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Epigenetic alterations of DNA play key roles in determining gene structure and expression. Methylation of the 5-position of cytosine is thought to be the most common modification of the genome in mammals. Studies have generally shown that hypermethylation in gene regulatory regions is associated with inactivation and reduced transcription and that alteration in established methylation patterns during development can affect embryonic viability. Changes in methylation have also been associated with aging and cellular senescence as well as tumorogenesis. DNA methyltransferase 1 (DNMT1) is thought to play an important role in maintaining already established methylation patterns during DNA replication and catalyzes the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the 5-position of cytosines in the CpG dinucleotide. Several studies illustrate changes in activity and transcription of DNMT1 during aging and here we show a comprehensive method of detection of DNMT1 mRNA transcription from senescing cells in culture.

Hel-N1, is an RRM protein which is a mammalian homologue of the Drosophila melanogaster RNA binding protein, ELAV (embryonic lethal abnormal vision). Hel-N1 binds to RNA containing short stretches of uridylates similar to those found in the 3' untranslated regions (3'-UTRs) of oncoprotein and cytokine mRNAs. The GLUT1 glucose transporter has an extensive 3' UTR that is AU-rich reminiscent of the 3'UTR of an oncogene mRNA. An in vitro RNA binding assay using Hel-N1 demonstrated binding to a specific portion of the GLUT1 3'UTR. Analysis of the folding pattern of this region depicted the retention of a stem loop structure, wherein the loop is composed of a stretch of uridylates. To further analyze the potential function of Hel-N1, stable transfectants were made in the 3T3-L1 cell line. The transfectants have been characterized, and the presence of the Hel-N1 DNA and protein verified. Data indicate Hel-N1 is a ligand for GLUT1 and its binding affects the stability and translatability of the GLUT1 message.

We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.