Interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct the inhibition of P-TEFb.
Transcription elongation of eukaryotic genes by RNA polymerase II depends on the positive transcription elongation factor b (P-TEFb). When sequestered into the large complex, P-TEFb kinase activity is inhibited by the coordinate actions of 7SK small nuclear RNA (7SK snRNA) and hexamethylene bisacetamide (HMBA)-induced protein 1 (HEXIM1). We found that the basic region in HEXIM1 directs its nuclear import via two monopartite and two bipartite nuclear localization sequences. Moreover, the arginine-rich motif within it is essential for its binding to 7SK snRNA, P-TEFb, and inhibition of transcription. Notably, the basic region interacts with the adjacent acidic regions in the absence of RNA. The removal of the positive or negative charges from these regions in HEXIM1 leads to its sequestration into the large complex and inhibition of transcription independently of the arginine-rich motif. Finally, the removal of the negative charges from HEXIM1 results in its subnuclear localization into nuclear speckles. We propose a model where the interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct its binding to P-TEFb and subcellular localization that culminates in the inhibition of transcription.
Barboric
Matjaz
M
Department of Medicine, Rosalind Russell Medical Research Center, University of California at San Francisco, San Francisco, CA 94143-0703, USA.
Kohoutek
Jirí
J
Price
Jason P
JP
Blazek
Dalibor
D
Price
David H
DH
Peterlin
B Matija
BM
D000081:Acetamides / Q000737:chemistry; D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012319:RNA Polymerase II / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D000081:Acetamides; D001426:Bacterial Proteins; C476123:HEXIM1 protein, human; D008164:Luminescent Proteins; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C054562:yellow fluorescent protein, Bacteria; D012313:RNA; D001120:Arginine; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase; D042925:Positive Transcriptional Elongation Factor B; D012319:RNA Polymerase II; C014026:hexamethylene bisacetamide; D005990:Glycerol