The Archaeal Lsm Protein Binds to Small RNAs

Abstract

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.Sm and Sm-like (Lsm) proteins constitute a large family of proteins known to be involved in RNA metabolism. Representatives of this family are found in all three domains: bacteria, archaea, and eukarya. All of them share a common bipartite sequence motif, known as the Sm domain, consisting of two conserved segments separated by a region of variable length and sequence. The bacterial family member is the Hfq protein (1, 2), which has a plethora of functions (3). Hfq is a highly conserved protein encoded within many bacterial genomes (4). Although the protein does not show a high similarity to the Lsm proteins on the primary structure level, it possesses striking similarities in both function and tertiary and quaternary structure to the eukaryotic Lsm proteins (3, 5). Hfq monomers assemble to form highly stable hexamers (6), which bind preferentially to A/U-rich sequences (7, 8) but have a relaxed RNA binding specificity and participate in many stages of RNA metabolism. It was therefore proposed that Hfq is an ancient, less specialized form of the Lsm proteins (9). One of the identified functions of Hfq is its interaction with sRNAs (10). It has been proposed that the protein acts as an RNA chaperone that might simultaneously recognize the sRNA and its target and facilitate its interaction. An Escherichia coli hfq insertion mutant showed pleiotropic phenotypes including decreased growth rates and yields, increased cell sizes, and an increased sensitivity to stress conditions (11-13). These defects are at least in part a reflection of the fact that Hfq is required for the function of several sRNAs including DsrA, RprA, Spot42, OxyS, and RhyB (14 -17).Eukaryotes have t...

The Lsm Proteins: Ring Architectures for RNA Capture

“…The diversity of biological activities of Lsm proteins compared to their more specialized Sm counterparts supports this two-step evolution model (Beggs, 2005;Khusial et al, 2005). The presence of up to three Lsm proteins in archaea, as well as an Hfq-like protein in archaeal M. jannaschii, further supports a common ancestor of eukaryotic and archaeal Lsm proteins (Fischer et al, 2011). A few multidomain proteins incorporating Lsm components have been observed (summarized, Figure 2).…”

“…Hfq controls gene expression either by rearranging the RNA secondary structure, or by increasing the concentration of RNA locally to promote RNA-RNA interactions (Moll et al, 2003;Lease & Woodson, 2004;Afonyushkin et al, 2005). A similar mode of binding to sRNA was recently observed for the archaeal Lsm from Haloferax volcanii (Fischer et al, 2011). As for the eukaryotic Lsm proteins, Hfq is required for deadenylation-dependent mRNA decay.…”

The Non-Coding Regulatory RNA Revolution in Archaea

“…To date, very few of the newly reported candidate sRNAs in the archaea have been functionally characterized [2,13,37] Co-immuno-precipitation with the Lsm protein, the archaeal homolog of Hfq, was used to "capture" sRNAs in vitro [6]. However, the role of Lsm -or any other RNA-binding proteinremains to be elucidated in the archaea.…”

“…sRNAs in bacteria and eukarya play essential roles in transcriptional regulation, chromosome replication, RNA processing and modification, mRNA stability and translation, and even protein degradation and translocation [3][4][5]. Recently, it was discovered that archaeal genomes encode for large numbers of sRNAs and that many of them are responsive to environmental stresses [1, 2,[6][7][8][9][10][11][12][13][14].…”

Contribution of Non‐Canonical Interactions to the Stability of Sm/LSm Oligomeric Assemblies

“…In general they are very stable and sometimes the presence of chaotropic agent is necessary for their disruption. [31,35,36] We have previously reported that contribution of hydrophobic interactions, hydrogen bonds and salt bridges to the stability of Sm oligomers. [37] In order to better understand the nature of these assemblies, in this study we have analyzed the contribution of non-canonical interactions to the stability of assemblies.…”