The role of the C-terminal helix of U1A protein in the interaction with U1hpII RNA.

Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a 'lure and lock' model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA-protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.

HuR/methyl-HuR and AUF1 regulate the MAT expressed during liver proliferation, differentiation, and carcinogenesis.

BACKGROUND & AIMS: Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine are tightly regulated by 2 genes: methionine adenosyltransferase 1A (MAT1A) and methionine adenosyltransferase 2A (MAT2A). MAT1A is expressed in the adult liver, whereas MAT2A expression primarily is extrahepatic and is associated strongly with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. METHODS: In silico analysis of the 3' untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AU-rich RNA binding factor 1 (AUF1) and HuR, respectively. We investigated the posttranscriptional regulation of MAT1A and MAT2A by AUF1, HuR, and methyl-HuR in the aforementioned biological processes. RESULTS: During hepatic de-differentiation, the switch between MAT1A and MAT2A coincided with an increase in HuR and AUF1 expression. S-adenosylmethionine treatment altered this homeostasis by shifting the balance of AUF1 and methyl-HuR/HuR, which was identified as an inhibitor of MAT2A messenger RNA (mRNA) stability. We also observed a similar temporal distribution and a functional link between HuR, methyl-HuR, AUF1, and MAT1A and MAT2A during fetal liver development. Immunofluorescent analysis revealed increased levels of HuR and AUF1, and a decrease in methyl-HuR levels in human livers with hepatocellular carcinoma (HCC). CONCLUSIONS: Our data strongly support a role for AUF1 and HuR/methyl-HuR in liver de-differentiation, development, and human HCC progression through the posttranslational regulation of MAT1A and MAT2A mRNAs.

DNA methylation-based biomarkers for early detection of non-small cell lung cancer: an update.

Lung cancer is the number one cancer killer in the United States. This disease is clinically divided into two sub-types, small cell lung cancer, (10-15% of lung cancer cases), and non-small cell lung cancer (NSCLC; 85-90% of cases). Early detection of NSCLC, which is the more common and less aggressive of the two sub-types, has the highest potential for saving lives. As yet, no routine screening method that enables early detection exists, and this is a key factor in the high mortality rate of this disease. Imaging and cytology-based screening strategies have been employed for early detection, and while some are sensitive, none have been demonstrated to reduce lung cancer mortality. However, mortality might be reduced by developing specific molecular markers that can complement imaging techniques. DNA methylation has emerged as a highly promising biomarker and is being actively studied in multiple cancers. The analysis of DNA methylation-based biomarkers is rapidly advancing, and a large number of potential biomarkers have been identified. Here we present a detailed review of the literature, focusing on DNA methylation-based markers developed using primary NSCLC tissue. Viable markers for clinical diagnosis must be detectable in 'remote media' such as blood, sputum, bronchoalveolar lavage, or even exhaled breath condensate. We discuss progress on their detection in such media and the sensitivity and specificity of the molecular marker panels identified to date. Lastly, we look to future advancements that will be made possible with the interrogation of the epigenome.

Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer death in men and women in the United States and Western Europe. Over 160,000 Americans die of this disease every year. The five-year survival rate is 15% - significantly lower than that of other major cancers. Early detection is a key factor in increasing lung cancer patient survival. DNA hypermethylation is recognized as an important mechanism for tumor suppressor gene inactivation in cancer and could yield powerful biomarkers for early detection of lung cancer. Here we focused on developing DNA methylation markers for squamous cell carcinoma of the lung. Using the sensitive, high-throughput DNA methylation analysis technique MethyLight, we examined the methylation profile of 42 loci in a collection of 45 squamous cell lung cancer samples and adjacent non-tumor lung tissues from the same patients. RESULTS: We identified 22 loci showing significantly higher DNA methylation levels in tumor tissue than adjacent non-tumor lung. Of these, eight showed highly significant hypermethylation in tumor tissue (p < 0.0001): GDNF, MTHFR, OPCML, TNFRSF25, TCF21, PAX8, PTPRN2 and PITX2. Used in combination on our specimen collection, this eight-locus panel showed 95.6% sensitivity and specificity. CONCLUSION: We have identified 22 DNA methylation markers for squamous cell lung cancer, several of which have not previously been reported to be methylated in any type of human cancer. The top eight markers show great promise as a sensitive and specific DNA methylation marker panel for squamous cell lung cancer.