ABSTRACT
Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.
Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Models, Molecular , Nucleic Acid Conformation , Polypyrimidine Tract-Binding Protein/chemistry , RNA/chemistry , Ribonucleoprotein, U1 Small Nuclear/chemistry , Binding Sites , Carbon Isotopes , Chromatography, High Pressure Liquid , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Polypyrimidine Tract-Binding Protein/genetics , Protein Binding , Ribonucleoprotein, U1 Small Nuclear/genetics , Software , Tandem Mass Spectrometry , Ultraviolet RaysABSTRACT
The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3'end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation.
