Journal of Molecular Biology
Crystal Structure of Lsm3 Octamer from Saccharomyces cerevisiae: Implications for Lsm Ring Organisation and Recruitment
Introduction
The Sm and Sm-like (Lsm) proteins are essential components of ribonucleoprotein (RNP) complexes involved in a multitude of RNA processing events including mRNA degradation and splicing, histone formation and telomere replication.1, 2, 3 The Sm members of the family were initially identified to be functional in small nuclear RNPs (snRNPs) U1, U2, U4/U6 and U5 involved in pre-mRNA splicing. Recently, our knowledge of their role has expanded and the L/Sm family is now viewed as a robust scaffold mediating a range of protein–RNA interactions.4 In eukaryotes, a ring comprising seven distinct proteins of the Lsm family associates with RNA at uridine-rich sequences to form the core of a variety of snRNP complexes. The exact protein constituents of the ring appear to determine the function of the complex. Thus, the canonical Sm proteins, SmB/B′, D1, D2, D3, E, F and G associate with spliceosomal U1, U2, U4 and U5 small nuclear RNA to carry out the splicing of pre-mRNA;5 a heptamer of Lsm proteins, Lsm[2–8], is involved in U6 snRNP biogenesis;6, 7 and a complex including the Lsm[1–7] heptameric ring leads to localisation in the cell cytoplasm and functioning in 5′-to-3′ mRNA decay.8, 9, 10
While Lsm proteins are found across all three domains of life, examination of the genomes of archaeal species reveals that only one to three complete Lsm genes are encoded, in contrast to the 16 or more found in eukaryotes.7, 11 Crystallographic studies have shown that these individual archaeal Lsm proteins can complex as homomeric rings of heptamers11, 12, 13, 14 or hexamers.15 These X-ray crystal structures, together with density from electron microscopy of mixed eukaryotic Lsm proteins and crystal structures of dimers of the human Sm complex, have led to models of a functional assembly of seven distinct Sm/Lsm proteins in a heteromeric ring.16, 17, 18 As yet, however, no structure of an intact eukaryotic Lsm heteromeric ring complex has been determined.
The L/Sm fold characteristic of this protein family consists of a highly curved five-stranded β-sheet generally preceded by a short N-terminal α-helix. The sequence of loop L4 linking strands β3 and β4 is extremely variable across the family, ranging in length from 3 to 30 residues (and even longer in Lsm1119). The curved β-sheet encloses a core of hydrophobic residues, which extends into adjacent protein monomers once the closed ring assembly is formed. The interface between subunits in the toroid also includes extension of the β-sheet hydrogen-bonding network, with strand β4 of one subunit aligned against strand β5 of its neighbour. These extensive contacts between subunits throughout the Lsm assembly result in an extremely stable ring complex organised so as to present one face incorporating the helices of each Lsm subunit (the ‘helix face’) on the opposite side of the toroid to loop L4 residues (the ‘loop face’). A U-rich sequence of RNA is thought to encircle the inner edge of the helix face to hydrogen-bond with specific residues of the exposed loops (L3 and L5) and possibly also to pass through the central pore.14, 20, 21, 22
Although most commonly grouped as heptameric complexes, structures incorporating hexameric organisation of Lsm subunits have also been observed. Homomeric complexes of hexamers of an archaeal Lsm protein and the bacterial homologue, Hfq, have been defined by X-ray crystallography.15, 23 There is also some evidence that a hexameric Lsm complex (incorporating Lsm[2–7]) may be functional, involved in pseudouridylation of rRNA.24 Furthermore, during crystallisation, Lsm rings assemble into higher-order quaternary structures, with interactions between rings occurring via helix face-to-helix face packing,11, 22, 25 loop face-to-loop face packing20 (also seen in Hfq23) or helix face-to-loop face stacking observed to form fibres.25 Recently, more complicated fibrillar structures have also been reported for Hfq.26
In this study, we have determined the crystal structure of a homomeric eukaryotic Lsm ring complex formed by the Lsm3 protein from the budding yeast Saccharomyces cerevisiae. At 89 residues, this is the smallest of the yeast Lsm proteins, lacking the charged extensions seen in the N- or C-terminal regions of most other Lsm sequences (Fig. 1a). Lsm3 is a close sequence relative of the Sm ring component SmD2 (20% identity) and is thought to partner proteins Lsm2 and Lsm6 within the heteromeric Lsm ring complexes in vivo.18, 27 Recombinant Lsm3 readily forms highly stable homomeric complexes in solution, and our crystal structure defines a novel ring arrangement of eight Lsm3 subunits. While the structure shows the subtle alteration of subunit interactions required for octahedral geometry, it also provides key insights into additional protein recruitment by Lsm complexes. These likely utilise longer loop L4 segments protruding from the non-RNA binding face of the mixed Lsm toroid.
Section snippets
Discrete complexes of yeast Lsm3 in solution
Recombinant expression of N-terminal His6-tagged Lsm3 produced soluble, folded protein. Size-exclusion chromatography revealed two distinct peaks for some Lsm3 preparations, fractions I and II, the former eluting at or near the void volume of the Superose-12 column used. The relative proportion of these two fractions was found to be pH-dependent (Fig. 1b). At pH 8.0, all Lsm3 eluted solely as the smaller species, and this constituted the source material for structural studies. However, when
Discussion
The crystal structure determined here for yeast Lsm3 shows the monomer unit to be closely similar to the five-stranded β-sheet structure observed within previously determined L/Sm protein complexes. Loop L4 comprises the most variable portion of the Lsm sequence, and the region around this loop accounts for the largest differences between Lsm3 and earlier structures. Excluding residues from this variable region, polypeptide main-chain atoms of Lsm3 are within 0.9–1.2 Å r.m.s.d. of those of
Protein expression and purification
Lsm3 was amplified from S. cerevisiae genomic DNA and cloned into the pET-based vector pCL774 (gift of Nick Dixon, University of Wollongong) to code for an N-terminal His6-tagged fusion product. Bacterial BL21(DE3) pLysS cells containing Lsm3 in pCL774 were grown at 37 °C in Luria broth containing ampicillin and chloramphenicol. Protein expression was induced by addition of isopropyl β-d-thiogalactopyranoside (to 0.5 mM) and cell growth continued at 25 °C (3 h). Pelleted cells were resuspended
Acknowledgements
We would like to thank Geoff Kornfeld (University of New South Wales) for genetic material, Liza Cubeddu for Lsm3 constructs and Karlie Neilson for mass spectrometry. This work was financed by research and travel grants from Macquarie University. N.N. acknowledges support of an Australian Government Postgraduate Award and B.S. the receipt of a Canadian Institute of Health and Research Fellowship.
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Cited by (26)
Archaeal Lsm rings as stable self-assembling tectons for protein nanofabrication
2017, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Naturally located within large ribonucleoprotein particles, Lsm complexes contribute core molecular machinery in vivo for RNA processing across eukaryotes, bacteria and archaea [18,19]. Quaternary ring structures formed by recombinant Lsm samples encompass hexameric, heptameric or octameric groupings, structures which have been well defined by x-ray crystallography [20–23]. The Lsmα protein from the archeon Methanobacterium occurs as a heptameric ring structure of diameter 6.5 nm, with a 1.5 nm inner pore [24].
Structural and functional control of the eukaryotic mRNA decapping machinery
2013, Biochimica et Biophysica Acta - Gene Regulatory MechanismsCitation Excerpt :While the Lsm1-7 complex is predicted to form a heteroheptameric ring [43,64–67], its subunits may form various subcomplexes. A crystal structure of Lsm3 from S. cerevisiae shows that it forms an octameric ring structure [68]. Recent crystallographic analysis combined with analytical ultracentrifugation of Lsm3, Lsm4 and a Lsm5/6/7 subcomplex from S. pombe indicates that these exist in solution as a heptamer, a monomer and a hexamer, respectively [69,70].
Structure of the LSm657 complex: An assembly intermediate of the LSm1-7 and LSm2-8 rings
2011, Journal of Molecular BiologyCitation Excerpt :Finally, differences between the crystal structure and the solution state were previously observed for the LSm3 protein. In that case, the crystal structure suggested the assembly of the LSm3 rings into complexes of very high molecular mass, a feature that was not observed in solution.22 From a technical point of view, it is not straightforward to study the hexameric LSm657 ring with a molecular mass of 60 kDa using high-resolution NMR spectroscopy.
The structures of mutant forms of Hfq from Pseudomonas aeruginosa reveal the importance of the conserved His57 for the protein hexamer organization
2010, Acta Crystallographica Section F: Structural Biology and Crystallization CommunicationsA structural biology view on the enzymes involved in eukaryotic mRNA turnover
2023, Biological Chemistry
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Present address: N. Naidoo, School of Physics, University of New South Wales, Sydney, NSW 2052, Australia.