Nuclear Speckle Is A Key Site For Regulating The Activity Of P-TEFb

Transcriptional regulation of eukaryotic genes is the basis of gene expression regulation networks in cells,and the transcription of the intracellular coding protein’s gene and the small Nuclear RNA(snRNA)depend mainly on RNA polymerase II.The transcription process is involved 5 steps,such as the assembly of the initiation complex,the initiation of transcription,the clearing of the promoter region,the enlongation of transcription and the termination of transcription.Due to the discovery of the positive transcription elongation factor B(P-TEFb),which has been suggested that the assembly of the initiation complex and the initiation of transcription are a key step in the regulation of transcription of eukaryotic genes.However,with in-depth exploration of P-TEFb transcriptional regulation,it is found that transcriptional elongation is tightly reguLated and also the rate limiting step of gene transcription.P-TEFb is present in the cells with two forms,inactive and active.Among them,the inactive P-TEFb is mainly "bound" into the 7SK snRNP complex,while the active P-TEFb consists of BRD4-P-TEFb complex and Super Elongation Complexs(SECs).Our previous studies found that the 7SK snRNP complex dephosphorylated the site of CDK9/Thrl86 by calcium dependent PP2B and PP1 signal pathway,leading to the release of P-TEFb from the 7SK snRNP complex.Also BRD4-P-TEFb and SECs complexs cooperatively reguLate RNA Pol Ⅱ pause release via different recruitment pathways.The dephosphorylation of CDK9/Thr186 must be reactivated,that is,the CDK9/Thr186 phosphorylation which could restore the activity of P-TEFb kinase.However,the mechanism of how to re assemble the phosphorylated CDK9/Thr186 of inactive P-TEFb has not been clarified.According to previous reports,Nuclear Speckle is a space for assembly,storage and modification of splicing factors.And SR protein(SRSF1,SRSF2,etc.),as the major splicing factor in Nuclear Speckle,can not only play a role in the modification of RNA,but also promote the transcription elongation of P-TEFb.On the basis of previous reports,we used the HeLa cells and F1C2 cells with stable expression of Flag-CDK9 as the research model,using HMBA as the drug stimuLation model.By the overexpression and silencing of genes,nucleic acid extraction and immunofluorescence experiments and so on.We found that the activity group of P-TEFb is in high salt and the inactivity group of P-TEFb distributes in low salt.However,P-TEFb related proteins are distributed in Nuclear Speckle by immunofluorescence assay,and the phosphorylated CDK9/Thr186 is also distributed in Speckle.At the same time,according to the principle of DNA damage induced by UV irradiation,with the degradation of Pol Ⅱ and the suspension of transcription,we found that the active P-TEFb still exists even if the chromatin is destroyed via IP of the high salt contents.And we also observed that the active P-TEFb related protein factors(CDK9,CyclinT,BRD4,SECs,etc.)were clustered in Speckle,which indicated that Nuclear Speckle is a key site for the regulation of active P-TEFb.In addition,through the knockout and overexpression experiments,we explored the BRD4,AFF1 and AFF4 are the key factor to raise the CDK9 to the Nuclear Speckle.And the CyclinT is located in the Nuclear Speckle through the ELL2.That is,CyclinT and CDK9 are recruited successively to the Nuclear Speckle by different protein factors.In other words,P-TEFb related components are dissociated before being raised to Speckle.With previous research we have found that HMBA stimuLation resuLted in the dephosphorylation of the CDK9/pT186,and P-TEFb released from the inactive compound.And meanwhile,CDK9 and Cyclin T are also dissociated,which will further reveal the molecuLar mechanism and plays an important role in improving the regulation of the P-TEFb.