The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.

The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNArecognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152-155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181-359). Consistently, point mutations in an evolutionarily conserved motif (aa 202-205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.

Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclindependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system. Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3,26,46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6).The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5Ј end of the viral transcript (4,38,59,66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein.The formation of a quaternary complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15,18,30,33,44,65).Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity...

Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.

Background. Enteroviruses are small non-enveloped viruses with (+) ssRNA genome with one open reading frame. Enterovirus protein 3C (or 3CD for some species) binds the replicative element oriL to initiate replication. The replication of enteroviruses features low fidelity, which allows the virus to adapt to the changing environment on the one hand, and requires additional mechanisms to maintain the genome stability on the other. Structural disturbances in the apical region of oriL domain d can be compensated by amino acid substitutions in positions 154 or 156 of 3C (amino acid numeration corresponds to poliovirus 3C), thus suggesting the co-evolution of these interacting sequences in nature. The aim of this work was to understand co-evolution patterns of two interacting replication machinery elements in enteroviruses, the apical region of oriL domain d and its putative binding partners in the 3C protein. Methods.To evaluate the variability of the domain d loop sequence we retrieved all available full enterovirus sequences (>6400 nucleotides), which were present in the NCBI database on February 2017 and analysed the variety and abundance of sequences in domain d of the replicative element oriL and in the protein 3C. Results.A total of 2,842 full genome sequences was analysed. The majority of domain d apical loops were tetraloops, which belonged to consensus YNHG (Y=U/C, N=any nucleotide, H=A/C/U). The putative RNA-binding tripeptide 154-156 (Enterovirus C 3C protein numeration) was less diverse than the apical domain d loop region and, in contrast to it, was species-specific. Discussion. Despite the suggestion that the RNA-binding tripeptide interacts with the apical region of domain d, they evolve independently in nature. Together, our data indicate the plastic evolution of both interplayers of 3C-oriL recognition. PeerJ reviewing PDF |

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Background: The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.

The positive transcription elongation factor P-TEFb controls the elongation of transcription by RNA polymerase II. P-TEFb is inactivated upon binding to HEXIM1 or HEXIM2 proteins associated with a noncoding RNA, 7SK. In response to the inhibition of transcription, 7SK RNA, as well as HEXIM proteins, is released by an unknown mechanism and P-TEFb is activated. New partners of 7SK RNA were searched for as potential players in this feedback process. A subset of heterogeneous ribonuclear proteins, hnRNPs Q and R and hnRNPs A1 and A2, were thus identified as major 7SK RNA-associated proteins. The degree of association of 7SK RNA with these hnRNPs increased when P-TEFb-HEXIM1-7SK was dissociated following the inhibition of transcription or HEXIM1 knockdown. This finding suggested that 7SK RNA shuttles from HEXIM1-P-TEFb complexes to hnRNPs. The transcription-dependent dissociation of P-TEFb-HEXIM1-7SK complexes was attenuated when both hnRNPs A1 and A2 were knocked down by small interfering RNA. As hnRNPs are known to interact transiently with RNA while it is synthesized, hnRNPs released from nascent transcripts may trap 7SK RNA and thereby contribute to the activation of P-TEFb.The positive transcription elongation factor P-TEFb is required to activate the transcription of most class II genes (48). P-TEFb comprises two subunits, CDK9, a cyclin-dependent protein kinase, and its corresponding cyclin T1 or cyclin T2. The activity of P-TEFb is regulated. It increases in response to the inhibition of transcription (45, 63) or cardiac cell hypertrophic stimulation (54). Previous studies have indicated that 7SK RNA associates with an inactive form of P-TEFb. 7SK RNA is an abundant (2 ϫ 10 5 -molecule-per-cell) noncoding nuclear RNA of 331 nucleotides (60, 68). The inhibition of P-TEFb activity relies upon the binding of HEXIM1 or HEXIM2 proteins to cyclin T1 or T2 (5,13,41,64). This process requires the association of 7SK RNA with HEXIM1 or HEXIM2 (40,65). Two hairpins in the 7SK RNA structure are involved in this association (14). 7SK binding to HEXIM proteins promotes a major conformational change allowing the C-terminal domains of the proteins to interact with the Nterminal domains of cyclin T's (41, 55). This transcriptiondependent regulation may constitute a feedback loop finetuning the efficiency of the elongation step in class II gene transcription.The molecular regulatory mechanism remains largely unknown. 7SK RNA is very stable even when it dissociates from HEXIM proteins; its degradation is unlikely to contribute to the dissociation. Posttranslational modifications of protein subunits in P-TEFb-HEXIM-7SK may determine their association. For instance, the phosphorylation of CDK9 on threonine residue T186 is required, but the in vivo regulation of this step has not been established (10,35,47). HEXIM proteins and 7SK RNA may be released when P-TEFb binds to components of the transcriptional machinery, such as transcription factors like NF-B (2), retinoblastoma protein (56), androgen receptor (34), aryl hydrocarbon r...