Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry.
Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m(3)G cap and snurportin 1. We were able to define previously unknown sites of protein-protein and protein-RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m(3)G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m(3)G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA.
Kühn-Hölsken
Eva
E
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Lenz
Christof
C
Dickmanns
Achim
A
Hsiao
He-Hsuan
HH
Richter
Florian M
FM
Kastner
Berthold
B
Ficner
Ralf
R
Urlaub
Henning
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D039381:RNA Cap-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides; D053719:Tandem Mass Spectrometry
D003432:Cross-Linking Reagents; D039381:RNA Cap-Binding Proteins; D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D013388:Succinimides; C405483:U1 small nuclear RNA; C072252:m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester