Identification of Cross-links from MS2-MBP Protein

Isolation of functional ribonucleoproteins in vitro is obtained through the combination of approaches like gel filtration with affinity chromatography employing bacteriophage MS2 coat protein. Spliceosome obtained so is used to determine the protein components of the spliceosome by mass spectrometry (Zhou & Reed, 2003). During the course of current studies, the RNP complex assembled by using HeLa nuclear extract was purified by MS2-MBP affinity purification method (Zhou & Reed, 2003). Data obtained by the MS analysis of the UV-cross-linked RNP complexes on LTQ Orbitrap Velos mass spectrometer was analysed by OpenMS. The affinity of the bacteriophage MS2 coat protein for its target MS2 RNA stem loops formed the basis of this method. The binding of MS2-MBP fusion protein to the MS2 RNA stem loops of target RNA has opened possibility to study the probable interaction sites of the aforementioned protein with RNA.

Direct interaction of MBP with nucleic acids has not been reported till now. Two peptides of MS2 coat protein and twelve peptides of MBP were found to be involved in cross-linking with oligonucleotides (Table 3.4 & Figure 3.13). Cross-linked amino acid residues for only seven peptides were screened out of fourteen cross-linked peptides. For the first time, the present studies have demonstrated that twelve peptides of MBP cross-link to uracil, whereby the lysine has been designated as the cross-linked amino acid residue.

Crystal structure of monomeric MBP (Figure 3.13) shows two distinct globular N and C domains, joined by three segments. Each domain is composed of central β-pleated sheet, flanked on both sides by α-helices (Quiocho et al., 1997). Most of the cross-linked peptides are found to be lying in the region of α-helices. Out of twelve cross-linked peptides, seven have been occupying N domain, four

137 occupying C domain and one occupying the third segment joining the two domains.

In the current studies, two peptides of MS2 protein were found to be cross-linked to uracil. The results are concomitant to those reported by Budowsky et al., 1976, according to which lysine residues in coat protein were found to cross-link to the genomic nucleic acid upon UV-irradiation.

Crystal structure of MS2 coat protein is composed of three coat protein dimers with a stretch of RNA (Figure 3.14). Each monomer is composed of five stranded β-sheet and two α-helical segments. The cross-linked lysine residues K⁴⁴² and K⁴⁴⁷ of peptides ⁴³⁸KYTIKVEVPK⁴⁴⁷ and ⁴⁴³VEVPKGAWR⁴⁵¹ respectively are found to be lying in β-sheet in closer proximity to cytosine at position −5 of nucleotides in RNA in the crystal structure. Grahn et al., 1999 reported that K⁴⁴² is among the probable amino acids that formed cross-links with nucleotides and the substitution of wild type uracil by cytosine at position −5 resulted in stronger binding of RNA hairpin to MS2 coat protein so from the present study, it can be anticipated that K⁴⁴² is the probable amino acid and uracil (in case of contamination)/cytosine (in case of bait RNA) at position −5 is the probable nucleotide taking part in cross-linking.

4.3.2 Identification of Uracil Fragments and Adducts

To determine the specific and definite cross-linking sites, mass spectrometric analysis of peptide oligonucleotide heteroconjugates is carried out. Cross-linking in combination to mass spectrometry provides an ideal paradigm to elucidate uncharacterized and unidentified RNA-protein cross-links (Kühn-Holsken et al., 2005). UV-induced cross-linking assists in generating direct zero-length cross links without intervening linker.

Keeping in view that the cross-links are additive in nature, the peptide-oligonucleotide cross-links were identified by using OpenMS. In addition to the peptide signals, the signals of characteristic marker ions produced by

138 fragmentation of the cross-linked nucleotides can also be observed in the MS2 spectra. Furthermore, the nucleotide fragments can be observed as adducts cross-linked to an amino acid residue resulting in the shifting of ion series within the spectra. The identification of these fragments and adducts may add to the authenticity of cross-linking data generated by the mass spectrometric analysis of the cross-links.

During the cross-linking analysis of RNP complex assembled by incubating labeled/non-labeled (PM5/MINX) pre-mRNA with HeLa nuclear extract proteins followed by the mass spectrometric and data analysis, revealed that the uracil nucleotide is the most frequently cross-linked nucleotide with the peptide moieties. In the previous studies numerous uracil nucleotide fragments have been reported (Table 3.5 & 3.6). However, in the current studies, there have been few signals in the MS2 spectra, which are assumed to be generated by the fragmentation of uracil. For this, the target signals generated by using isotopically labeled uracil (¹³C, ¹³C¹⁵N and 5-D1, ribose-3‘, 4‘, 5‘, 5‘-D4 labeled uracil) were compared to estimate the elemental composition.

The peptide ³⁷⁵DYAFVHFEDR³⁸⁴ derived from heterogeneous nuclear ribonucleoprotein R protein was found to be cross-linked to uracil nucleotide.

Upon CID fragmentation of nucleotides, the loss of phosphate group and neutral loss of water are usually observed. By keeping all these points in view the elemental composition of the uracil nucleotide fragment of interest can be predicted i.e. C8H6N2O3 generated via combined loss of two water (H2O) molecules, one phosphate group (HPO3) and formaldehyde (CH2O) molecule (Figure 3.17). This result is in accordance with the previous studies conducted on the pseudouridine fragmentation in which the loss of two water molecules and CH2O from the sugar moiety give rise to a product ion at m/z 179.

The peptide ²¹⁵YQVIGK²²⁰ of mitochondrial endonuclease G is found to be cross-linked to uracil nucleotide with the loss of H2O molecule. Its MS2 spectrum shows relatively high intensity ion signal at m/z 175.0714, which is expected to be generated by the fragmentation of cross-linked uracil nucleotide. The

139 comparative analysis among the MS2 spectra of the same cross-linked peptide with labeled and non-labeled uracil shows that the elemental composition of the target ion signal is C6H10N2O4 (Figure 3.18 & 3.19).

The MS2 spectrum of the cross-linked peptide ²¹⁵YQVIGK²²⁰ with U-H2O shows a shift in b ion series with the mass of 208 Da. When the MS2 spectra of the same cross-linked peptide along with differentially labeled and non-labeled uracil have been compared, the elemental composition of the target adduct was deduced to be C9H8N2O4 which was probably generated due to the loss of two H2O and one HPO3 molecules from the intact uracil nucleotide (Figure 3.18 & 3.20). In 1969, Rice & Dudek have also reported a similar fragment of 208 Da generated by the fragmentation of pseudouridine however in the current studies it has been observed by the fragmentation of uracil.

4.3.3 Identification of RNPs Isolated from HeLa Nuclear Extract