Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

Abstract

Reversible phosphorylation of Pol II and accessory factors helps order the 12 transcription cycle. Here we define two kinase-phosphatase switches that operate 13 at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes 14 inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' 15 ends of genes, respectively, and have overlapping but distinct specificities for 16Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-17 proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 18 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between KOW motifs 4 19 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation 20 decreases as transcription complexes pass the cleavage and polyadenylation 21 signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in 22In fission yeast, chemical-genetic inhibition of Cdk9 led to rapid, nearly complete 130 dephosphorylation of the Spt5 CTD (T1/2 ~20 sec); the rate of decay decreased ~4-fold in 131 dis2 mutant strains, suggesting that the fast kinetics in dis2 + cells were partly due to the 132 concomitant activation of Dis2 (PP1) when Cdk9 is inactivated 27 . In HCT116 cells, both 133 pThr806 and a phosphorylation outside the CTRs, pSer666, were lost rapidly upon 134 treatment with 250 nM NVP-2 (T1/2 ~10 min), consistent with a similar, reinforcing effect 135 of kinase inhibition and phosphatase activation (Fig. 1d). 136 137

Understanding transcription across scales: From base pairs to chromosomes

“…RNA polymerase II and the elongation factor SPT5 (the homolog of bacterial NusG) are phosphorylated on long repetitive elements that are found on the C terminus of the RNA polymerase II subunit RPB1 and the C terminus of SPT5 during active elongation (Bataille et al, 2012;Eick and Geyer, 2013;Yamada et al, 2006). These phosphorylations are maintained in gene bodies, and their loss is associated with termination (Cortazar et al, 2019;Kecman et al, 2018;Krajewska et al, 2019;Parua et al, 2018Parua et al, , 2020. Current data support a combined role of the allosteric and torpedo models in RNA polymerase II termination.…”

Fast-acting chemical tools to delineate causality in transcriptional control

“…Metabolic labeling-based nascent RNA-seq after 30-min NVP-2 treatment revealed global downregulation of transcription, which supports the role of CDK9 as a master regulator of RNA Pol II pause release (Muhar et al, 2018). In another study, a 1to 45-min NVP-2 time course revealed rapid loss of SPT5 phosphorylation, strengthening SPT5 as a direct target of CDK9 and leading to mechanistic dissection of distinct CDK9/phosphatase switches that regulate RNA Pol II pause release and termination (Parua et al, 2020).…”

CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation

“…We have generated new phosphoantibodies for SPT5 T806P and SF3B1 T142P, as these two proteins are known to have functions at the 3’end of protein-coding genes, either at the transcriptional level (SPT5) or through splicing and definition of the last exon (SF3B1) (Kyburz et al, 2006, Cortazar et al, 2019, Tellier et al, 2020a, Parua et al, 2020). Kinase-phosphatase switches have recently been shown to regulate transcription at the 3’ end of gene and CDK9 and PP1 activities play a major role in controlling SPT5 T806 phosphorylation and pol II elongation close to poly(A) sites (Parua et al, 2018, Cortazar et al, 2019, Parua et al, 2020). In addition, PP2A is known to dephosphorylate SPT5 S666P (Parua et al, 2018, Huang et al, 2020, Parua et al, 2020).…”

“…While the functions of transcriptional kinases are becoming clearer, thanks in part to the development of cell lines with analog-sensitive kinases (Bishop et al, 2000), the role of phosphatases in this process is still poorly understood. Several CDK9 targets, including SPT5 and Xrn2, are dephosphorylated by the protein phosphatases (PP)1, PP2A, and PP4 (Parua et al, 2018, Huang et al, 2020, Parua et al, 2020, Vervoort et al, 2021). These PPs are involved in numerous cellular processes (Shi, 2009) and whilst their roles in splicing are well known (Mermoud et al, 1992, Shi et al, 2006), their function in transcription and other co-transcriptional processes is only beginning to emerge.…”