Journal of Molecular Biology
Volume 377, Issue 5, 11 April 2008, Pages 1357-1371
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Crystal Structure of Lsm3 Octamer from Saccharomyces cerevisiae: Implications for Lsm Ring Organisation and Recruitment

https://doi.org/10.1016/j.jmb.2008.01.007Get rights and content

Abstract

Sm and Sm-like (Lsm) proteins are core components of the ribonucleoprotein complexes essential to key nucleic acid processing events within the eukaryotic cell. They assemble as polyprotein ring scaffolds that have the capacity to bind RNA substrates and other necessary protein factors. The crystal structure of yeast Lsm3 reveals a new organisation of the L/Sm β-propeller ring, containing eight protein subunits. Little distortion of the characteristic L/Sm fold is required to form the octamer, indicating that the eukaryotic Lsm ring may be more pliable than previously thought. The homomeric Lsm3 octamer is found to successfully recruit Lsm6, Lsm2 and Lsm5 directly from yeast lysate. Our crystal structure shows the C-terminal tail of each Lsm3 subunit to be engaged in connections across rings through specific β-sheet interactions with elongated loops protruding from neighbouring octamers. While these loops are of distinct length for each Lsm protein and generally comprise low-complexity polar sequences, several Lsm C-termini comprise hydrophobic sequences suitable for β-sheet interactions. The Lsm3 structure thus provides evidence for protein–protein interactions likely utilised by the highly variable Lsm loops and termini in the recruitment of RNA processing factors to mixed Lsm ring scaffolds. Our coordinates also provide updated homology models for the active Lsm[1–7] and Lsm[2–8] heptameric rings.

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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    Present address: N. Naidoo, School of Physics, University of New South Wales, Sydney, NSW 2052, Australia.

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