MAQ1 and 7SK RNA Interact with CDK9/Cyclin T Complexes in a Transcription-Dependent Manner

Abstract

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...

The breast cell growth inhibitor, estrogen down regulated gene 1, modulates a novel functional interaction between estrogen receptor alpha and transcriptional elongation factor cyclin T1

“…This may explain why minor repression by the FLAG-EDG1 (D150-177) NLS deletion mutant was observed in reporter assays (Figure 2c). Although previous studies have strongly implicated the EDG1 NLS region in nuclear localization (Michels et al, 2003;Ouchida et al, 2003), our data suggest that unknown factors, in Figure 3 Subcellular localization of EDG1 and EDG1 deletion mutants and their interaction with the E/F domain of ERa in vitro. (a) CHO cells were transfected with expression vectors for FLAG-EDG1 or FLAG-EDG1 deletion mutants and subjected to immunostaining as described in 'Materials and methods'.…”

“…We have shown that the NLS region of EDG1 was at least partially responsible for the interaction with ERa (Figure 3b), and others reported that cyclin T1 interaction involved the C-terminus EDG1/HEXIM1 (Michels et al, 2003;Yik et al, 2003). Since cyclin T1 and ERa appeared to interact with different regions of EDG1, some of the interactions observed between GST-EDG1 and cyclin T1 when ERa is present could be due to interaction of ERa with both GST-EDG1 and cyclin T1.…”

“…During preparation of this manuscript, three additional manuscripts were published on the EDG1 homologue, HEXIM1 (Michels et al, 2003;Ouchida et al, 2003;Yik et al, 2003). EDG1/HEXIM1 was shown to repress NF-kB-dependent transcription via its C-terminus and interaction with NF-kB also did not appear to be sufficient for transcriptional repression (Ouchida et al, 2003).…”

“…Both ERa and EDG1 bind the cyclin T1 subunit of P-TEFb and compete with each other for cyclin T1. Since EDG1 was demonstrated to be a inhibitory factor for the functions of P-TEFb (Michels et al, 2003;Yik et al, 2003), it is likely that EDG1 inhibits ERa transcription via sequestering cyclin T1 from ERa. Our proposed model is shown in Figure 7.…”

“…Two specific kinases involved in the phosphorylation of CTD include cyclin-dependent kinase 7 (CDK7)/cyclin H of TFIIH and P-TEFb (Positive transcription elongation factor b) (CDK9/cyclin (T1, T2a, or T2b)), which preferentially phosphorylate Ser 5 and Ser 2 of the heptapeptide repeats, respectively (Spencer et al, 1997). Recent studies further demonstrated that EDG1/HEXIM1 binds to cyclin T1 through its carboxy terminus and functions as an inhibitor of PTEFb activity (Michels et al, 2003;Yik et al, 2003).…”