title: Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.
abstract: Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Elad
authors.initials: E
authors.lastname: Eliahoo
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Phyana
authors.initials: P
authors.lastname: Litovco
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Ron
authors.initials: R
authors.lastname: Ben Yosef
authors.affiliation: Smoler Proteomics Center, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Keren
authors.initials: K
authors.lastname: Bendalak
authors.affiliation: Smoler Proteomics Center, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Tamar
authors.initials: T
authors.lastname: Ziv
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel. Electronic address: manor@tx.technion.ac.il.
authors.forename: Haim
authors.initials: H
authors.lastname: Manor
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004271:DNA, Fungal; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C110506:Srp1 protein, S pombe; C093803:TraX protein, bacteria; C502800:tsn1 protein, S pombe; D017856:Phosphotransferases (Phosphate Group Acceptor); C099438:inositol hexakisphosphate kinase
keywords: Affinity purification; Mass spectrometry; Protein–protein interactions; RNA-binding proteins; SR proteins