RIP-Seq Suggests Translational Regulation by L7Ae in Archaea

Abstract

L7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius. A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5′ untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea. Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin, suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria.

RNA stabilization in hyperthermophilic archaea

“…These L7Ae‐associated mRNAs encode for the ribosomal proteins S17e, L39e, L31e, LX, and L7Ae; the C/D box sRNA‐associated proteins Nop5 and fibrillarin; the methyltransferase TRM1; and the translation initiation factor IF‐6. The observed binding of L7Ae to its mRNA indicates that its translation can be regulated by a negative‐feedback loop mechanism . Lilley and collaborators solved the crystal structure of Archeoglobus fulgidus L7Ae bound to its mRNA 5ʹ UTR region.…”

“…These experiments identified more than hundred different RNA interactors of L7Ae, including 59 C/D box sRNAs, RNase P, and SRP RNA. In addition, 32 mRNA targets were discovered and potential k‐turn structures were identified in the vicinity of the coding sequences . These L7Ae‐associated mRNAs encode for the ribosomal proteins S17e, L39e, L31e, LX, and L7Ae; the C/D box sRNA‐associated proteins Nop5 and fibrillarin; the methyltransferase TRM1; and the translation initiation factor IF‐6.…”

“…In addition, 32 mRNA targets were discovered and potential k-turn structures were identified in the vicinity of the coding sequences. 41 These L7Ae-associated mRNAs encode for the ribosomal proteins S17e, L39e, L31e, LX, and L7Ae; the C/D box sRNA-associated proteins Nop5 and fibrillarin; the methyltransferase TRM1; and the 42 translation initiation factor IF-6. The observed binding of L7Ae to its mRNA indicates that its translation can be regulated by a negative-feedback loop mechanism.…”

“…The observed binding of L7Ae to its mRNA indicates that its translation can be regulated by a negative-feedback loop mechanism. 41 Lilley and collaborators solved the crystal structure of Archeoglobus fulgidus L7Ae bound to its mRNA 5ʹ UTR region. This interaction stabilizes the standard k-turn conformation and promotes ribosome binding site occlusion.…”

Multipartite genomes and the sRNome in response to temperature stress of an Arctic Pseudoalteromonas fuliginea BSW20308

“…High throughput RNA-seq has been widely used to investigate gene transcription under different treatments. RNA deep sequencing has also been recently applied to discover the sRNomes (the complete set of small RNAs in the organism) involved in gene regulation (Daume et al, 2017;Zorgani et al, 2016). Small RNAs (sRNAs) are usually noncoding RNA molecules of less than 500 nucleotides in length, and function as regulators involved in modulating a wide range of physiological responses such as stresses and pathogenesis (Waters and Storz, 2009).…”