Alterations of the Transcriptome of Sulfolobus acidocaldarius by Exoribonuclease aCPSF2

Abstract

Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso), which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2) group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2) was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

RNA stabilization in hyperthermophilic archaea

“…Crystal structures were solved for aCPSF1 variants from three different archaea, Pyrococcus horikoshii , Methanosarcina mazei , and Methanothermobacter thermautotrophicus , revealing a tripartite structure that enables exoribonucleolytic activity with 5ʹ monophosphorylated (5ʹp) RNA substrates . In S. solfataricus , Sso‐aCPSF2 was demonstrated to act as a 5ʹ to 3ʹ exonuclease with a preference for RNA molecules with 5ʹp ends over RNAs with 5ʹppp termini . The translation initiation factor a/eIF2 can bind mRNAs with 5ʹppp ends (but not RNAs with 5ʹp or 5ʹpp ends) to provide protection from Sso‐aCPSF2 degradation, and in vivo experiments showed that the overproduction of a/eIF2(γ) increases the stability of different mRNAs .…”

Binding of the 5′-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus

“…We have put forward a model specifying that either trimeric SsoIF2 or SsoIF2γ alone binds to the 5′-terminal end of mRNAs and protects them from 5′ to 3′ directional decay during nutrient limitation, for example, when they grow chemolithotrophically [12]. This hypothesis was supported by the discovery of the S. solfataricus RNase aCPSF2 displaying 5′-to-3′ directional mRNA decay, which was shown to be impeded in vitro by SsoIF2γ bound to the 5′-terminal triphosphate end of RNA substrates [24,25]. This would result in 5′-end protection of the respective mRNAs under conditions when translation is decreased.…”

RNase J is required for processing of a small number of RNAs inRhodobacter sphaeroides

“…This is in contrast to B. subtilis , where under severe depletion conditions of RNase J1 roughly 30% of all mRNAs were affected 21 . Just recently an RNA-seq approach with the Crenarchaeon Sulfolobus acidocaldarius revealed global alterations of the transcriptome by deletion of aCPSF2 an archaeal homolog of RNase J 37 . The identification of RNA fragments exclusively accumulating in the RNase J deletion strain 2.4.1∆ rnj and virtually not detectable in the wild type leads to a number of questions regarding different aspects of RNA metabolism.…”