HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells.
P-TEFb regulates eukaryotic gene expression at the level of transcription elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein HEXIM1 or HEXIM2. In an effort to determine the minimal region of 7SK needed to interact with HEXIM1 in vitro, we found that an oligo comprised of nucleotides 10-48 sufficed. A bid to further narrow down the minimal region of 7SK led to a surprising finding that HEXIM1 binds to double-stranded RNA in a sequence-independent manner. Both dsRNA and 7SK (10-48), but not dsDNA, competed efficiently with full-length 7SK for HEXIM1 binding in vitro. Upon binding dsRNA, a large conformational change was observed in HEXIM1 that allowed the recruitment and inhibition of P-TEFb. Both subcellular fractionation and immunofluorescence demonstrated that, while most HEXIM1 is found in the nucleus, a significant fraction is found in the cytoplasm. Immunoprecipitation experiments demonstrated that both nuclear and cytoplasmic HEXIM1 is associated with RNA. Interestingly, the one microRNA examined (mir-16) was found in HEXIM1 immunoprecipitates, while the small nuclear RNAs, U6 and U2, were not. Our study illuminates novel properties of HEXIM1 both in vitro and in vivo, and suggests that HEXIM1 may be involved in other nuclear and cytoplasmic processes besides controlling P-TEFb.
Li
Qintong
Q
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Cooper
Jeffrey J
JJ
Altwerger
Gary H
GH
Feldkamp
Michael D
MD
Shea
Madeline A
MA
Price
David H
DH
D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D014364:Tryptophan / Q000737:chemistry
D004277:DNA, Single-Stranded; C476123:HEXIM1 protein, human; D035683:MicroRNAs; D012330:RNA, Double-Stranded; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D012313:RNA; D014364:Tryptophan; D004247:DNA; D042925:Positive Transcriptional Elongation Factor B