Crystal Structures of the Pyrococcus abyssi Sm Core and Its Complex with RNA: COMMON FEATURES OF RNA BINDING IN ARCHAEA AND EUKARYA

Stéphane Thore,Claudine Mayer,Claude Sauter,Stephen Weeks,Dietrich Suck

doi:10.1074/jbc.m207685200

Abstract

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.

Highlights

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences
They facilitate its biogenesis as Sm proteins do with other U snRNAs, and second, they increase the recycling of the di-small nuclear RNPs (snRNPs) U4/U6 after splicing has occurred [18]
Because P. abyssi contains only one type of Sm protein, the free and RNA-complexed structures are providing the first model of a complete Sm core

Summary

COMMON FEATURES OF RNA BINDING IN ARCHAEA AND EUKARYA*

Anococcus jannashii genome does not contain any open reading frame encoding an Sm protein but does contain a protein related to the bacterial Hfq. Crystal structures of several eukaryotic [26] and archaeal [27,28,29] Sm proteins have shown that the amino acid signature sequence defines a specific Sm-fold composed of a strongly bent 5-stranded ␤-sheet preceded by a short ␣-helix. In the Archaeoglobus fulgidus Sm1 protein (AFSm1), three residues, namely His-37 and Asn-39 from the first part and Arg-63 from the second part of the Sm domain, form a uridine-specific binding pocket [29] These residues are highly conserved within the Sm/Lsm family, and UV crosslinking experiments show that Phe-37 of the human SmG protein (equivalent to His-37 in AF-Sm1) contacts the first uridine of the Sm site [30], suggesting that the RNA binding site has been conserved between Archaea and Eukaryotes. The model may help to understand the regulatory function of the individual Sm protein subunits within the Sm core because the binding to the U-rich sequence composing the Sm binding site can be distinguished from the binding to surrounding sequences

Protein Purification

Gel Shift Assays

Structure Determination and Refinement

RESULTS AND DISCUSSION

CONCLUSION