Nuclear Speckle Is A Key Site For The Formation Of Transcriptionally Active P-TEFb

Positive transcription elongation factor b(P-TEFb),composed of CDK9 and CyclinT1,is able to promote RNA transcription elongation and pre-mRNA processing.To adapt to different growth environments and transcriptional requirements,considerable P-TEFb is "confined" in the inactive complex 7SK snRNP,and P-TEFb is released from 7SK snRNP by two signaling pathways PP2B and PPla to form transcriptionally active P-TEFb with proteins such as BRD4 and AFF1,recruited to Pol? in the suspended state via related proteins on the transcription machinery,and phosphorylates the respective substrates,thereby promoting transcription machinery restart and transcription elongation continues.Our laboratory has done a lot of research on the release of P-TEFb from inactive complexes and the recruitment mechanism of transcriptionally active P-TEFb,and has achieved good research results,but there is still a link between release and recruitment that remains to be studied,that is,how and where transcriptionally active P-TEFb is formed.This paper focuses on the site of transcriptionally active P-TEFb formation.To explore this issue,we used HeLa Strain as a model and established a model of HMBA-induced transcriptional pause release,and successfully found and validated that each subunit of transcriptionally active PTEFb is localized in Nuclear Speckle using techniques such as mass spectrometry,coimmunoprecipitation,western blot,and immunofluorescence.On this basis,we found that when Nuclear Speckle is disrupted,it leads to the inability to form transcriptionally active P-TEFb,and therefore,identified that Nuclear Speckle is the site of transcriptionally active P-TEFb formation.Immediately after,we destroyed Nuclear Speckle and found that P-TEFb could not be recruited to Pol ? through three recruitment channels on the transcription machine,the transcriptional machinery cannot be restarted,full length mRNA cannot be formed,and the overall transcriptional level of the cell is inhibited.Nuclear Speckle plays an important role in epigenetic regulation,transcriptional activation and repression,termination,splicing,3 'end processing,mRNA modification,and mRNA packaging and transport.This study not only discovers the site of transcriptionally active P-TEFb formation,but also gives us a new understanding of the role of Nuclear Speckle in the transcription.