Engineered Rings of Mixed Yeast Lsm Proteins Show Differential Interactions with Translation Factors and U-Rich RNA
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. Biochemistry
(Impact Factor: 3.02).
03/2010; 49(11):2335-45. DOI: 10.1021/bi901767w
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.
Available from: Alexander William Tarr
- "We observe that LSm1 and LSm2 have similar effects on miR-122 regulation of HCV IRES-driven translation and on HCV replication, whereas LSm3 does not affect this process (Figure 2 and Supplementary Figure S4). Alternative LSm multimers can assemble in vitro (51). Our results suggest that LSm1 may function in a complex other than LSm1–7 to stimulate HCV IRES-driven translation in a manner that involves miR-122. "
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ABSTRACT: The P body protein LSm1 stimulates translation and replication of hepatitis C virus (HCV). As the liver-specific microRNA-122
(miR-122) is required for HCV replication and is associated with P bodies, we investigated whether regulation of HCV by LSm1
involves miR-122. Here, we demonstrate that LSm1 contributes to activation of HCV internal ribosome entry site (IRES)-driven
translation by miR-122. This role for LSm1 is specialized for miR-122 translation activation, as LSm1 depletion does not affect
the repressive function of miR-122 at 3′ untranslated region (UTR) sites, or miR-122–mediated cleavage at a perfectly complementary
site. We find that LSm1 does not influence recruitment of the microRNA (miRNA)-induced silencing complex to the HCV 5′UTR,
implying that it regulates miR-122 function subsequent to target binding. In contrast to the interplay between miR-122 and
LSm1 in translation, we find that LSm1 is not required for miR-122 to stimulate HCV replication, suggesting that miR-122 regulation
of HCV translation and replication have different requirements. For the first time, we have identified a protein factor that
specifically contributes to activation of HCV IRES-driven translation by miR-122, but not to other activities of the miRNA.
Our results enhance understanding of the mechanisms by which miR-122 and LSm1 regulate HCV.
Available from: Matthew W Bowler
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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.
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