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.
- SourceAvailable from: InTechRNA Processing, 08/2011; , ISBN: 978-953-307-557-0
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ABSTRACT: Sm-like (Lsm) proteins are ubiquitous and function in many aspects of RNA metabolism, including pre-mRNA splicing, nuclear RNA processing, mRNA decay and miRNA biogenesis. Here three crystal structures including Lsm3, Lsm4 and Lsm5/6/7 sub-complex from S. pombe are reported. These structures show that all the five individual Lsm subunits share a conserved Sm fold, and Lsm3, Lsm4, and Lsm5/6/7 form a heptamer, a trimer and a hexamer within the crystal lattice, respectively. Analytical ultracentrifugation indicates that Lsm3 and Lsm5/6/7 sub-complex exist in solution as a heptamer and a hexamer, respectively while Lsm4 undergoes a dynamic equilibrium between monomer and trimer in solution. RNA binding assays show that Lsm2/3 and Lsm5/6/7 bind to oligo(U) whereas no RNA binding is observed for Lsm3 and Lsm4. Analysis of the inter-subunit interactions in Lsm5/6/7 reveals the organization order among Lsm5, Lsm6 and Lsm7.PLoS ONE 05/2012; 7(5):e36768. DOI:10.1371/journal.pone.0036768 · 3.53 Impact Factor
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ABSTRACT: The exoRNase Eri1 inhibits RNA interference and trims the 5.8S rRNA 3' end. It also binds to the stem-loop of histone mRNAs, but the functional importance of this interaction remains elusive. Histone mRNAs are normally degraded at the end of S phase or after pharmacological inhibition of replication. Both processes are impaired in Eri1-deficient mouse cells, which instead accumulate oligouridylated histone mRNAs. Eri1 trims the mature histone mRNAs by two unpaired nucleotides at the 3' end but stalls close to the double-stranded stem. Upon oligouridylation of the histone mRNA, the Lsm1-7 heteroheptamer recognizes the oligo(U) tail and interacts with Eri1, whose catalytic activity is then able to degrade the stem-loop in a stepwise manner. These data demonstrate how degradation of histone mRNAs is initiated when 3' oligouridylation creates a cis element that enables Eri1 to process the double-stranded stem-loop structure.Nature Structural & Molecular Biology 12/2012; DOI:10.1038/nsmb.2450 · 11.63 Impact Factor