Alternative promoter usage and splicing of ZNF74 multifinger gene produce protein isoforms with a different repressor activity and nuclear partitioning

We have previously shown that ZNF74, a candidate gene for DiGeorge syndrome , encodes a developmentally expressed zinc finger gene of the Kruppel-assoc iated box (KRAB) multifinger subfamily. Using RACE, RT-PCR, and primer exte nsion on human fetal brain and heart mRNAs, we here demonstrate the existen ce of six mRNA variants resulting from alternative promoter usage and splic ing. These transcripts encode four protein isoforms differing at their N te rminus by the composition of their KRAB motif. One isoform, ZNF74-I, which corresponds to the originally cloned cDNA, was found to be encoded by two a dditional mRNA variants. This isoform, which contains a KRAB motif lacking the N terminus of the KRAB A box, was devoid of transcriptional activity. I n contrast, ZNF74-II, a newly identified form of the protein that is encode d by a single transcript and contains an intact KRAB domain with full A and B boxes, showed strong repressor activity. Deconvolution immunofluorescenc e microscopy using transfected human neuroblastoma cells and nonimmortalize d HS68 fibroblasts revealed a distinct subcellular distribution for ZNF74-I and ZNF74-II, In contrast to ZNF74-I, which largely colocalizes with SC-35 in nuclear speckles enriched in splicing factors, the transcriptionally ac tive ZNF74-II had a more diffuse nuclear distribution that is more characte ristic of transcriptional regulators. Taken with the previously described R NA-binding activity of ZNP74-I and direct interaction with a hyperphosphory lated form of the RNA polymerase IT participating in pre-mRNA processing, o ur results suggest that the two ZNF74 isoforms exert different or complemen tary roles in RNA maturation and in transcriptional regulation.