Function of Sm proteins in Sulfolobus solfataricus

Sm and Sm-like proteins involved in different aspects of RNA metabolism are conserved throughout all kingdoms of life. In E. coli the Sm-like protein Hfq mediates post-transcriptional regulation, including riboregulation together with small non-coding RNAs (ncRNAs). Binding of ncRNAs to mRNAs can either activate or silence gene expression. In Eukaryotes Lsm2-8 is localized in the nucleus, where it is involved in RNA processing and degradation of nuclear RNAs. The Lsm1-7 complex is localized in cytoplasmic processing bodies (P-bodies), binds to deadenylated mRNAs and promotes de-capping, which precedes 5-> 3directional mRNA decay. Despite the availability of several 3D-structures of archaeal Sm proteins, their function has remained elusive. The aim of this study is two-fold. It aims at defining a role for the Sm proteins in the crenarchaeal model organism Sulfolobus solfataricus (Sso), and at elucidating the molecular mechanism(s) underlying ncRNA-mediated gene regulation in Sso. One part of the study builds on two previously identified Sso ncRNAs and their predicted target mRNAs. Our preliminary data confirmed the expression of these ncRNAs and revealed that they form duplexes with the 3 un- translated region of the respective putative target mRNA, which is reminiscent to the mode of action of eukaryotic miRNAs. Genetic and biochemical studies will be performed to assign a role for these ncRNAs in post- transcriptional regulation, viz whether these ncRNAs affect the translational efficiency and/or transcript stability, as well as the possible involvement/role of the Sso Sm-proteins in this process(es). To study the function of the two Sm proteins in Sso, two routes will be taken: We will attempt to create knock out mutants in Sm1 and Sm2. If successful, the effect of the deletions will be assessed by microarray analysis under different stress conditions. These studies have the potential to reveal common as well as different functions of both Sm proteins in Sso RNA metabolism. In a complementary approach we will identify Sso RNAs bound to His- tagged variants of Sm1 and Sm2 by deep sequencing. It is anticipated that the combinatorial analysis of the microarray- and sequencing data will enable us to make an educated guess on ncRNAs/mRNAs pairs as well as other "RNAs" targeted by Sm proteins in Sso. In addition, attempts will be made to identify protein factors potentially involved in Sso riboregulation. The candidate proteins will be tested for their ability to interact directly and/or indirectly (via RNA) with either Sso Sm protein. It is anticipated that these studies contribute to a better understanding of post-transcriptional regulatory events in Archaea, and in particular that they shed light on evolutionary aspects of ncRNA-mediated regulation.

Sm and Sm-like proteins, which are conserved throughout all kingdoms of life, are involved in different aspects of RNA metabolism. In contrast to Bacteria and Eukarya, the molecular function of archaeal Sm proteins is poorly understood. The aims of this study were to define a role for the Sm proteins in the crenarchaeal model organism Sulfolobus solfataricus (Sso), and to shed more light on post-transcriptional regulation by non-coding (nc) RNAs.We were able to report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sso the transposon derived paralogous RNAs, RNA-2571-4, exhibit extended complementarity to the 3UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation resulted in decreased RNA-2571 and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA was faster degraded in vitro upon duplex formation with RNA-2571. Insertion of the 1183 3UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.We have identified short RNA motifs in mRNAs that bind specifically to Sso Sm2 protein. We have been able to determine the structure of Sm2, and to identify by mass spectrometry residues in Sm2 that interact with a derived Sm2 RNA consensus motif. In addition, we were able to show that the Sm2 RNA motif can affect the longevity when transplanted onto a reporter mRNA.We have searched for protein binding partners of Sm1 and Sm2. Among others, we have identified components of the exosome. We could demonstrate a direct interaction of Sm1/2 via the exosomal subunit DnaG. Moreover, using in vitro cleavage assays, we showed that Sm1/2 negatively impact on exosome function by interfering with its 3 to 5 exonucleolytic activity.We have identified several ncRNA candidates that bind either to Sso Sm1 and / or Sm2. We have created deletion mutants in two candidate ncRNA genes. In one case the deletion inhibited growth, whereas deletion of the second ncRNA gene increased the growth rate ~two-fold. Comparative transcriptomic approaches are currently underway to identify target genes of the latter ncRNA.We have recently obtained a deletion mutant in the Sm1 protein gene. Currently, transcriptional profiling- and follow-up studies are performed to assess the effect of Sm1 on bulk RNA metabolism in Sso.