Direct Interaction of the KRAB/Cys2-His2Zinc Finger Protein ZNF74 with a Hyperphosphorylated Form of the RNA Polymerase II Largest Subunit

Abstract

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys 2 -His 2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with premRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.Zinc finger proteins of the TFIIIA/Kruppel type belong to the largest known family of transcription factors (1, 2). These proteins are characterized by Cys 2 -His 2 zinc finger motifs often repeated in tandem that fold around zinc ions and function as nucleic acid binding domains (3-6). About one-third of mammalian Cys 2 -His 2 zinc finger proteins contain a conserved domain of approximately 75 amino acids called KRAB (Kruppelassociated box) (7). The KRAB domain, located at the N terminus of Cys 2 -His 2 multifinger proteins, can confer strong distance-independent transcriptional repression of both activated and basal RNA polymerase II promoter activity (8 -14). Since they encode a repression motif and a potential DNA binding domain, members from the large KRAB/Cys 2 -His 2 protein family are presently thought to function as transcriptional regulators of gene expression.We previously cloned ZNF74, a gene that encodes a KRAB/ Cys 2 -His 2 protein (15). ZNF74 lies a few kilobases proximal to a polymorphic CA repeat (D22S264) (16) that was shown to be a distal marker for 22q11.2 deletions associated with increased susceptibility to schizophrenia (17). ZNF74 is also one of the few genes found hemizygously deleted in the majority of patients with the DiGeorge syndrome, a microdeletion disorder associated with a wide variety of congenital malformations including cardiac defects, thymic hypoplasia, and hypocalcemia (18 -20). To date, however, both the role of embryologically expressed ZNF74 in the DiGeorge syndrome (19) and its biochemical and cellular functions remain unclear.Although the presence of a KRAB motif and of 1...

The C2H2 zinc-finger protein SYD-9 is a putative posttranscriptional regulator for synaptic transmission

“…Interestingly, both ZNF74 and Glis2, the human proteins containing zinc-finger domains highly homologous to those in SYD-9 proteins, also localize to discrete speckles in nuclei. ZNF74 directly interacts with the hyperphosphorylated form of RNA polymerase II and colocalizes with splicing factors in the nuclear matrix, implicating its potential role in mRNA processing (54,55). We speculate that at least certain isoforms of SYD-9 function as splicing factors or premRNA processors that specifically regulate the splicing and translation of RNA transcripts that are essential to mediate endocytosis.…”

“…Many RNA-binding proteins and proteins that regulate pre-mRNA processing display speckle-like patterns located at the interchromatin regions (56). Biochemical studies in mammalian systems have identified several C2H2 zinc-finger motif-containing proteins that localize to nuclear speckles and play a role in transcription or posttranscriptional mRNA processing events, (54,55,61). Interestingly, both ZNF74 and Glis2, the human proteins containing zinc-finger domains highly homologous to those in SYD-9 proteins, also localize to discrete speckles in nuclei.…”

“…3C) that can potentially mediate interactions with DNA, RNA, and protein domains (22). These motifs are most homologous to zinc fingers in human proteins ZNF574, Glis2 (54,55), and ZNF74 (Fig. 8, which is published as supporting information on the PNAS web site).…”

Phosphorylation and functions of the RNA polymerase II CTD

“…Interestingly, genetic studies (Jona et al 2001) revealed interactions between genes encoding Elongator subunits and CTK1, the gene encoding the catalytic subunit of CTDK-I. A number of other proteins of disparate function have been shown to bind directly to the PCTD, including the KRAB/Cys2-His2 zinc finger protein ZNF74 (Grondin et al 1997), and the splicing and transcription-associated proteins PSF and p54 nrb /NonO (Emili et al 2002). Interestingly, it should be noted that a pool of nontranscribing RNAPII carries a PCTD and is associated with certain transcription and processing factors in potential assembly areas called "transcriptosomes" (cf.…”

Hyperphosphorylated C-terminal Repeat Domain-associating Proteins in the Nuclear Proteome Link Transcription to DNA/Chromatin Modification and RNA Processing

“…RNA processing proteins known to bind directly to the phosphoCTD include the following: subunits of capping enzyme (21)(22)(23), subunits of 3Ј end formation factors (24,25), splicing factors (9), splicing factor-like proteins (26 -29), and an apparent small nucleolar RNA 3Ј end factor (30). A few other proteins of disparate function have been shown to bind directly to the phosphoCTD, including the prolyl isomerase ESS1 (31), the histone acetyltransferase p300/CBP-associated factor (32), and the KRAB/Cys 2 -His 2 zinc finger protein ZNF74 (33). Significantly, however, the currently known phosphoCTD-associating proteins (PCAPs) were not, for the most part, discovered in a systematic way, and it is therefore very likely that numerous PCAPs remain to be discovered.…”