Heterogeneous nuclear ribonucleoprotein DO contains transactivator and DNA-binding domains

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiqu itous protein that binds RNA and DNA sequences specifically, and has been i mplicated in the transcriptional regulation of the human complement recepto r 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, pro viding direct proof that hnRNP D0 possesses a transactivator domain. We fou nd, using truncated hnRNP D0proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, usi ng a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA -binding domains alone were not able to bind double-stranded DNA. Neverthel ess, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the imp ortance of the 24-amino-acid region in DNA binding, and suggested the invol vement 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 recombinan t hnRNP D0E and D0D bound DNA, only the B isoform recognized DNA in vivo. W e propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA- binding transactivator and has distinct transactivator and DNA-binding doma ins.