title: Engineered rings of mixed yeast Lsm proteins show differential interactions with translation factors and U-rich RNA.
abstract: The Lsm proteins organize as heteroheptameric ring assemblies capable of binding RNA substrates and ancillary protein factors. We have constructed simplified Lsm polyproteins that organize as multimeric ring structures as analogues of the functional Lsm complexes. Polyproteins Lsm[2+3], Lsm[4+1], and Lsm[5+6] incorporate natural sequence extensions as linker peptides between the core Lsm domains. In solution, the recombinant products organize as stable ring oligomers (75 A wide, 20 A pores) in discrete tetrameric and octameric forms. Following immobilization, the polyproteins successfully act as affinity pull-down ligands for proteins within yeast lysate, including native Lsm proteins. Interaction partners were consistent with current models of the mixed Lsm ring assembly in vivo but also suggest that dynamic rearrangements of Lsm protein complexes can occur. The Lsm polyprotein ring complexes were seen in gel shift assays to have a preference for U-rich RNA sequences, with tightest binding measured for Lsm[2+3] with U(10). Polyprotein rings containing truncated forms of Lsm1 and Lsm4 were found to associate with translation, initiation, and elongation protein factors in an RNA-dependent manner. Our findings suggest Lsm1 and/or Lsm4 can interact with translationally active mRNA.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
authors.forename: Meghna
authors.initials: M
authors.lastname: Sobti
authors.forename: Liza
authors.initials: L
authors.lastname: Cubeddu
authors.forename: Paul A
authors.initials: PA
authors.lastname: Haynes
authors.forename: Bridget C
authors.initials: BC
authors.lastname: Mabbutt
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020815:Polyproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D015202:Protein Engineering; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012996:Solutions; D013379:Substrate Specificity; D014498:Uracil
chemical_list: D020815:Polyproteins; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D014498:Uracil