Investigating chemical crosslinking of protein-RNA complexes by mass spectrometry

by Alexander Wulf
Doctoral thesis
Date of Examination:2021-11-22
Date of issue:2022-08-12
Advisor:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Patrick Cramer
Persistent Address: http://dx.doi.org/10.53846/goediss-9406

 

 

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Abstract

English

RNA plays a quintessential role in cellular processes such as gene expression, transcription, RNA processing, and translation. Proteins carrying out those diverse functions are inherently flexible to accommodate dynamic RNA structures, leading to a structurally adjustable RNA-binding protein (RBP) complex that often evades crystallographic or cryo-EM analyses. To elucidate protein-RNA interfaces of such RBPs, crosslinking coupled with mass spectrometry (XL-MS) can provide direct evidence of interacting amino acid residues and nucleotides. XL-MS captures transient interactions that are stabilized through the crosslinking event, and enable peptide-centric analyses that also shed light on the crosslinked nucleotide. Here, a chemical crosslinking workflow for the chemical crosslinkers 1,2,3,4-diepoxybutane (DEB), nitrogen mustard (NM), and 2-iminothiolane (2-IT) is presented that allows for specific, reliable, and unambiguous identification of protein-RNA interfaces at amino acid resolution. The workflow consists of 6 steps: chemical crosslinking, RNA and protein digestion, C18 chromatography, TiO2 enrichment, ESI-LC-MS/MS, and data analysis using the RNPxl/Nuxl software. Acquired data from chemical crosslinking coupled with mass spectrometry (cXL-MS) of model protein Hsh49 with various synthetic RNAs features detectable nucleic acid fragments to all four nucleotides. Moreover, insights from this cXL-MS data demonstrate that certain crosslinkers display a nucleotide preference and that cXL-MS generally identifies complementary cross-link sites to canonical UV-crosslinking. Here, lists of mass shifts that are specific to each nucleotide and crosslinker combination are reported, allowing spectral annotation to identify crosslinked spectrum matches (CSMs) by the RNPl/Nuxl software. The Hsh49 model system is expanded by its cognate protein Cus1 and U2 RNA, forming a part of the yeast spliceosomal complex, and analyzed by cXL-MS, revealing RNA-binding activity in both RNA recognition motifs. Additionally, methyltransferase Dnmt2 and the negative elongation factor complex (NELF) are analyzed by the developed method. This revealed previously unknown alternative confirmations of Dnmt2 and substantial RNA-binding events within the tentacle regions of the NELF complex, adding valuable information on the protein-RNA interplay in these complexes. When expanding cXL-MS of protein-RNA complexes to live cells, multiple RBPs from E. coli, B. subtilis, and human HeLa cells were identified in states bound to their cognate RNAs in vivo, which illustrates the powerful application of this method to complex samples.
Keywords: Crosslinking mass spectrometry; protein-RNA complex; Ion mobility mass spectrometry; Methode development; E. coli
 
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