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 transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3′ and 5′ ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.

Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination

“…Upon recognition of the poly(A) signal, RNAPII decelerates and continues transcribing beyond PAS until the 5′-3′ exonuclease XRN2 degrades the cleaved RNAPII-tethered nascent transcript and catches up to RNAPII, evicting it from DNA ( Cortazar et al., 2019 ; Parua et al., 2018 , 2020 ). The observed increase in 4sU-labeled reads downstream of PAS suggests that either transcription termination or degradation of the post-PAS transcript is impaired upon acute SPT6 depletion.…”

Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle

“…Thus, we hypothesized that these three genes may be more relevant to adipose tissue regulation than other members. Indeed, CDK4 can phosphorylate IRS2 and Rb to promote adipogenesis [ 16 , 17 ], whereas CDK9, a component of positive transcription elongation factor b (P-TEFb), can phosphorylate the C-terminal domain of RNA polymerase II and regulate the transcription of target genes by facilitating transcriptional elongation [ 32 ]. In 3 T3-L1 cells, CDK9 increased the adipogenic potential by phosphorylating PPARγ directly and inducing its transcriptional activity [ 33 ].…”

CDK9 keeps RNA polymerase II on track

“…CDK9 activity is required to maintain the phosphorylation status of Spt5 upstream of the poly(A) site, most likely by preventing Spt5 dephosphorylation by PP1. Indeed, PP1 is a target of CDK9 in both yeast and human [30,31] and this phosphorylation is inhibitory [30,64]. Thus, CDK9 Fig.…”

“…Phosphorylation of Spt5 occurs in its repetitive C-terminal repeat region (CTR) that, like the RNAPII CTD, harbors the typical Ser/Thr-Pro motif found in most CDK substrates [59]. CDK9 strengthens SPT5 phosphorylation by also inhibiting protein phosphatase 4 (PP4), a phosphatase that stabilizes promoter-proximal pausing by keeping SPT5 unphosphorylated in the 5′ region of genes [64] (Fig. 1).…”

“…The instability of this paused RNAPII is due to the recruitment of the integrator complex, whose endonucleolytic activity triggers cleavage of nascent mRNA transcripts and promoterproximal termination [139]. Recent findings showed that two protein phosphatases, PP2A and PP4, can keep Spt5 unphosphorylated in the promoter-proximal region, thus promoting pausing by counteracting CDK9-mediated phosphorylation [64,141,142]. A non-canonical PP2A enzyme, associated with the Integrator complex, may also dephosphorylate the RNAPII CTD [142,143].…”