PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca²⁺ signaling

Abstract

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV-or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca 2+ )-calmodulin-protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca 2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1␣ (PP1␣) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1␣ releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca 2+ signaling, PP2B and PP1␣ alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription.[Keywords: P-TEFb; CDK activation; transcriptional elongation; Ca 2+ signal transduction; protein phosphatases] Supplemental material is available at http://www.genesdev.org. Received November 21, 2007; revised version accepted March 31, 2008. In eukaryotes, the transcription of protein-coding genes is performed by RNA polymerase (Pol) II in a cyclic process consisting of several tightly regulated stages (Sims et al. 2004). During the elongation stage, the C-terminal domain of the largest subunit of Pol II is phosphorylated by the positive transcription elongation factor b (P-TEFb) (Peterlin and Price 2006;Zhou and Yik 2006). This modification is crucial for Pol II to change from abortive to productive elongation and produce full-length RNA transcripts. Consisting of Cdk9 and cyclin T1 (or the minor forms T2 and K), P-TEFb is considered a general transcription factor required for the expression of a vast array of protein-coding genes (Chao and Price 2001;Shim et al. 2002). Not only is P-TEFb critical for cellular gene transcription, it is also a specific host cofactor for the HIV-1 Tat protein. Tat recruits P-TEFb to the TAR RNA element located at the 5Ј end of nascent viral transcripts, allowing P-TEFb to phosphorylate stalled Pol II and enhance HIV-1 elongation (Peterlin and Price 2006).However, not every P-TEFb in the nucleus is in a transcriptionally active state. A major reservoir of P-TEFb (∼50% of total P-TEFb in HeLa cells) actually exists in an inactive complex termed 7SK snRNP that also contains the 7SK snRNA (Nguyen et al. 2001;Yang et al. 2001) and three nuclear...

Bromodomain and Extra-terminal (BET) Bromodomain Inhibition Activate Transcription via Transient Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein

“…We also found that HMBA, SAHA, and JQ1 share a common mechanism of transcriptional activation (16,17,34). SAHA also affects histone acetylation, and JQ1 affects the "readout" of these marks by BRD4.…”

“…JQ1, HMBA, and SAHA Have Comparable Effects on HIV Transcription-This and previous studies suggested that HMBA, SAHA, and JQ1 share a common mechanism of transcriptional activation (16,17,34). To this end, we treated J⌬K cells with HMBA or SAHA alone or in combination with JQ1 for 24 h and measured the production of HIV by p24 capsid ELISA.…”

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

“…Such contact between Hexim1 and the Cdk9 activation segment accounts for its inhibition. Importantly, phosphorylation of S175 and T186 in the "T-loop" is required for P-TEFb assembly with Hexim1 and 7SK RNA (21,48,49). Like Hexim1, the HIV viral protein Tat interacts mainly with cyclin T1 and is known to compete with Hexim1 for association with P-TEFb (5,22).…”

Disparity between microRNA levels and promoter strength is associated with initiation rate and Pol II pausing

“…However, the outcome of the slow polymerase was an even greater decrease in miRNA synthesis. It is therefore anticipated that under certain physiological conditions in which PolII processivity is altered 40,41 or transcription re-initiation rate is regulated, 42 the effect on miRNA expression would be differential.…”