Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122 during Xenopus Development

Abstract

CBTF 122 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 CBTF 122 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 RNAbinding domains that inhibit RNA binding also affect DNA binding in vitro. In addition, these mutations alter the ability of CBTF 122 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 CBTF 122 (also known as 4F and ubp4) belongs to a family of double-stranded RNA-binding domain (dsRBD) 1 containing proteins implicated in a variety of cellular processes. CBTF 122 and its splice variant CBTF 98 have been identified as subunits of CBTF, a transcription factor that activates the GATA-2 gene both in oocytes and zygotically at the start of gastrulation (1, 2). Homologues of CBTF 122 have been identified mainly through their affinity for RNA and include NF90 (also known as DRBP76 and NFAR1), NF110, TCP80, ILF3 and MPP4 in humans (3), SPNR and ILF3 in mice (4), and p74 and ILF3 in rats (5). It is now clear that many of the human homologues arise from alternative splicing of transcripts from the ILF3 locus (6, 7).The functions assigned to these related proteins include the regulation of both transcription and mRNA stability. For example, the transcript levels of more than 90 genes, most of which are known to be regulated by type I interferons, were altered by expression of NF90, suggesting that NF90 has a role in mediating the antiviral response (8). However, it is unclear whether these changes are a result of transcriptional or posttranscriptional mechanisms. By contrast, specific regulation of interleukin-2 levels by NF90 is clearly dependent upon its stabilization of interleukin-2 mRNA (9). In transcriptional terms, the human homologues of CBTF 98

Phosphorylation of the NFAR proteins by the dsRNA-dependent protein kinase PKR constitutes a novel mechanism of translational regulation and cellular defense

“…In Xenopus, NFAR-like proteins CBTF 98 and CBTF 122 or 4F.1 and 4F.2 have been reported to associate with translationally quiescent mRNP complexes and regulate embryo development (Orford et al 1998;Brzostowski et al 2000;Scarlett et al 2004). Of interest is that T188 and T315 are evolutionarily conserved in CBTF 98 and CBTF 122 and undergo phosphorylation in response to virus infection.…”

NF90 Regulates Cell Cycle Exit and Terminal Myogenic Differentiation by Direct Binding to the 3′-Untranslated Region of MyoD and p21WAF1/CIP1 mRNAs

“…At the midblastula transition, degradation of maternal RNA releases CBTF for translocation into the nucleus, followed 4 h later by development of CCATT DNA binding activity and transcriptional activation of GATA-2 (20). The dsRBMs of CBTF are capable of binding both DNA and RNA (21), and intact dsRBMs are required for transcriptional control (15).…”

APTE: identification of indirect read-out A-DNA promoter elements in genomes

“…However, DNA can exist in a number of other conformations, the major types being A-form, Z-form and tetraplex, all of which have been implicated in gene regulation [8–10]. A-form is the canonical dsRNA structure with right-handed helices but with only 2.6 Å between bases and a 20-degree base tilt, while the sugar in A-form is in the c-3′ endo position in contrast to the c-2′ endo position observed for B-form.…”

“…We have previously shown that this mechanism is based on an interaction requiring both DNA base specific (direct read-out) and DNA structure specific (indirect read-out) interactions [8, 6]. The CBTF complex is composed of approximately eight sub-units of which the ilf3 protein is currently the only published component; however, this subunit is critical for CBTF activity.…”

“…Ilf3 contains two double stranded RNA binding domains (dsRBDs) and these domains are required for transcriptional activation in vivo and DNA binding in vitro [8]. The RNA binding activity of ilf3, and other dsRBD containing proteins, has been well characterised, indeed ilf3 was first identified through its interaction with RNA [13].…”