Natural antisense transcripts (NATs) are a class of genes whose role in controlling gene expression is becoming more and more relevant. We describe the identification of eight novel mouse NATs associated with transcription factors (Pax6, Pax2, Six3, Six6, Otx2, Crx, Rax and Vax2) that play an important role in eye development and function. These newly identified NATs overlap with the mature processed mRNAs or with the primary unprocessed transcript of their corresponding sense genes, are predicted to represent either protein coding or non-coding RNAs and undergo extensive alternative splicing. Expression studies, by both RT-PCR and RNA in situ hybridization, demonstrate that most of these NATs, similarly to their sense counterparts, display a specific or predominant expression in the retina, particularly at postnatal stages. We found a significant reduction of the expression levels of one of these NATs, Vax2OS (Vax2 opposite strand) in a mouse mutant carrying the inactivation of Vax2, the corresponding sense gene. In addition, we overexpressed another NAT, CrxOS, in mouse adult retina using adeno-associated viral vectors and we observed a significant decrease in the expression levels of the corresponding sense gene, Crx. These results suggest that these transcripts are functionally related to their sense counterparts and may play an important role in regulating the molecular mechanisms that underlie eye development and function in both physiological and pathological conditions.

The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.T ranscription elongation is highly processive, yet the rate of nucleotide addition varies significantly for different ternary elongation complexes (TECs). In the normal elongation pathway, each nucleotide addition is followed by translocation of RNA polymerase (RNAP) along DNA in single-nucleotide increments. This process transfers the RNA 3′ end from the i + 1 site to the i site of the active center, thus allowing binding of the next NTP and subsequent phosphodiester bond formation. Translocation is thought to be a stochastic, rapid, and fully reversible process and not rate-limiting for elongation (1, 2). Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3, 4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5-9).Immediately following bond formation, a catalytically inactive and highly pyrophosphorolytic (pretranslocated) state is formed. In this state, the 3′ RNA end remains in the i + 1 site of the active center. The 3′ RNA thus prevents NTP binding and generates a substrate for pyrophosphorolysis. To bind the next NTP, RNAP must translocate 1-bp forward along the DNA register to form a catalytically active and pyrophosphate-resistant (posttranslocated) state. Stationary RNAP has been suggested to "ratchet" between the two states via Brownian motion (5). NTP bound to the transient posttranslocated state acts as a pawl that interferes with backward translocation, thereby favoring the formation of a phosphodiester bond. Retention of this NTP in the posttranslocated TEC is facilitated by isomerization of the active site that blocks substrate exit, and aligns it for catalysis (10, 11)...

We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.Addition of methyl groups mostly at the CpG dinucleotide represents the major epigenetic modification of eukaryotic genomes heritable by somatic cells after cell division. DNA methylation plays an essential role in mammalian development (24) and in different biological processes such as tissue-specific gene expression (40), X-chromosome inactivation (17), genomic imprinting (2, 33), and repression of transposable elements (46). Abnormal methylation is a cause of human genetic diseases, including ICF syndrome (16), and is involved in carcinogenic processes primarily through aberrant hypermethylation of tumor suppressors' promoter regions (12,21,22).Methylated DNA is generally associated with transcriptional silencing (6, 7, 32), but how DNA methylation silences gene expression is not well understood. Studies are now focusing on the different components of the DNA methylation system and particularly on the mechanisms by which methyl-CpG signal is targeted, read, and maintained. Recently, a family of five mammalian methyl-CpG-binding proteins (MeCP2, MBD1, MBD2, MBD3, and MBD4) has been found to be essential to interpret the methylation patterns and to mediate the biological consequences of DNA methylation (1, 15, 18). All methyl-binding proteins share a common structural stretch of 60 to 80 amino acids called the mCpG-binding domain (MBD) and, with the exception of MBD4 (19, 34), mediate transcription...

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