Critical factors for precise and efficient RNA cleavage by RNase Y in Staphylococcus aureus

Abstract

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.

Fig 1. RNase Y cleaves the model transcripts.

A) Sequence alignment of S. aureus and B. subtilis transcripts with the published RNase Y cleavage sites indicated [5,19,21]. B) Schematic representation of the Sa-gapR, Sa-glnR, Bs-cggR and Bs-glnR operons in S. aureus and B. subtilis. TSS indicates the transcription start site, and the native RNase Y cleavage sites are indicated by blue dotted lines. Coloured lines show the regions cloned in front of the transcription terminator (TT) of the pEB01 vector (green). The location of the P1 probe is indicated. C) The four constructs from panel B were transformed into the WT and ΔY strains, and RNase Y cleavage was detected by Northern blotting using the P1 probe to detect the full-length (black arrowhead) and the smaller RNase Y cleavage product (grey arrowhead). A probe against 5S rRNA (5S) is used as loading control. pSaGap was also introduced into the S. aureus strain BsY, which expresses B. subtilis RNase Y from the S. aureus rny promoter at the native S. aureus chromosomal rny locus. D) EMOTE data for cleavage of the four wild-type vector constructs. Columns indicate the proportion of detected RNA molecules with 5’-ends at each position within the shown window (the sum of all columns is 1). WT (black columns): S. aureus WT, BsY (white columns): S. aureus with SaRNase Y coding sequence substituted with the BsRNase Y coding sequence. EMOTE data from the S. aureus ΔY strain is shown in S3A Fig.

Fig 2

S. aureus RNase Y complements a B. subtilis Δrny strain. A). Northern blots of total B. subtilis RNA, showing the disappearance of the uncleaved transcripts (glnRA and atpIBE, black arrows) after rifampicin addition, accompanied by the appearance of the downstream cleavage products (glnA and atpBE, grey arrows). “WT”: B. subtilis wild-type strain. “Δrny”: B. subtilis with the RNase Y gene deleted. “Δrny + SaY”: Δrny strain with S. aureus RNase Y expressed from the amyE locus. “Δrny + BsY”: Δrny strain with B. subtilis RNase Y expressed from the amyE locus. 16S rRNA was used as loading control. B). Half-lives of the two full-length transcripts (glnRA and atpIBE) in the four B. subtilis strains. B. subtilis Δrny was the only strain where the half-lives were long enough to be measured accurately. C). RNA decay curves of glnRA and atpIBE transcripts in the B. subtilis Δrny strain, showing the percentage of remaining RNA at the different time-points.

Fig 3. The upstream G promotes precise cleavage.

A) Northern blots using probe P1 on transcripts from wild-type pSaGap, and versions where the G upstream of the cleavage site has been mutated to U, C and A (pSaGap[G258U], pSaGap[G258C] and pSaGap[G258A]), respectively. Each plasmid was introduced into the WT, ΔY and BsY strains. The full-length transcript and the downstream cleavage product are indicated by black and grey arrowheads, respectively. A 5S rRNA probe was used as loading control. B) EMOTE analysis of the exact cleavage position for S. aureus RNase Y. The sequence below each graph shows the nucleotides surrounding the natively preferred cleavage site (blue dotted line). The mutated nucleotide for each plasmid is shown in red. Columns indicate the proportion of detected RNA molecules with 5’-ends at each position within the shown window (the sum of all columns is 1). Two biological replicates were analysed, and the error-bars indicate the standard deviation. C) Same as panel B, but where B. subtilis RNase Y has replaced S. aureus RNase Y at the native rny locus.

Fig 4. Deleting sectors of the pSaGap transcript in the region surrounding the RNase Y cleavage.

A) Predicted secondary structures of positions +117 to +504 in the pSaGap vector transcript. We divided the region into six sectors (I-VI), shown in different colours and with black and grey lines below the nucleotide sequence. The native RNase Y cleavage site (Y-cut) is located between sector II and sector III, and the positions of the stop codon of gapR and the start codon of gapA are indicated. Sectors II, III and IV (marked with yellow, green and cyan background, respectively) are highly important for RNase Y cleavage. Sector I (light green) can be deleted without affecting RNase Y cleavage. Only one of sectors V and VI (orange and blue, respectively) can be deleted without abolishing cleavage. The KpnI and AcsI restriction sites used for cloning the +177 to +504 gap operon fragment are shown in red. The predicted minimum free energy for each secondary structure is indicated, but it should be noted that translation will continuously disrupt the secondary structures in sectors I, V and VI. B) Northern blots of the pSaGap transcripts deleted of the various sectors. Black arrowheads denote full-length transcripts and grey arrowheads denote the downstream cleavage products. 5S rRNA was used as loading control. C) EMOTE data showing the exact location of 5’ ends in the strains used for Northern blotting in panel B. The green “-4” indicates the 4 nt upstream shift of the cleavage position in the pSaGap[ΔIII] transcript. EMOTE data is presented as number of detected molecules with a given 5’-end divided by the total number of detected molecules within the chosen window. EMOTE data from the ΔY strain can be found in S3B Fig. Arrows indicate that the EMOTE data was obtained for the vector where both sector I and sector VI were deleted (see panel D). D) EMOTE data showing the exact location of 5’ ends in the strain carrying the vector where both sector I and sector VI were deleted (pSaGap[ΔIΔVI]), EMOTE data from the ΔY strain can be found in S3C Fig.

Fig 5. Generating an RNase Y cleavage site in an exogenous transcript.

A) Overview of the two vectors pfliM and pfliM::II-V, where regions from S. aureus are in purple, regions from E. coli are in light blue and regions in green are from the vector backbone. TT indicates the transcription terminator and P1 shows the location where the P1 probe hybridises. gapII-V indicates the 103 bp from sector II to V in the Sa-gapR operon. B) Northern blot of the fliM transcript. The full-length uncleaved RNA is indicated by a black arrowhead. 5S rRNA was used as loading control. C) Northern blot of the fliM::II-V transcript, where the uncleaved RNA and downstream cleavage products are indicated by black and grey arrowheads, respectively. 5S rRNA was used as loading control. D) EMOTE data for WT and ΔY strains carrying the pfliM::II-V vector. The data from the WT strain (black) shows two distinct peaks at the native RNase Y cleavage site (Y) as well as two nucleotides upstream, whereas the data from the ΔY strain (grey) only shows a single peak two nucleotides upstream of the native RNase Y site. EMOTE data is presented as number of detected molecules with a given 5’-end divided by the total number of detected molecules within the chosen window.

Fig 6. RNase Y cleavage position depends on hairpin composition.

A) Reverse transcription error rates, normalised with error rates from mock-treated RNA samples, for individual nucleotides around the cleavage site in the gap transcript (blue Y). G and U are not affected by DMS and have an average normalised error rate of 1.29 (white columns). A and C (blue and orange columns, respectively) have increased error rates when unprotected by duplex formation. The sectors are indicated below the graph. Standard deviation bars come from three different RNA preparation. B) The predicted S. aureus gapR hairpin structure immediately downstream of the cleavage site, correlated with the data from panel A. Protected and unprotected bases are shown in red and green, respectively. Yellow indicates an intermediate signal for bases that presumably are protected in some RNA molecules but not in others. C) B. subtilis Bs-cggR hairpin structure immediately downstream of the cleavage site, with the RNase Y cleavage position shown in green. D) The hairpin structure immediately downstream of the cleavage site in the pSaGap construct was exchanged with the hairpin from Bs-cggR to obtain pSaGap[ΔIV::cggHP]. Mutated nucleotides are shown in red. E) Northern blot to detect the RNase Y-dependent cleavage of the pSaGap[ΔIV::cggHP] transcript. F) EMOTE signal from the pSaGap[ΔIV::cggHP] transcript. The native cleavage positions of pSaGap and pBsCgg are shown in blue and green, respectively. Mutated nucleotides are in red. EMOTE data is presented as number of detected molecules with a given 5’-end divided by the total number of detected molecules within the chosen window. EMOTE data from the ΔY strain can be found in S3D Fig.

Fig 7. Hairpin G-C base-pairing promotes cleavage in pSaGap, pBsCgg and pSaGln transcripts.

A). Putative secondary structures of the pSaGap sector IV hairpin with mutated G-C base-pair (mutated nucleotides in red). Northern blots revealed efficient cleavage when the G-C pair was inverted (pSaGap[G268C,C276G]) but only weak cleavage when either the G or the C were mutated (green arrowheads). EMOTE data for these constructs can be found in S7 Fig. B). Putative secondary structures of the pBsCgg downstream hairpin with mutated G-C base-pairs (mutated nucleotides in red). Northern blots revealed efficient cleavage by S. aureus RNase Y when the G-C pairs were inverted (pBsCgg[GtoC,CtoG]) but only weak cleavage when the three Gs were mutated to Cs (green arrowhead). C). Putative secondary structures of the pSaGln downstream hairpin with mutated G-C base-pairs (mutated nucleotides in red). Northern blots revealed efficient cleavage when the G-C pairs were inverted (pSaGln[GtoC,CtoG]) but only weak cleavage when CGUGG was mutated to GCUCC (pSaGln[GtoC]). EMOTE data for these constructs can be found in S7 Fig.

Fig 8. Modifications of the putative hairpin stem in sector IV.

A) Putative secondary structures of sector IV and derivatives. pSaGap: shown with the upstream and downstream hairpin “arms” in yellow and orange, respectively. pSaGap[InvStem]: the “arms” of the hairpin inverted. pSaGap[InvStemGC]: the “arms” of the hairpin, excluding the G-C base-pair, inverted. pSaGap[U280A]: the third nucleotide in the downstream “arm” mutated to an A, disrupting the potential base-pairing with A264 of the upstream “arm”. pSaGap[A264U]: third nucleotide of the upstream “arm” mutated to U, disrupting the potential base-pairing with U280 of the downstream “arm”. pSaGap[A264U,U280A]: Both A264 and U280 are mutated as above, resulting in a reconstituted putative base-pairing. The native RNase Y cleavage position is indicated with a blue dotted line. B) EMOTE cleavage profiles of the mutants illustrated in panel A. EMOTE data is presented as number of detected molecules with a given 5’-end divided by the total number of detected molecules within the chosen window. The native RNase Y cleavage position is indicated with a blue dotted line. EMOTE data from the ΔY strain can be found in S3E Fig C) Northern blots of pSaGap, pSaGap[InvStem] and pSaGap[InvStemGC]. Black and grey arrowheads indicate the positions of full-length transcripts and cleavage product, respectively. The cleavage products of pSaGap[InvStem] and pSaGap[InvStemGC] are barely detectable.

Fig 9. The three nucleotides downstream of the cleavage position strongly influence cleavage efficiency.

A) The putative hairpin in the Sa-gapR transcript, with the randomly mutagenised trinucleotide marked with red N’s. The native RNase Y cleavage site is indicated by a light blue Y. B) The relative cleavage efficiency, measured with EMOTE, of each of the 64 possible combinations for the three nucleotides downstream of the RNase Y cleavage site. The wild-type trinucleotide (AGA) is shown in green and is set to 1 on the arbitrary scale. All other sequences were compared to the wild-type cleavage efficiency, using T-tests with correction for multiple testing. * = p<0.05, ** = p<0.01 and *** = p<0.001. The experiment was repeated four times. Trinucleotides mentioned in the text are highlighted with grey arrows. C) Northern blot of constructs with selected trinucleotides in sector III (pSaGap[IIIGGA], pSaGap[IIIAUU], pSaGap[IIICUA] and pSaGap[IIICGA]) showing the lack of cleavage with AUU. D) The potential for prolonging the hairpin stem when the trinucleotide is either AUU or GUU. R indicates a purine.

Fig 10. Schematic summary of elements defining an RNase Y cleavage site.

Blue arrows denote effect that were identified in the SaGap transcript and green arrows denote effect that were also identified in other transcripts. A hairpin structure is often found a few nucleotides after the RNase Y cleavage position. Cleavage efficiency is defined by the primary sequence of the three nucleotides after the cleavage position as well duplex-formation in the downstream hairpin. The region upstream of the cleavage (sector II) is also important for efficiency, but its sequence appears to be unimportant. Cleavage positioning is defined by a G right before the cleavage position as well as distance to sequence elements within the stem of the downstream hairpin.