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 [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492–5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B 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 D0B 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. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.
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March 1999
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Research Article|
February 22 1999
Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specificDNA-binding protein
Mate TOLNAY;
Mate TOLNAY
1
*Department of Clinical Physiology, Walter Reed Army Institute of Research, 14th and Dahlia Streets, Bldg. 40, Washington, DC 20307-5100, U.S.A.
†Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, U.S.A.
1To whom correspondence should be addressed, at the Walter Reed Army Institute of Research (e-mail mtolnay@hotmail.com).
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Lyudmila A. VERESHCHAGINA;
Lyudmila A. VERESHCHAGINA
*Department of Clinical Physiology, Walter Reed Army Institute of Research, 14th and Dahlia Streets, Bldg. 40, Washington, DC 20307-5100, U.S.A.
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George C. TSOKOS
George C. TSOKOS
*Department of Clinical Physiology, Walter Reed Army Institute of Research, 14th and Dahlia Streets, Bldg. 40, Washington, DC 20307-5100, U.S.A.
†Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, U.S.A.
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Biochem J (1999) 338 (2): 417–425.
Article history
Received:
July 20 1998
Revision Received:
November 09 1998
Accepted:
December 08 1998
Citation
Mate TOLNAY, Lyudmila A. VERESHCHAGINA, George C. TSOKOS; Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specificDNA-binding protein. Biochem J 1 March 1999; 338 (2): 417–425. doi: https://doi.org/10.1042/bj3380417
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