The SmAP2 RNA binding motif in the 3′UTR affects mRNA stability in the crenarchaeum Sulfolobus solfataricus

Abstract

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro. Residues in SmAP2 interacting with the SmAP2-cRBM were mapped by UV-induced crosslinking in combination with mass-spectrometry, and verified by mutational analyses. The RNA-binding site on SmAP2 includes a modified uracil binding pocket containing a unique threonine (T40) located on the L3 face and a second residue, K25, located in the pore. To study the function of the SmAP2-RBM in vivo, three authentic RBMs were inserted in the 3′UTR of a lacS reporter gene. The presence of the SmAP2-RBM in the reporter-constructs resulted in decreased LacS activity and reduced steady state levels of lacS mRNA. Moreover, the presence of the SmAP2-cRBM in and the replacement of the lacS 3′UTR with that of Sso2194 encompassing a SmAP2-RBM apparently impacted on the stability of the chimeric transcripts. These results are discussed in light of the function(s) of eukaryotic Lsm proteins in RNA turnover.

RNA stabilization in hyperthermophilic archaea

“…Finally, Hfq proteins were also shown to stabilize RNA molecules by inhibiting 3ʹ to 5ʹ polynucleotide phosphorylase (PNPase) exonuclease activity . Recent studies of two Sm‐like archaeal proteins (Lsm1 and Lsm2) in the model crenarchaeon S. solfataricus P2 revealed that they are capable of interacting with a large subset of sRNAs and suggested their involvement in tRNA/rRNA processing and the biogenesis of s(no)RNAs . It was also observed that these proteins interact with the archaeal exosome .…”

“…50,51 Recent studies of two Sm-like archaeal proteins (Lsm1 and Lsm2) in the model crenarchaeon S. solfataricus P2 revealed that they are capable of interacting with a large subset of sRNAs and suggested their involvement in tRNA/rRNA processing and the biogenesis of s(no)RNAs. [52][53][54] It was also observed that these proteins interact with the archaeal exosome. 55 Most archaeal genomes contain orthologs coding for subunits of the exosome complex.…”

Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing

“…One of these, SmAP2, appears to be essential, as knockout attempts failed repeatedly (U. Bläsi, unpublished data). Even though the physiological function of the SmAP2 is still elusive, 53 mRNA substrates were recently identified that all carried a specific motif that is tightly bound by SmAP2 in vitro [39,40]. To aid the characterization of the in vivo role of SmAP2, we sought to study the phenotype of the SmAP2-depletion in S. solfataricus P1.…”

Novel roles for Sm-class RNAs in the regulation of gene expression

“…Similarly to eukaryotic Sm proteins, Hfq binds to U-rich sequences present in regulatory small RNAs (sRNAs) that also base-pair with target mRNAs to regulate their stability and translation [64] (Figure 3). Also, recent reports suggest that archaeal Sm-like proteins have a role in mRNA stability [65,66] Viruses usually acquire host factors and modify them to exploit their normal cellular functions for their own benefit. HVS contains several open reading frames with striking homology to cellular genes that are thought to have been pirated from the host, including genes related to nucleotide metabolism, cell cycle control, apoptosis, and innate or adaptive immune response [2].…”