The human and murine MOK2 proteins are factors able to recognize both DNA and RNA through their zinc finger motifs. This dual affinity of MOK2 suggests that MOK2 might be involved in transcription and post-transcriptional regulation of MOK2 target genes. The IRBP gene contains two MOK2-binding elements, a complete 18 bp MOK2-binding site located in intron 2 and the essential core MOK2-binding site (8 bp of conserved 3'-half-site) located in the IRBP promoter. We have demonstrated that MOK2 can bind to the 8 bp present in the IRBP promoter and repress transcription from this promoter by competing with the CRX activator for DNA binding. In this study, we identify a novel interaction between lamin A/C and hsMOK2 by using the yeast two-hybrid system. The interaction, which was confirmed by GST pull-down assays and co-immunolocalization studies in vivo, requires the N-terminal acidic domain of hsMOK2 and the coiled 2 domain of lamin A/C. Furthermore, we show that a fraction of hsMOK2 protein is associated with the nuclear matrix. We therefore suggest that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription.
Background information. hsMOK2 (human MOK2) is a DNA-binding transcriptional repressor. For example, it represses the IRBP (interphotoreceptor retinoid-binding protein) gene by competing with the CRX (cone-rod homeobox protein) transcriptional activator for DNA binding. Previous studies have shown an interaction between hsMOK2 and nuclear lamin A/C. This interaction could be important to explain hsMOK2 ability to repress transcription.Results. In the present study, we have tested whether missense pathogenic mutations of lamin A/C, which are located in the hsMOK2-binding domain, could affect the interaction with hsMOK2. We find that none of the tested mutations is able to disrupt hsMOK2 binding in vitro or in vivo. However, we observe an aberrant cellular localization of hsMOK2 into nuclear aggregates when pathogenic lamin A/C mutant proteins are expressed.Conclusions. These results indicate that pathogenic mutations in lamin A/C lead to sequestration of hsMOK2 into nuclear aggregates, which may deregulate MOK2 target genes.
The human and murine MOK2 orthologue genes encode Krü ppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain as MOK2, we have suggested that PAX3 and IRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention on IRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.The human and murine MOK2 orthologue genes, which are preferentially expressed in brain and testis tissues, encode two different Krü ppel/TFIIIA-related zinc finger proteins. The human and murine genes have been localized to band q13.2-q13.3 of chromosome 19 and chromosome 6, respectively (1, 2). The human hsMOK2 protein shows substantial differences with the murine MOK2 protein. The mouse MOK2 protein contains seven tandem zinc finger motifs with only five additional amino acids at its COOH-terminal end (3). The seven fingers motifs are highly similar to one another but are distinct from those of other zinc finger proteins. The structural feature of murine MOK2 protein is also found at the end of human hsMOK2 protein. Furthermore, the human protein contains three additional zinc finger motifs in tandem with the others and a nonfinger acidic domain of 173 amino acids at the NH 2 -terminal end (2). We have previously shown that human MOK2 RNA maturation results in three mRNAs with different 5Ј-untranslated exons. One of these three mRNAs encodes a smaller MOK2 protein (hsMOK2⌬) containing 10 zinc finger motifs and a small NH 2 -acidic domain made up of 76 amino acids. We have shown that the human and murine MOK2 proteins are able to recognize both DNA and RNA through their zinc finger motifs (4). Electron microscopy and specific RNA homopolymer binding activity showed clearly that the murine and human MOK2 proteins are RNA-binding proteins th...
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