AMPA receptor-mediated, calcium-dependent CREB phosphorylation in a subpopulation of auditory neurons surviving activity deprivation.

Although dependence on afferent synaptic activity has been shown for central neurons in every sensory system, the mechanisms of afferent maintenance of target sensory neurons are not understood. Neurons in the cochlear nucleus (CN) require afferent activity for maintenance and survival. One of the earliest changes seen after activity deprivation is an increase in intracellular calcium that leads to the death of 30% of the neuronal population. Sixty minutes after deafferentation, the surviving neurons show increased phosphorylation of the transcription factor calcium/cAMP response element-binding protein (CREB). CREB phosphorylation in activity-deprived CN neurons is dependent on increased intracellular calcium resulting from influx through AMPA receptors and is mediated by calcium/calmodulin-dependent kinases and protein kinase A. We conclude that in CN neurons, the deafferentation-induced increase in calcium activates at least two kinase pathways that phosphorylate CREB in surviving neurons. We hypothesize that this phosphorylation results in the transcription of genes containing the calcium/cAMP response element within their promoter regions, and these genes code for proteins that allow the neurons to compensate for their hypercalcemic, activity-deprived state.

An RNA model system for investigation of pseudouridine stabilization of the codon-anticodon interaction in tRNALys, tRNAHis and tRNATyr.

The nucleoside conformation of pseudouridine (psi) was investigated in a series of RNA oligonucleotides and compared with the same sequences containing the parent, unmodified uridine nucleoside. 1H NMR spectroscopy was used to determine the glycosyl conformational preference in pseudouridine systems at the nucleoside level; these experiments were extended to trimers, and ultimately to RNA tetraloop hairpins that are models for the codon-anticodon interaction in tRNA. ROESY 1D and 2D NMR experiments were used to measure the nucleoside conformational preference as a function of temperature. The thermodynamic stability of the RNA tetraloops was also analyzed using UV monitored Tm experiments which established that pseudouridine has a very strong stabilizing effect on double-stranded, base pairing interactions when the modification is located within a base-paired region. This was shown for a tetraloop hairpin model of the codon-anticodon interaction in tRNA(Tyr) which contains a psi at position 35. Pseudouridine also stabilizes double-stranded RNA when the psi modification is in a single-stranded region adjacent to a duplex region as occurs for psi at positions 38 or 39 in tRNA(Lys) and tRNA(His). These results establish that pseudouridine modification of RNA is a powerful and versatile mechanism for stabilizing local RNA structure in both single-stranded and double-stranded regions. Previously postulated roles for pseudouridine as a "conformational switch" are unlikely in light of the increased barrier to rotation about the glycosyl bond upon modification of uridine to pseudouridine. The Tm and NMR data show that local RNA stacking stabilization as a result of psi will stabilize adjacent double-stranded RNA regions such as the codon-anticodon interaction in tRNA.