Abstract
We previously cloned ZNF74, a developmentally expressed zinc finger gene commonly deleted in DiGeorge syndrome. Here, the intron/exon organization of the human gene and the functional properties of the expressed protein are presented. This zinc finger gene from the transcription factor IIIA/Kruppel family contains three exons. A truncated Kruppel-associated box (KRAB) located at the N terminus of the predicted 64-kDa zinc finger protein is encoded by exon 2. The remainder of the protein including the zinc finger domain as well as the 3'-untranslated region (UTR) is encoded by exon 3. Both 5'-UTR (exon 1) and 3'-UTR contain repetitive Alu elements. In vitro translation of a cDNA encoding the entire ZNF74 coding region produced a 63-kDa protein as determined on sodium dodecyl sulfate-polyacrylamide gel. A bacterially expressed fusion protein shown to bind tightly to 65zinc was used to test the nucleic acid binding properties of ZNF74. By RNA binding assays, ZNF74 was found to bind specifically to poly(U) and poly(G) RNA homopolymers. The restricted binding to these homopolymers and not to poly(A) and poly(C) suggested that ZNF74 displays RNA sequence preferences. RNA binding was mediated by the zinc finger domain. Immunofluorescence studies on transfected cells revealed ZNF74 nuclear localization. The labeling pattern observed in the nuclei clearly excluded the nucleoli. The zinc finger region lacks a classical nuclear localization signal but was found to be responsible for nuclear targeting. Subcellular and in situ sequential fractionations further showed that ZNF74 is associated with the nuclear matrix. The RNA binding properties of this protein and its tight association with the nuclear matrix, a subnuclear compartment involved in DNA replication as well as RNA synthesis and processing, suggest a role for ZNF74 in RNA metabolism.
Highlights
RNA binding was mediated by the zinc finger domain. genes on the chromosome or a single gene
A KRAB motif was confirmed to be in frame at the Nterminal extremity of ZNF74 protein owing to the use of various modifications of traditional sequencing methods to track banding artefacts in extremely GC-rich regions of ZNF74 –1 cDNA [49, 50] (Fig. 1)
We report that ZNF74 gene encodes a KRAB zinc finger protein with RNA binding properties
Summary
Plasmid Constructs—The previously isolated 2.4-kb ZNF74 –1 cDNA cloned in pBluescript SK (Stratagene, La Jolla, CA) was used for preparing the various ZNF74 constructs [9]. The amylose resin (50 l/assay) with immobilized fusion protein (0.5–2 g) was incubated with 100 ng of radioactive homopolymer RNA ((2 ϫ 103) Ϫ (1 ϫ 104) cpm/ng) in a total volume 400 l of binding buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 50 M ZnCl2, 10 mM -mercaptoethanol, 10% glycerol) in the presence or absence of competitor RNA or DNA (sonicated salmon sperm) for 30 min at room temperature. The binding of 100 ng of homopolymer RNA to increasing concentrations of fusion protein was quantified (up to 40 – 60% of input RNA could be retained after washing), and the amount of MBP-ZNF74-(aa 106 –572) protein used in the described experiments was fixed to have always less than 10 –20% of the input labeled RNA bound. Monolayers of extracted cells at different steps of the nuclear matrix preparation were fixed and treated for immunofluorescence as described above
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