

Proper regulation of gene expression is critical for organismal development and survival. Promoter-proximal pausing of RNA Polymerase II (RNAPII) and regulated release into processive elongation prevents precocious gene synthesis and provides a platform for cellular signal integration. Paused RNAPII is stabilized by pausing factors DSIF and NELF. Phosphorylation of RNAPII, DSIF, and NELF by the kinase P-TEFb triggers pause release and the transition to processive elongation. While previous studies focused on mechanisms that license RNAPII to enter productive elongation, it has recently become appreciated that paused polymerase can undergo an alternate fate, namely premature termination. However, the factors inducing premature termination remain underexplored. In this thesis, I describe how two multi-subunit complexes, Integrator and the Rixosome, drive promoter-proximal termination to restrict RNA synthesis by RNAPII. Both complexes were previously reported to have roles in noncoding RNA biogenesis. This work, however, extends their functions to protein-coding genes and RNAPII transcription more broadly. First, I show that Integrator recruits the Protein Phosphatase 2A (PP2A) to RNAPII. PP2A dephosphorylates key residues on the paused complex, opposing the stimulatory effect of P-TEFb. Modulation of PP2A association with Integrator causes RNAPII hyperphosphorylation and increased processive elongation. Second, I used rapid protein depletion and nascent transcriptomics to show that the Integrator endonuclease subunit INTS11, which facilitates transcription termination through nascent RNA cleavage, acts much more broadly than previously appreciated, driving termination at all RNAPII loci. I conclude that Integrator globally balances RNAPII elongation and termination promoter-proximally. Interestingly, loss of INTS11 does not affect the phosphatase activity of Integrator-PP2A, highlighting the modular nature of Integrator. Finally, my analysis indicates that in mammalian cells, the Rixosome is recruited to Polycomb Repressive Complex (PRC) target genes. PRC compacts chromatin to represses genes until the correct developmental timepoint. At these sites, the Rixosome endonuclease subunit LAS1L cleaves nascent RNA, which facilitates RNAPII termination and targets RNA for degradation. This prevents expression of developmentally inappropriate transcripts. In sum, this work highlights promoter-proximal transcription termination as a crucial mechanism for regulating gene expression.