The Heptameric SmAP1 and SmAP2 Proteins of the Crenarchaeon Sulfolobus Solfataricus Bind to Common and Distinct RNA Targets

Abstract

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism.

The SmAP1/2 proteins of the crenarchaeon Sulfolobus solfataricus interact with the exosome and stimulate A-rich tailing of transcripts

“…The genes encoding the SmAP1 and SmAP2 proteins were abutted at the 3΄end with a 10×His-tag encoding sequence. Transcription of the genes from the virus-derived pMJ05-vector was controlled by an arabinose-inducible promoter in Sso PH1-16 ( 26 ). When compared with endogenous protein levels, this expression system permits a 3- to 10-fold overproduction of the respective proteins ( 48 , 55 ).…”

“…The Sso strains PH1-16(pMJ05-SmAP1-His), PH1-16(pMJ05-SmAP2-His) ( 26 ) and PH1-16(pMJ05-DnaG-His) were generated as described ( 48 ) ( Supplementary Data ). For expression of the plasmid borne genes, the strains PH1-16(pMJ05), harboring the backbone vector (mock control), PH1-16(pMJ05-SmAP1-His), PH1-16(pMJ05-SmAP2-His) and PH1-16(pMJ05-DnaG-His) were grown at 75°C in arabinose-containing Brock's medium.…”

“…The SmAP1 coding gene was cloned as described in Supplementary Data . DnaG-His, SmAP1-His and SmAP2-His were purified as previously described ( 26 , 49 ). For Co-IP with purified components, 50 pmol of DnaG-His was either incubated alone (mock control) or SmAP1-His and SmAP2-His were incubated together with 50 pmol DnaG-His, respectively, in 200 μl Co-IP buffer (50 mM Tris–HCl pH 6.0, 100 mM KCl; 5% glycerol, 1 mM MgCl 2 and 0.1% Triton X-100) for 10 min at 65°C.…”

“…While bacterial and eukaryotic Lsm family members have been studied in more detail ( 2 , 4 , 5 , 14 ), the function of Sm-like archaeal proteins (SmAPs) remains poorly understood. Like other Sm/Lsm proteins, the SmAPs are composed of an N-terminal α-helix and five β-strands forming a continuous Sm-fold ( 22 – 26 ). However, the homo-heptameric SmAPs display characteristic differences from bacterial and eukaryotic Lsm proteins, e.g.…”

“…SmAP1 and SmAP2 show 50% similarity, whereas they share only 30% similarity with SmAP3. In Sso different classes of non-coding RNAs and mRNAs were identified that interact either with SmAP1 or SmAP2 or with both proteins ( 26 ). The large number of associated intron-containing tRNAs and rRNA modifying RNAs suggested as well a role of these SmAPs in tRNA/rRNA processing ( 26 ).…”