Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

摘要: Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding molecular details of these processes requires identification characterization protein-RNA interactions. Over years various approaches have been used to investigate interactions, computational analyses look for RNA binding domains, gel-shift mobility assays on recombinant mutant proteins as well co-crystallization NMR studies structure elucidation. Here we report a more specialized direct approach using UV-induced cross-linking coupled with mass spectrometry. This permits cross-linked peptides moieties can also pin-point exact contact sites within protein. The power this method is illustrated application different single- multi-subunit RNP belonging prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, identified RNA-binding three Cas7 protein homologs mapped results reveal structurally conserved - interfaces. These demonstrate strong potential spectrometry analysis identify interaction proteins.