Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains.
Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.
Tolnay
M
M
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. mtolnay@hotmail.com
Baranyi
L
L
Tsokos
G C
GC
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018604:Epitope Mapping; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011487:Protein Conformation; D012261:Ribonucleoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D010455:Peptides; D012261:Ribonucleoproteins; D015534:Trans-Activators; D004247:DNA