ABSTRACT
SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.
Subject(s)
Mutation , Neoplasm Proteins/chemistry , Oncogene Proteins/chemistry , Phosphoproteins/chemistry , RNA Splicing Factors/chemistry , Spliceosomes/chemistry , Splicing Factor U2AF/chemistry , Amino Acid Sequence , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Gene Expression , Genes, Tumor Suppressor , HeLa Cells , Humans , Models, Molecular , Moths , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Splicing , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spliceosomes/metabolism , Spliceosomes/ultrastructure , Splicing Factor U2AF/genetics , Splicing Factor U2AF/metabolismABSTRACT
The universally conserved GTPase HflX is a putative translation factor whose GTPase activity is stimulated by the 70S ribosome as well as the 50S but not the 30S ribosomal subunit. However, the details and mechanisms governing this interaction are only poorly understood. In an effort to further elucidate the functional mechanism of HflX, we examined its interaction with the 70S ribosome, the two ribosomal subunits (50S and 30S), as well as its ability to interact with guanine nucleotides in the respective ribosomal complexes using a highly purified in vitro system. Binding studies reported here demonstrate that HflX not only interacts with 50S and 70S particles, but also with the 30S subunit, independent of the nucleotide-bound state. A detailed pre-steady-state kinetic analysis of HflX interacting with a non-hydrolyzable analog of mant-GTP, coupled with an enzymatic probing assay utilizing limited trypsinolysis, reveal that HflX·GTP exists in a structurally distinct 50S- and 70S-bound form that stabilizes GTP binding up to 70 000-fold and that may represent the "GTPase-activated" state. This activation is likely required for efficient GTP-hydrolysis, and may be similar to that observed in elongation factor G. Results reported here address the surprising low affinity of free HflX for GTP and suggest that cellular HflX will mainly exist in the HflX·GTP·ribosome-bound form. A minimal model for the functional cycle of HflX is proposed.
