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

Abstract

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

The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation

“…This motif is mirrored by the RREs of CNBP found by in vitro selection (Ray et al 2016), as well as by PAR-CLIP in this study. RNA-protein crosslinking in 4SU-PAR-CLIP, HiTS-CLIP, and other CLIP-seq procedures occurs predominantly at uridines (Kramer et al 2014; Sharma et al 2015) and therefore requires the presence of uridine bases within a few nucleotides of the binding site. Our unbiased motif enrichment analysis revealed CNBP’s G-rich RRE mitigating concerns that results from UV-crosslinking based protocols are disproportionately skewed towards U-rich RREs.…”

The SmAP2 RNA binding motif in the 3′UTR affects mRNA stability in the crenarchaeum Sulfolobus solfataricus

“…The UV induced protein–RNA crosslinking and enrichment of crosslinked hetero-conjugates was performed as described ( 32 , 33 ). Briefly, 1 nmol of SmAP2-cRBM and 1 nmol of (SmAP2) 7 protein were mixed in a 1:1 molar ratio in a total volume of 100 μl buffer containing 50 mM HEPES (pH 7.0), 50 mM KCl, 5 mM MgCl 2 and 10 mM β-mercaptoethanol.…”

sRNA41 affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1

“…In preparation for the mass-spectrometry (MS) 800 mg of protein extracts from M. mazei wild type (Mm_wt) and M. mazei DsRNA 41 (Mm_Ds41) were precipitated in ethanol as described previously (Sharma et al, 2015). Protein extracts were digested in-solution in the presence of 8 M Urea (in 100 mM TEAB).…”