Coordination of the Fe-S cluster biogenesis network by the sRNA RyhB in E. coli

Description of the data and file structure

We present evidence suggesting the sRNA RyhB directly regulates the hscBA-fdx-iscX section of the ISC-HSC operon, as well as the complete SUF operon particularly under conditions of iron (Fe) starvation. Strikingly, RyhB organizes a three-fold pattern of expression of the iscR-SUA-hscBA-fdx-iscX operon. The sRNA promotes (i) increased levels of iscR and (ii) constant expression of iscSUA, which encodes the scaffold for Fe-S cluster formation in the absence of Fe. However, the third part of the operon, hscBA-fdx-iscX, which encodes a chaperone that facilitates Fe-S cluster transfer to receptor Apo-proteins, is (iii) considerably repressed by RyhB. Additional western blots and enzymatic assays indicate that RyhB coordinates the HSC-dependent shift from HscBA-IscU group of proteins to IscA-dependent proteins. Furthermore, RyhB represses the entire sufABCDSE transcript under low Fe conditions, which partially impedes Fur derepression of the operon. Overall, our data suggests that the sRNA RyhB is essential for ensuring the adaptative expression of ISC-HSC machineries according to Fe levels and helps to switch to SUF machinery during Fe depletion conditions or oxidative stress.

Files and variables

File: Supplementary Data Prevost 2025.docx

Materials and Methods

     Strains and plasmids

     In vitro transcription

     Electrophoretic mobility shift assays

     Supplementary References

Supplementary Figures

Figure S1. Identification of new RyhB targets with MAPS. Visualization of MS2-RyhB and RyhB MAPS reads of (A) iscRSUA-hscBA-iscX-fdx or (B) sufABCDSE using the UCSC microbial genome browser (6). IntaRNA-predicted (hsc) or CopraRNA-predicted (suf) sites are represented below each graphical representation of RNA-seq reads. Related to Fig. 1 and Table 1.

Figure S2.  Identification and degradation of polycistronic mRNA targets by RyhB.  Northern blot analysis of polycistronic mRNA targets identified by MAPS. At an OD600nm=0.5 in LB, RyhB was overexpressed from pBAD-ryhB by the addition of 0.1% arabinose (ARA) in a (A) ΔryhB background (hcsB, hscA, iscX, iscR, and ryhB probes) or (B) ΔryhB Δfur background (sufA, sufB, sufC, sufE, and ryhB probes). 16S rRNA was used as loading control and Millenium marker as ladder.

Figure S3.  Description of all translational and transcriptional lacZ fusions used in this study for (A) iscRSUA-hscBA-fdx-iscX or (B) sufABCDSE operon. SD, Shine-Dalgarno sequence; -/+ from transcription start site. Related to Material and Methods.

Figure S4. Two sites in the 5’-UTR of hscBA mRNA are targeted by RyhB in vitro. Lead acetate (PbAc) probing of γ-hscBA with RyhB. γ-hscBA was incubated for 15 min in the absence (-) or in the presence (+) of RyhB (0.1μM) prior to addition of PbAc. Ctrl; non-reacted samples, OH; alkaline ladder, T1; RNase T1 ladder. Numbers on the left indicate nucleotide position relative to +1 transcriptional start site. Putative RyhB pairing site is indicated with a (A) purple line for pairing site 1 and a (B) blue line for the pairing site 2. AUG is indicated with green line. Related to Fig. 2D and E.

Figure S5. RyhB regulates hscBA through direct base pairing.  (A) In vitro RyhB pairing sites on the hscBA mRNA. Mutations introduced in RyhB to obtain RyhBGGAA are indicated in gray. Compensatory mutations introduced in hscB­A to obtain hscBAmutBS1 or hscBAmutBS2 are indicated in purple and blue, respectively. Electrophoretic mobility shift assay (EMSA) of (B) γ-hscBA:RyhB complex, top panel, and hscBA:RyhBGGAA, low panel, (C) γ-hscBAmutBS1:RyhB, and (D) γ-hscBAmutBS2:RyhB complex (5 nM : 0-500 nM, incubation 15 min at 37°C). (E) Densitometry analysis plot of the fraction of γ-hscBA, γ-hscBAmutBS1, or γ-hscBAmutBS2 bound to RyhB or RyhBGGAA. Data are representative of two independent experiments. (F) β-galactosidase assay of HscBA-LacZ with RyhB or RyhBGGAA induced by addition of 0.1% arabinose at OD600nm=0.1 Samples (N=3, mean ± SD) were taken at OD600nm=1.0. P= 0.0004, ns: P> 0.05, one-way ANOVA test. Related to Fig. 2D and E.

Figure S6.  Coding sequence of the sufAB mRNA is targeted by RyhB in vitro.  (A) Lead acetate (PbAc) probing of γ-sufAB with RyhB. γ-sufAB was incubated for 15 min without (-) or with (+) RyhB (0.1μM) prior to addition of PbAc. Ctrl, non-reacted samples; OH, alkaline ladder; T1, RNase T1 ladder. Numbers on the left indicate nucleotide position relative to the +1 transcriptional start site. Putative RyhB pairing site is indicated with a blue line. AUG is indicated with a green line. Related to Fig. 2F and G.

Figure S7. RyhB regulates sufAB through direct base pairing. (A) In vitro RyhB pairing site on the sufAB mRNA. Mutations introduced into RyhB to obtain RyhBmut are indicated in gray. Compensatory mutations introduced in sufAB to obtain sufABmut are indicated in purple and blue. Electrophoretic mobility shift assay (EMSA) of (B) γ-sufAB:RyhB complex, top panel, and sufAB:RyhBmut, low panel, (C) γ-sufABmut:RyhB and sufABmut:RyhBmut complex (5 nM : 0-500 nM, incubation 15 min at 37°C). (D) Densitometry analysis plot of the fraction of γ-sufAB and γ-sufABmut bound to RyhB or RyhBmut. Data are representative of two independent experiments. (E) β-galactosidase assay of SufAB-LacZ with RyhB or RyhBmut induced by addition of 0.1% arabinose at OD600nm=0.1 Samples (N=3, mean ± SD) were taken at OD600nm=1.0. P< 0.0001, ns: P> 0.05, one-way ANOVA test. 

Figure S8. Expression of RyhB, hscBA-fdx-iscX, and sufABCDSE in different conditions. (A).  Northern blot analysis of hscBA-fdx-iscX (hscA probe) and sufABCDSE (sufE probe) mRNAs in LB at an OD600nm of 0.5, in WT, ΔryhB, Δfur, and ΔfurΔryhB backgrounds. Both 16S rRNA and 5S rRNA were used as loading controls. Data are representative of two independent experiments. (B) Northern blot analysis of RyhB sRNA (OD=0.5) in M63 0,2% glucose without (-) and with FeSO4 added at 0.1 µM, 0.5 µM, or 1 µM, in a WT or ΔryhB background. 5S rRNA was used as a loading control. Data are representative of two independent experiments. Related to Fig. 4A, B, C, and D.

Figure S9. Fe-S biogenesis, scaffold, and carrier proteins (A). Simplified scheme of SUF Fe-S biogenesis under low iron conditions in WT background. Apo-IscR will increase through the action of RyhB and will activate sufABCDSE transcription. Concurrently, RyhB is expressed and degrades sufABCDSE mRNA to maintain iron homeostasis. (B) In the ΔryhB background SUF system will be highly expressed.

File: Supplemental Tables S1 S2 S3 Prevost 2025.xlsx

Description: 

Dataset S1. Target mRNAs enriched by RyhB-MS2 MAPS

Dataset S2. List of all strains and plasmids used in this study

Dataset S3. List of all oligonucleotides used in this study

Access information

Data was derived from the following sources:

We have used previously published data (GEO: GSE66519) and reanalyzed them (National Library of Medicine: PRJNA1273173) with the Fragments Per Million mapped reads (FPM) normalization method.