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. 2018 Nov 13;115(46):11814-11819.
doi: 10.1073/pnas.1809663115. Epub 2018 Oct 31.

In Vivo 3'-to-5' Exoribonuclease Targetomes of Streptococcus pyogenes

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Free PMC article

In Vivo 3'-to-5' Exoribonuclease Targetomes of Streptococcus pyogenes

Anne-Laure Lécrivain et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5' or 3' end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3'-to-5' exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3'-to-5' exoRNase for RNA turnover in S. pyogenes In particular, PNPase is responsible for the degradation of regulatory elements from 5' untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3'-to-5' exoRNases.

Keywords: 3′-end sequencing; 3′-to-5′ exoRNase; 5′-end sequencing; RNA degradation; Streptococcus pyogenes.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of 3′-to-5′ exoRNase processing sites. (A) Number of transcript end positions more abundant in the WT (red) or in the Δrnase (green). (B) Schematic representation of RNA sequencing profiling with annotated 3′-to-5′ exoRNase trimming start (more abundant in the Δrnase) and trimming stop (more abundant in the WT).
Fig. 2.
Fig. 2.
YhaM trims a few nucleotides of transcript ends generated by transcription termination or endoRNase cleavages. The YhaM sequence logo is shown based on the sequence alignment of transcript 3′ ends more abundant in WT compared with ΔyhaM, located at either a maximal distance of 10 nt (A) or further from 10 nt (C) from predicted terminators. The average of the minimal free energy (25-nt length sequences) at each position surrounding YhaM processing sites (vertical dotted line) is shown. (B and D) Models for YhaM in vivo processing of transcript 3′ ends. YhaM trims 3 nt, on average, of transcripts harboring intrinsic terminators (B) or generated from the processing by an endoRNase (scissors) (D). (E) Validation of YhaM short processivity in vivo by Northern blotting analyses in WT and ∆yhaM (also SI Appendix, Fig. S4).
Fig. 3.
Fig. 3.
PNPase degrades intermediate decay fragments. (A) Model for PNPase in vivo degradation activity. The intermediate decay fragments are generated by one or several endoRNases (scissors) and are fully degraded by PNPase (pacman) in the WT. (B) Model for in vivo PNPase degradation of 5′ UTRs and Northern blotting analyses of T-boxes and putative riboswitches in WT and ∆pnpA. An endoRNase (scissors) cleaves in the 5′ UTR of the transcript, further degraded by PNPase (pacman) up to the TSS in the WT. (C) Northern blotting analysis of dpr 5′ UTR in WT and ∆pnpA. In B and C, RNA stability was investigated up to 8 or 30 min after addition of rifampicin to the medium to stop transcription (RNA sequencing coverage, loading control, and half-life measurements are shown in SI Appendix, Fig. S8). Regulatory 5′ UTR full lengths (FL) are indicated on the left side of the blots. TPP, thiamine pyrophosphate.
Fig. 4.
Fig. 4.
glyQ T-box decay relies on RNase R and PNPase. RNA stability of the glyQ T-box was investigated in WT, ∆pnpA, and ∆rnr up to 8 min after addition of rifampicin to the medium to stop transcription. The full-length (FL) T-box and the intermediate fragment of degradation (1) are indicated on the left side of the blot (half-life measurements are shown in SI Appendix, Fig. S8B).

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References

    1. Hui MP, Foley PL, Belasco JG. Messenger RNA degradation in bacterial cells. Annu Rev Genet. 2014;48:537–559. - PMC - PubMed
    1. Apirion D. Degradation of RNA in Escherichia coli. A hypothesis. Mol Gen Genet. 1973;122:313–322. - PubMed
    1. Andrade JM, Pobre V, Silva IJ, Domingues S, Arraiano CM. The role of 3′-5′ exoribonucleases in RNA degradation. Prog Mol Biol Transl Sci. 2009;85:187–229. - PubMed
    1. Oussenko IA, Abe T, Ujiie H, Muto A, Bechhofer DH. Participation of 3′-to-5′ exoribonucleases in the turnover of Bacillus subtilis mRNA. J Bacteriol. 2005;187:2758–2767. - PMC - PubMed
    1. Vincent HA, Deutscher MP. Substrate recognition and catalysis by the exoribonuclease RNase R. J Biol Chem. 2006;281:29769–29775. - PubMed

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