Regulation of cyclin T1 expression and function by an alternative splice variant that skips exon 7 and contains a premature termination codon.
Cyclin T1 (CCNT1), a gene containing nine exons, forms the positive transcription elongation factor b (P-TEFb) complex and regulates a wide variety of biological processes including transcription. We discovered a novel splice variant of CCNT1 that lacks exon 7 (dE7). RT-PCR analysis revealed that the dE7 transcript was detected in almost all tissues examined. The dE7/FL transcript ratio was high in quiescent peripheral blood mononuclear cells (PBMC) and in tissues poor in cell division; however, it was low in activated PBMC and in tissues with high cell proliferative potential. These results suggest that exon 7 skipping is linked to cell cycle progression. Increasing the dE7/FL transcript ratio resulted in the reduction of CCNT1 protein levels, indicating that the expression of CCNT1 protein is controlled by exon skipping. Exon 7 skipping yields a +1 frameshift at exon 8, which generates a premature termination codon (PTC). The dE7 transcript levels increased when cells were treated with the protein synthesis inhibitor cycloheximide (CHX) or a kinase inhibitor wortmannin (WORT), whilst the FL transcript levels were unchanged, suggesting that the dE7 transcript is a target of nonsense-mediated decay (NMD). Importantly, reduction of dE7 transcript by WORT correlated well with the decrement of CCNT1 protein expression. The dE7 transcript would produce an approximately 23kDa protein that covers approximately 70% of the cyclin box. The ectopically expressed dE7 protein physically interacted with CDK9 and competed with FL CCNT1 for CDK9, thus should act dominant-negatively on FL CCNT1. The replication of human immunodeficiency virus type 1 (HIV-1), heavily dependent on the CCNT1 function, was inhibited by dE7 protein through the attenuation of Tat/long terminal repeat (LTR)-driven transcription. Taken together, these results suggest that dE7 is a novel splice variant that regulates the expression and function of CCNT1.
AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Emiko
E
Urano
Kosuke
K
Miyauchi
Reiko
R
Ichikawa
Yuko
Y
Futahashi
Jun
J
Komano
D017398:Alternative Splicing / Q000187:drug effects / Q000502:physiology*; D000730:Androstadienes / Q000494:pharmacology; D002460:Cell Line; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism*; D056906:Cyclin T / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000235:genetics / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D005091:Exons / Q000502:physiology*; D005260:Female; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D008297:Male; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077191:Wortmannin; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D000730:Androstadienes; C503684:CCNT1 protein, human; D018388:Codon, Terminator; D056906:Cyclin T; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D054322:tat Gene Products, Human Immunodeficiency Virus; D003513:Cycloheximide; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D000077191:Wortmannin