LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated

Abstract

Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.

Transcription control by long non-coding RNAs

“…83,[92][93][94] 7SK can also function cooperatively with the chromatin regulator HMGA1 to control transcription in both P-TEFb-dependent and -independent modes. 95,96 These cases exemplify a vast range of new mechanistic possibilities by which a regulatory RNA may control transcription by interacting with different protein complexes.…”

“…76,79,[81][82][83][84][85][86] Even though 7SK does not seem to tightly bind chromatin, recent findings link this RNA with the transcription machinery and localized regulation at promoters, implying a more dynamic role in its recruitment to target genes.…”

“…87 Tat also binds to the 5' stem-loop of 7SK, apparently triggering a conformational change in the RNA, but it is unclear if RNA-binding is required for P-TEFb displacement or if it occurs after P-TEFb and Hexim1 are removed from the 7SK snRNP. 83,[88][89][90][91] A prominent model is that Tat captures active P-TEFb from a large pool of inactive 7SK-bound P-TEFb complexes via this competition mechanism and then delivers it to the HIV promoter by binding to TAR. 87 Recent ChIP analyses on an integrated HIV promoter indicate that the 7SK snRNP proteins Larp7 and Hexim1, along with cyclin T1 and Cdk9, the formation of DNA-DNA-RNA triple helical structures as in the case of DHFR and pRNA, or by contacting factors already bound to chromatin.…”

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“…It plays a crucial role in increasing transcriptional elongation from RNA Pol II-dependent promoters, including many key developmental and response genes, as well as the vast majority of protein coding genes [48]. Activity of CDK9 is controlled by associations with other proteins, most notably its cyclin partners, Cyclin T and Cyclin K, but also other activatory cellular proteins, such as c-myc, NF-κB, androgen receptor; and inhibitory complexes, such as the 7SK small nuclear RNA containing complex [49][50][51][52][53][54].…”

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“…23,25,38 The two newly identified factors favor the sequestration of P-TEFb away from chromatin DNA. Indeed, the methylphosphate capping enzyme MEPCE and the RNAbinding protein LARP7 associate with and stabilize the 7SK snRNA, which, in association with inhibitory proteins, termed HEXIMs, binds to P-TEFb and prevents its recruitment to transcribing RNAPII complexes 15,18 (see Figure 3, lower part, for a schematic representation). The formation of transcriptionally active P-TEFb requires its dissociation from the HEXIM-7SK inhibitory complex.…”