PMC

MALDI-MS/MS analysis of cross-linked peptides derived from reconstituted [U1 snRNP-SPN1] complexes. (A) MALDI product-ion spectrum of a peptide–peptide cross-link at m/z 2557.3 derived from the cross-linked proteins U1-70K–SmD2 after in-gel trypsinization (B, band I). The sequences of the cross-linked peptides (U1-70K, HHNQPYCGIAPYIR; Sm-D2, SKPVNK) with their specific y-type ions are listed within the spectrum. (B) MALDI product-ion spectrum of a peptide–peptide cross-link at m/z 1499.7 derived from band II in B. MS analysis identified SPN1 cross-linked to SmB/B′. The cross-linked sequences from SPN1 (SGYCVNR) and from SmB/B′ (IKPK) are given as insets within the spectrum. Sequences were determined according to the y-type fragment ions.

IMAC enrichment for the analysis of peptide–RNA oligonucleotide cross-links derived from UV-irradiated [U1 snRNP-SPN1] complexes. (A) MALDI-MSMS analysis of isolated cross-linked precursor (MW = 2042.3 Da). The fragmentation of the cross-link in the MALDI reveals that the extra mass is at least partly due to nucleic acids, as the loss of phosphate and ribose is seen clearly within the spectrum. Peptide-specific fragmentation is observed as well. (B) MALDI MS analysis of an enriched and semi-preparative purified cross-link species (see ‘Materials and Methods’ section) encompassing the cross-linked SPN1 tryptic fragment GSSHSPDHPGCLMoxEN. The MALDI MS spectrum shows mass signals for the non-cross-linked oxidized peptide and various corresponding cross-linked peptide-oligoribonucleotides. The differences in mass between the non-cross-linked and cross-linked species that correspond to nucleotides are listed; they reveal the cross-linked RNA moiety (AAAUp). The cross-link still has an extra mass of ∼152 Da that cannot be explained (see also text).

Location of protein–protein cross-linking sites identified in Sm proteins. Sequence alignment of the Sm1 and Sm2 motifs of the human Sm proteins B/B′, D2, D1, D3, E, F and G are according to Hermann et al. (). The topology of the secondary structure was adapted from the crystal structures of SmB-D3 and SmD1-D2 protein complexes (). Conserved amino acids are highlighted as follows: uncharged, hydrophobic residues (L, I, V, A, F, W, Y, C, M) are in light red; acidic and basic amino acids (D, E, R, K) are in green, and 100% conserved amino acids in dark red. Secondary structure elements in the Sm proteins are depicted as follows: α = helix; β = beta-sheet, L = loop. Arrows mark the cross-linked amino acids in the variable loop 4 of the protein sequences of SmD2 and SmB and in the C-terminal part of SmD3.

Detection and structural analysis of [U1 snRNA-SPN1] cross-links by LC/MS/MS. (A) Precursor-ion-scanning experiments for m/z 79 (PO⁻₃) in negative-ion mode to determine the molecular weight of putative cross-linked species (see text and ‘Materials and Methods’ section for details). Observed m/z values of PO⁻₃-generating precursors are plotted against their retention time. The corresponding molecular weights listed (MW = 2042.8 Da, 2284.0 Da, 2371.9 Da and 2613.9 Da) are selected in a second experiment for MRM-triggered MSMS analysis in positive-ion mode (see text and ‘Materials and Methods’ section for details). (B) MRM-triggered product-ion scan of a cross-linked precursor (MW = 2042.3 Da) derived from SPN1. MRM was triggered by the monitoring production of specific RNA-base fragment ions, i.e. [M + nH]ⁿ⁺ → [{111, 112, 135, 151} + H]⁺. The sequence of the cross-linked peptide (GSSHSPDHPGCLMEN) with the corresponding fragment ions is given within the deconvoluted spectrum. The deciphered sequence shows an extra mass of 476 Da that cannot be explained by any combination of RNA mono- and/or dinucleotides.

(A) Purification of the [U1snRNP-SPN1] complex. SPN1 and U1 snRNPs mixed in a 7 to 1 molar ratio were incubated on ice for 10 minutes prior to removal of excess of unbound SPN1 by size exclusion chromatography (SE). The fractions obtained were analyzed by SDS–PAGE and visualized by Silver staining. Upper Panel: Chromatogram of the SE elution. Lower panel: Proteins in the respective fractions were separated by SDS–PAGE. Fractions 2 and 3 containing the [U1snRNP-SPN1] complex were used for subsequent cross-linking experiments. Of note, U1 70K protein escapes detection by silver staining (). (B) Protein–protein cross-linking of reconstituted [SPN1–U1 snRNP] complexes using sulfo-MBS. Sulfo-MBS-induced protein–protein cross-links were analyzed by SDS-PAGE and subsequently stained with Coomassie. Lane 1, non-cross-linked [U1 snRNP-SPN1]; lane 2, cross-linked [U1 snRNP] using 5 µM sulfo-MBS; lane 3, cross-linked [U1 snRNP-SPN1] using 5 µM sulfo-MBS. The positions of the individual U1 snRNPs proteins are indicated. Previously identified U1 70K–SmD2 (marked with I), U1 70K-Sm B/B′, and SmB–SmD3 cross-linked species are indicated (*). A potential cross-link of U1 70K with presumably U1 C protein is indicated as well. Arrows II and III mark additional putative protein–protein cross-links that were not observed in previous studies () and may thus be expected to be specific for SPN1. Lane 4, molecular-weight marker (MW).

Overall locations of protein–protein and protein–RNA cross-linking sites in Sm proteins, SPN1 and within the U1 particle. (A) Location of the protein–protein cross-linking sites in SmD2, SmD3, SmB/B′. The Sm proteins are depicted as a heptameric ring structure in various colors. Images were generated using PYMOL, and the coordinates of the respective proteins within the published U1 snRNP structure () available in the PDB (PDBid: 3CW1). No coordinates are available for loop 4 of SmD2, SmB/B′ or for the C-terminus of SmD3. Note that only coordinates from the α-C atoms were available in the PDB database. Yellow spheres mark the N- and C-terminal borders of loop 4 in SmD2, which was found to be cross-linked to U1-70K. Green spheres mark the borders of the corresponding loop region 4 in SmB/B′ protein, which was found to cross-linked to SNP1. The dark red sphere marks the last C-terminal amino acid in SmD3 for which coordinates were available. The subsequent amino acids in SmD3 were found to be cross-linked to SPN1. (B) Domain structures of SPN1 with cross-linking sites. Images were generated using PYMOL and the coordinates of SPN1 in the PDB (PDBid: 1XK5). SPN1 is shown with its co-crystallized cap-analogue structure (,). The orientation of the domains of SPN1 is as found in complex with CRM1 (PDBid: 3GJX) with the structured C-terminus of SPN1. The N-terminal IBB domain is colored in light grey, and the m3-Cap binding domain of SPN1 is shown in dark grey with the TMG cap in red superposed form (PDBid: 1XK5); the cross-linking sites to SmB/B′ and SmD3 are marked in orange with the actual cross-linked amino acid shown as an orange sphere. The cross-linking site to stem-loop III of U1 (SL III) is shown in red, and the actual cross-linked amino acid as a red sphere. The flexible region linking the central domain to the C-terminal residues is indicated as black dashed line. (C) Location of protein–protein and protein–RNA cross-linking sites identified on U1snRNPs (according to PDBid: 3CW1). The RNA is shown in black, with the area of cross-link in SL III and TMG are depicted as red spheres. The stem-loops I (SL I), SL III, and SL IV are indicated. Note that stem-loop II (SL II) has been truncated in order to obtain crystal structure (). The Sm proteins are shown as in panel A using the same color code, whereas the U1-C and U1-70k are depicted in light and dark grey, respectively. The missing residues in U1-70k are indicated as dotted red line. Beginning and end point of cross-linked peptides are shown in colored spheres according to panel A.