Heterogeneous nuclear ribonucleoprotein DOB is a sequence-specific DNA-binding protein

Abstract

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response, the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene. We cloned the cDNA encoding this protein and found it to be identical with hnRNP DOB. a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP DOB displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent K(d) of hnRNP DOB for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP DOB displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP DOB for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250-400 nM, indicating that the protein binds to the CR2 promoter in vivo. Go-precipitation of a complex formed in vivo between hnRNP DOB and the TATA-binding protein demonstrates that hnRNP DOB interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP DOB that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.