Nucleotide-amino acid π-stacking interactions initiate photo cross-linking in RNA-protein complexes.

Photo-induced cross-linking is a mainstay technique to characterize RNA-protein interactions. However, UV-induced cross-linking between RNA and proteins at "zero-distance" is poorly understood. Here, we investigate cross-linking of the RBFOX alternative splicing factor with its hepta-ribonucleotide binding element as a model system. We examine the influence of nucleobase, nucleotide position and amino acid composition using CLIR-MS technology (crosslinking-of-isotope-labelled-RNA-and-tandem-mass-spectrometry), that locates cross-links on RNA and protein with site-specific resolution. Surprisingly, cross-linking occurs only at nucleotides that are π-stacked to phenylalanines. Notably, this π-stacking interaction is also necessary for the amino-acids flanking phenylalanines to partake in UV-cross-linking. We confirmed these observations in several published datasets where cross-linking sites could be mapped to a high resolution structure. We hypothesize that π-stacking to aromatic amino acids activates cross-linking in RNA-protein complexes, whereafter nucleotide and peptide radicals recombine. These findings will facilitate interpretation of cross-linking data from structural studies and from genome-wide datasets generated using CLIP (cross-linking-and-immunoprecipitation) methods.

Properties of N(4)-methylated cytidines in miRNA mimics.

Experiments conducted with micro RNA (miRNA) mimics often result in subtle phenotypic changes and hence require careful controls. A commonly used type of control reagent in the antisense/RNA interference fields is the mismatched sequence. However, it is difficult to use mismatch controls for miRNAs, mainly because base permutation in the seed region may generate a new miRNA seed with its own associated target transcripts. We incorporated N(4)-methylcytidine and N(4),N(4)-dimethylcytidine into a series of RNAs using the convertible nucleoside approach and measured their effects on hybridization affinity with complementary RNAs, and on miRNA-mediated and small interfering RNA (SiRNA)-mediated silencing. We report here that incorporation of a single N(4),N(4)-dimethylcytidine into the seed region of miRNAs can be used as a new class of negative miRNA control which (1) does not constitute a new seed sequence; (2) is accepted by the RNA-induced silencing complex (RISC); (3) causes a significant loss of binding affinity to target RNAs; and (4) is synthesized conveniently into oligoribonucleotides.

Electron relay race in peptides.

A peptide assay was developed that allows the measurement of electron-transfer (ET) efficiencies in peptides. It turns out that two-step ET processes are faster than single-step reactions. This requires relay amino acids with appropriate redox potentials. Not only aromatic but also sulfur-containing aliphatic amino acids can act as stepping stones for the charge. With tryptophan, histidine, and cysteine the reaction is a more complex proton-coupled ET.