Eukaryotic genomes encode a large number of RNA-binding proteins, which play critical roles in many aspects of gene regulation. To functionally characterize these proteins, a key step is to map their interactions with target RNAs. UV crosslinking and immunoprecipitation coupled with high-throughput sequencing has become the standard method for this purpose. Here we describe the detailed procedure that we have used to characterize the protein-RNA interactions of the mRNA 3′ processing factors. © 2014 Springer Science+Business Media, LLC.

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.

AMPA receptors-mediators of fast, excitatory transmission and synaptic plasticity in the brain-achieve great functional diversity through interaction with different auxiliary subunits, which alter both the trafficking and biophysical properties of these re

Magnetized high energy density physics offers new opportunities for observing magnetic field-related physics for the first time in the laser-plasma context. We focus on one such phenomenon, which is the ability of a laser-irradiated magnetized plasma to amplify a seed magnetic field. We performed a series of fully kinetic 3D simulations of magnetic field amplification by a picosecond-scale relativistic laser pulse of intensity $4.2\times 10^{18}$ W/cm$^2$ incident on a thin foil. We observe axial magnetic field amplification from an initial 0.1 kT seed to 1.5 kT over a volume of several cubic microns, persisting hundreds of femtoseconds longer than the laser pulse duration. The magnetic field amplification is driven by electrons in the return current gaining favorable orbital angular momentum from the seed magnetic field. This mechanism is robust to laser polarization and delivers order-of-magnitude amplification over a range of simulation parameters.