Alternative Promoter Usage and Splicing ofZNF74Multifinger Gene Produce Protein Isoforms with a Different Repressor Activity and Nuclear Partitioning

Abstract

We have previously shown that ZNF74, a candidate gene for DiGeorge syndrome, encodes a developmentally expressed zinc finger gene of the Kruppel-associated box (KRAB) multifinger subfamily. Using RACE, RT-PCR, and primer extension on human fetal brain and heart mRNAs, we here demonstrate the existence of six mRNA variants resulting from alternative promoter usage and splicing. These transcripts encode four protein isoforms differing at their N terminus 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 additional mRNA variants. This isoform, which contains a KRAB motif lacking the N terminus of the KRAB A box, was devoid of transcriptional activity. In contrast, ZNF74-II, a newly identified form of the protein that is encoded by a single transcript and contains an intact KRAB domain with full A and B boxes, showed strong repressor activity. Deconvolution immunofluorescence microscopy using transfected human neuroblastoma cells and nonimmortalized 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 active ZNF74-II had a more diffuse nuclear distribution that is more characteristic of transcriptional regulators. Taken with the previously described RNA-binding activity of ZNF74-I and direct interaction with a hyperphosphorylated form of the RNA polymerase II participating in pre-mRNA processing, our results suggest that the two ZNF74 isoforms exert different or complementary roles in RNA maturation and in transcriptional regulation.

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

“…For example, the involvement of classical ZFs in RNA metabolism might occur primarily in development (which will not be reflected in differentiated cancer cell lines). Indeed the RNA-related functions of TFIIIA (63), WT1 (9) and ZNF74 (10) do appear to play developmental roles. It is also possible that classical ZF proteins that play dual roles as DNA- and RNA-binding proteins (again, such as TFIIIA and WT1) are not easily recovered in pulldown assays of this type if they partition mostly or partly with DNA.…”

“…Isoforms lacking a three amino acid (KTS) insertion between the third and fourth fingers bind Igf-2 exon 2 DNA, whilst those bearing the KTS insertion preferentially bind the corresponding RNA; this latter activity is proposed to regulate levels of alternatively spliced Igf-2 transcripts (7,8). Similarly, the classical ZFs of human ZNF74 have been suggested to be involved in both transcriptional regulation and mRNA metabolism depending on splicing context (9,10). …”

The Zinc Finger Protein ZNF658 Regulates the Transcription of Genes Involved in Zinc Homeostasis and Affects Ribosome Biogenesis through the Zinc Transcriptional Regulatory Element

“…Second, the ZTREs in the rRNA precursor sequence are clustered not around the TSS but, most densely, several hundred bases upstream of the sequence of the first mature transcript, 28S rRNA. It is not implausible that ZNF658 (possibly expressed as different splice variants) has a dual role, regulating both (Pol II-mediated) transcription of specific target genes and RNA processing given that other members of the KRAB-domain containing ZNF protein family appear to have such a dual role ( 21 , 26 – 28 ). An effect on rRNA abundance of changing posttranscriptional processing could be due to differences in turnover rates of the precursor versus the mature transcripts.…”

KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions

“…Among these, ZNF74 and ZNF224 have been shown to exist as multiple protein isoforms generated through alternative promoter usage and splicing. Both ZNF74 and ZNF224 isoforms exhibit differences in their transcriptional repression abilities and nuclear partitioning, with the isoforms devoid of the KRAB box being found in splicing speckle domains and able to bind RNA [52-54]. By contrast, the ZNF74 and ZNF224 isoforms containing the full KRAB A and B boxes exhibit strong transcriptional repression and localize in a diffuse pattern throughout the nucleus.…”