Assessing the Utility of Thermodynamic Features for microRNA Target Prediction under Relaxed Seed and No Conservation Requirements

BACKGROUND: Many computational microRNA target prediction tools are focused on several key features, including complementarity to 5'seed of miRNAs and evolutionary conservation. While these features allow for successful target identification, not all miRNA target sites are conserved and adhere to canonical seed complementarity. Several studies have propagated the use of energy features of mRNA:miRNA duplexes as an alternative feature. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement. METHODOLOGY/PRINCIPAL FINDINGS: We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide sites of AGO protein interaction. This trend is confirmed on datasets that assay the effect of miRNAs on mRNA and protein expression changes, and a simple linear regression model leads to significant correlation of predicted versus observed expression change. Compared to 6-mer seed matches as baseline, application of our energy-based model leads to ∼3-5-fold enrichment on highly down-regulated targets, and allows for prediction of strictly imperfect targets with enrichment above baseline. CONCLUSIONS/SIGNIFICANCE: In conclusion, our results indicate significant promise for energy-based miRNA target prediction that includes a broader range of targets without having to use conservation or impose stringent seed match rules

BRUGADA SYNDROME AND CONDUCTION SYSTEM DISEASE ARE LINKED TO A SINGLE SODIUM CHANNEL MUTATION.

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na(+) channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (DeltaK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and DeltaK1500 mutant channel. The mutation shifted V(1/2)h( infinity ) to more negative membrane potentials and increased k(h) consistent with a reduction of inactivation valence of 1. The shift in h( infinity ) was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). V(1/2)m was shifted to more positive potentials, and k(m) was doubled in the DeltaK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, -1 and -2, respectively). For both mutations, V(1/2)h was shifted back toward control; however, V(1/2)m shifted progressively to more positive potentials. The late component of Na(+) current was increased in the DeltaK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation

Improved adenoviral vector for vascular gene therapy : beneficial effects on vascular function and inflammation.

First-generation, E1-deleted adenoviral vectors (E1-AV) can transduce the vascular endothelium with high efficiency, but their use is limited by the resulting acute endothelial injury and the long-term development of intimal hyperplasia. To reduce the impact of viral proteins on the gene-modified cells, a second-generation adenoviral vector with an additional pair of deletions in the E4 region was developed. To determine whether this E1/E4-AV vector would be useful for vascular gene transfer, we directly compared the efficiency of gene transfer to uninjured rabbit carotid arteries using either an E1/E4-AV or an E1-AV vector encoding beta-galactosidase. Both vectors efficiently transduced vascular endothelium; however, the E1/E4-AV vector gene-modified vessels showed higher beta-galactosidase expression 10 days after gene transfer. Importantly, the E1/E4-AV vector produced substantially less endothelial cell activation, less inflammation, and reduced neointimal hyperplasia compared with the E1-AV vector-treated vessels. The E1-AV vector-transduced vessels also demonstrated significantly impaired endothelium-dependent relaxation whereas the E1/E4-AV vector did not impact vasomotor function, even at doses of virus in 5-fold excess of the amount required for >90% transduction of the endothelium. We conclude that the E1/E4-AV vector is superior to the E1-AV vector for vascular gene therapy because of the prolonged transgene expression, reduced vascular inflammation, reduced intimal hyperplasia, and maintenance of normal vasomotor function

The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †

Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased γ-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process

Targeting PVR (CD155) and its receptors in anti-tumor therapy

Poliovirus receptor (PVR, CD155) has recently been gaining scientific interest as a therapeutic target in the field of tumor immunology due to its prominent endogenous and immune functions. In contrast to healthy tissues, PVR is expressed at high levels in several human malignancies and seems to have protumorigenic and therapeutically attractive properties that are currently being investigated in the field of recombinant oncolytic virotherapy. More intriguingly, PVR participates in a considerable number of immunoregulatory functions through its interactions with activating and inhibitory immune cell receptors. These functions are often modified in the tumor microenvironment, contributing to tumor immunosuppression. Indeed, increasing evidence supports the rationale for developing strategies targeting these interactions, either in terms of checkpoint therapy (i.e., targeting inhibitory receptors) or in adoptive cell therapy, which targets PVR as a tumor marker