Abstract
CBTF122 is a subunit of the Xenopus CCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122 containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (an in vivo target of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA binding in vitro. In addition, these mutations alter the ability of CBTF122 fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 gene in vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding both in vitro and in vivo.
Highlights
The 122-kDa subunit of the Xenopus CCAAT box transcription factor CBTF122 belongs to a family of double-stranded RNA-binding domain1 containing proteins implicated in a variety of cellular processes
Mutations in the double-stranded RNA-binding domain (dsRBD) of CBTF122EN Lead to a Loss of Transcriptional Regulation—The engrailed transcription repression domain (EN) when fused to a transcription activator leads to transcription repression of the target gene [34]
We confirmed that this effect was a result of changes at a transcriptional level and not a failure of CBTF122EN to be incorporated into the full CBTF complex, or an effect on mRNA turnover, by testing the strong transcription up-regulating CBTF122 form, CBTF122VP16
Summary
Mutations in the RNAbinding domains that inhibit RNA binding affect DNA binding in vitro These mutations alter the ability of CBTF122 fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 gene in vivo. These data support the hypothesis that the doublestranded RNA-binding domains of this family of proteins are important for their DNA binding both in vitro and in vivo. The 122-kDa subunit of the Xenopus CCAAT box transcription factor CBTF122 ( known as 4F and ubp4) belongs to a family of double-stranded RNA-binding domain (dsRBD) containing proteins implicated in a variety of cellular processes. It is clear that many of the human homologues arise from alternative splicing of transcripts from the ILF3 locus [6, 7]
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