4. Discussion
4.3 Considerations in the identification of protein-RNA interactions by UV induced cross-
A major part of the presented work focused on the investigation of protein-RNA interactions in crRNP complexes using UV induced protein-RNA cross-linking and MS. Some important considerations and potentials of the protein-RNA cross-linking approach will be discussed in this section in reference to the results obtained during this work.
1. Fragmentation mode: The fragmentation of peptide-RNA heteroconjugates was performed using high-energy collision dissociation (HCD) in Orbitrap instruments where the MS/MS fragmentation occurs in the Orbitrap, therefore benefiting from the high-mass accuracy. In addition the limitations of ion traps could be overcome because here the spectra obtained do not exhibit a low mass cut-off and the important ions like RNA marker ions, a2-b2 pairs and
126
immonium ions (usually located in the lower m/z region) were detected. Furthermore, as the yield of a cross-linking reaction is low and cross-links have a poor ionization efficiency compared to linear peptides, the MS analysis were performed using instruments with high resolution, high mass accuracy and better sequencing speeds such as LTQ-Orbitrap Velos and Q Exactive HF. This would provide a stronger likelihood that a peptide-RNA precursor would be picked for fragmentation out of a complex sample.
2. Identification of cross-link: The spectra for cross-links are poor quality compared to standard peptides because of low signal intensities. The automated proteomics search engines are directed toward peptide identification considering a cross-linked RNA moiety as a modification on the peptide. Also, a large number of potential RNA oligonucleotides can form a cross-link and together these two aspects make the data analysis difficult. The basic principle applied in cross-link identification was that the mass of a peptide-RNA cross-link precursor is purely additive of the masses of peptide and RNA moieties [160]. A new data analysis tool, RNPxl (Section 2.2.9.3) was used for the analysis which followed a precursor variant approach for automated identification of cross-linked heteroconjugates by subtraction of calculated RNA masses from the experimental precursor mass. The results were further checked manually to validate the cross-link identification.
3. Cross-linked nucleotides: The cross-linked nucleotide is determined by calculating the mass difference between the mass of cross-linked heteroconjugate and the mass of cross-linked peptide. There is a substantial difference in the reactivity of uridine and other nucleotides as observed in the cross-links reported in this work, in previous studies related to protein-RNA cross-linking in the Urlaub lab and also in the studies where the cross-linking yields of different amino-acids and nucleotides were investigated by Shetlar et al. [59, 108, 109, 160-163].
Conversely, if a linked heteroconjugate comprise adenosine, guanosine or cytidine linked to the amino acid residue it is less likely to be identified by the MS analysis. In the cross-links observed for the E. coli Cascade complex, the peptide 51SGYYAQNIGESSLRTIHLAQLR72 was observed cross-linked to UG-H2O, where G nucleotide was identified as the cross-linked nucleotide residue (Appendix, Figure 6.2 I). This was the only example of a cross-link where a nucleotide other than uracil was observed to be cross-linked. Considering that uracil is the most reactive nucleotide, some of the cross-linking experiments in this work were also performed with polyU oligonucleotide, but the aim was only to identify regions on the protein level that
4. DISCUSSION 127 interact with RNA. In addition, the information derived from protein-RNA cross-linking studies is limited when attempting to unambiguously map the cross-linked nucleotide at the level of RNA because in most cases the cross-linked RNA was a single nucleotide residue. Nonetheless, there were cross-links identified with longer RNA moieties, but the current cross-linking workflow only provides information about the composition of the cross-linked RNA moiety and not their position in the sequence.
4. RNA marker ions: A typical observation in the cross-link spectra reported in the work was presence of distinct marker ions of the intact nucleotides with a neutral loss of water (C = 306.05, U = 307.03, A = 330.06, G = 346.05) or the nucleic acid bases (C’ = 112.05, U’ = 113.03, A’ = 136.06, G’ = 152.06) in the lower m/z region of the spectrum. In previous studies fragmentation of pure RNA also has been observed to produce nucleobases due to the cleavage of N-glycosidic bond [164]. When only one nucleotide was cross-linked to a peptide, no marker ion signal is visible and the MS/MS spectra are dominated by peptide fragments. Some of the examples where marker ions were observed include A’ marker ion as depicted in C.
thermocellum Cas6b peptide 184MIGFK188 cross-linked UGA and G’ and C’ marker ions in M.
maripaludis Cas6b peptide 182NQNM(ox)VGFR189 cross-linked to UUGC-PO3 and (Figure 3.5).
5. Cross-linked amino acids: In the previous studies all amino acids except E, N and D have been demonstrated to cross-link with RNA moieties [59, 108, 161]. In this work 12 different amino acids have been reported to cross-link with RNA and these include M, Y, L, P, F, W, K, C, V, G, Q and R as shown in different examples in the Appendix, Figure 6.1-6.5. The aromatic residues are the most commonly observed cross-linked amino acid residues. Usually the cross-linked amino acid residues were identified when the fragment ions comprising this amino acid within the b- or y- series show a mass-shift corresponding to the mass of an RNA adduct and the other fragment ions are observed without any mass shifts e.g., amino acid residue M83 in the peptide
82LMAVTR87 cross-linked to uracil (Figure 6.1 A). In addition, when the fragment ion information was not sufficient, the immonium ion of the cross-linked amino acid was observed with a mass-shift corresponding to mass of RNA adducts e.g., amino acid residue W346 in the peptide
346WVEELKGGGQK356 linked to uracil (Figure 6.1 C). However, for nine peptide-RNA cross-links out of the 48 reported in this work, the cross-linked amino acid residue could not be identified e.g., the peptide 136VEDVHPISERPQYFSGDGK154 cross-linked to uracil (Figure 6.2 B).
128
When all considerations above are taken into account, the MS approaches have been very helpful in identifying interactions which are otherwise very difficult to do. In this work, approximately 50 protein-RNA cross-links and 126 inter-protein cross-links were identified and the stoichiometries of Type I-B Cascade complexes from H. volcanii and C. thermocellum were determined. These results when integrated with other structural techniques in collaboration resulted in three dimensional views of the complexes, as demonstrated by the more than five joint publications.