New molecular insights into an archaeal RNase J reveal a conserved processive exoribonucleolysis mechanism of the RNase J family

Abstract

RNase J, a prokaryotic 5'-3' exo/endoribonuclease, contributes to mRNA decay, rRNA maturation and post-transcriptional regulation. Yet the processive-exoribonucleolysis mechanism remains obscure. Here, we solved the first RNA-free and RNA-bound structures of an archaeal RNase J, and through intensive biochemical studies provided detailed mechanistic insights into the catalysis and processivity. Distinct dimerization/tetramerization patterns were observed for archaeal and bacterial RNase Js, and unique archaeal Loops I and II were found involved in RNA interaction. A hydrogen-bond-network was identified for the first time that assists catalysis by facilitating efficient proton transfer in the catalytic center. A conserved 5'-monophosphate-binding pocket that coordinates the RNA 5'-end ensures the 5'-monophosphate preferential exoribonucleolysis. To achieve exoribonucleolytic processivity, the 5'-monophosphate-binding pocket and nucleotide +4 binding site anchor RNA within the catalytic track; the 5'-capping residue Leu37 of the sandwich pocket coupled with the 5'-monophosphate-binding pocket are dedicated to translocating and controlling the RNA orientation for each exoribonucleolytic cycle. The processive-exoribonucleolysis mechanism was verified as conserved in bacterial RNase J and also exposes striking parallels with the non-homologous eukaryotic 5'-3' exoribonuclease, Xrn1. The findings in this work shed light on not only the molecular mechanism of the RNase J family, but also the evolutionary convergence of divergent exoribonucleases.

aCPSF1 controlled archaeal transcription termination: a prototypical eukaryotic model

“…The 5′-3′ eukaryotic exoribonuclease, yeast Rat1 or human Xrn2, has been determined to be recruited to the 5′-end of the cleaved downstream RNA that are still loaded by RNAP, and rapidly ‘chew’ its way along the RNA to catch up and dismantle the preceding RNAP, therefore working as a “torpedo” for eukaryotic RNAP dissociation and termination 8, 9, 12, 29 . Although no homologs of Rat1 or Xrn2 were found in Archaea, 5′-3′ exoribonucleases aRNase J, aCPSF2 and even aCPSF1 itself were indeed identified 19, 20, 38, 39 . They may play comparable function in aRNAP dissociation and termination in Archaea (Figure 6e).…”

“…7) and the intrinsic nature of endoribonuclease and exoribonuclease activities 19–21 could support its role in regular RNA turnover. As no exosome subunits were identified in methanococcal species 40 , aCPSF1 as well as other ribonucleases of β-CASP family were supposed to play major roles in RNA turnover 39, 41, 42 . While this work provides evidence for the involvement of aCPSF1 in RNA degradation.…”

“…Plasmid pSB1s carrying His-SUMO- aCPSF1 was then transformed into E. coli BW25113 and the transformants were induced by 0.1% L-arabinose. After a 16 h-induction at 22°C, cells were harvested, and the protein was purified as described previously 39 . Next, His-SUMO tags were removed by incubated with His-tagged Ulp1 protease at 30°C for 2h, and Mmp -aCPSF1 protein was purified through a His-Trap HP column to remove His-tagged SUMO and His-tagged Ulp1.…”

Plant Ribonuclease J: An Essential Player in Maintaining Chloroplast RNA Quality Control for Gene Expression

“…The domain structure, length, amino acid sequences, and catalytic mechanism of RNase J and related CPSF proteins are mostly conserved. These proteins contain the seven signature motifs of the metallo-β-lactamase (MBL) and β-CASP domains, I (D), II (HxHxDA), III (H), IV (D), A (D/G), B (H), and C (H) (Figure 1), that together participate in the coordination of two catalytic Zn 2+ ions [44,45]. RNase J is active as a dimer or a tetramer, and the amino acid sequence responsible for oligomerization is located at the C-terminus.…”

“…Most RNase Js display both 5′→3′ exonucleolytic and endonucleolytic activities when tested in vitro. Chlamydomonas reinhardtii RNase J (CrRNase J) is one of only three family members reported to exhibit exclusively Crystal structures of bacterial and archaeal RNase J predict a combination of 5 →3 exonuclease and endonuclease activities, both of which have been observed biochemically in vitro, with the exonuclease activity being dependent on the 5 end phosphorylation state [44][45][46][47][48]. Most RNase Js display both 5 →3 exonucleolytic and endonucleolytic activities when tested in vitro.…”