Article Figures & Data
Figures
- FIG 1
rG4 can be detected and visualized in vitro and in live bacterial cells. (A) rG4 was detected by the rG4-specific dye QUMA-1 in total RNAs. Tris-HCl buffer (10 mM, pH 7.4) with 100 mM KCl without QUMA-1 or with LiCl was used as the negative control. The fluorescence intensity at 670 nm was detected when the sample was excited at 555 nm in a Synergy 2 plate reader (BioTek). The relative fluorescence intensity value was normalized by using the ratio between fluorescence intensity value and the concentration of RNAs (per ng). (B) rG4 was detected by the rG4-specific dye QUMA-1 in live bacterial strains. The fluorescence intensity at 670 nm was detected when the sample was excited at 555 nm in a Synergy 2 plate reader (BioTek). The fluorescence intensity value was normalized by using the ratio between fluorescence intensity value and OD600 (per OD600). (C) rG4 structures were visualized in E. coli. (D) rG4 structures were visualized in P. aeruginosa. All the experiments were performed in at least three repetitions. Digital images were recorded by using an Eclipse Ni-E microscope (Nikon) with a 100× lens objective.
- FIG 2
rG4-seq revealed the rG4 functions in E. coli. (A) The percentage of rG4s in different rG4 subtypes. (B) rG4 density per kilobase in different regions of mRNA. Each gene was divided into three regions, 5′ UTR, CDS, and 3′ UTR. We normalized the length of each part of each gene to the corresponding bin. The size of each bin is 1; 0 to 1 means 5′ UTR, 1 to 2 means CDS region, and 2 to 3 means 3′ UTR. The region between two vertical red lines is the CDS region. The curve indicates the density of the RTS site at each relative position. As this graph shows, the density curve has two peaks and one valley in the middle, indicating that most of the RTS sites are located on two sides of the midline of the CDS region. (C) The functional classification of rG4 sites in E. coli.
- FIG 3
rG4 positively regulated the expression of HemL in E. coli. (A) mRNA transcripts harboring rG4 in hemL. (B) The mutation of rG4 in hemL CDS. (C) QUMA-1 specifically bound to the rG4 formed in hemL mRNA; the ligand-enhanced fluorescence spectra showed an enhanced fluorescence for the wild type under K+ compared to Li+ conditions. The rG4 mutant was used as a control. (D) ThT specifically bound to the rG4 in the hemL mRNA, producing an enhanced fluorescence signal for the wild type under the K+ conditions compared to the Li+ conditions. The rG4 mutant was used as a control. (E) The folded rG4 structure positively regulated the expression of hemL. All the experiments were performed in at least three repetitions. Significance is indicated as follows: *, P < 0.5; **, P < 0.01; ***, P < 0.001. Results represent means ± SD. (F) The folded rG4 structure positively regulated the protein expression of HemL. All the experiments were performed in at least three repetitions. EV, pAK1900 empty vector. (G) The schematic diagram of inserting hemL-rG4-WT into the 5′ UTR (−20 from ATG) of hemL-rG4-WT and hemL-ΔrG4-WT. (H) Immunoblot probed for the translation products of the HemL-5′ UTR (−20)-WT-rG4 and HemL-5′ UTR (−20)-Δ-rG4 constructs with rG4 inserted into the 5′ UTR of HemL. All the experiments were performed in at least three repetitions. **, P < 0.01. Results represent means ± SD.
- FIG 4
rG4-seq revealed the rG4 functions in P. aeruginosa. (A) The percentage of rG4s in different rG4 subtypes. (B) rG4 density per kilobase in different regions of mRNA. We plotted this graph by the same method described for Fig. 2B. The region between two vertical red lines is the CDS region. As this graph shown, RTS sites are mainly located in the middle part of the CDS region, especially in the position near the 3′ UTR. (C) The functional classification of rG4 sites in P. aeruginosa.
- FIG 5
rG4 positively regulated the functions of BswR in motility and biofilm formation in P. aeruginosa. (A) mRNA transcripts harboring rG4 in the bswR gene. (B) The mutation of rG4 in BswR CDS. (C) QUMA-1 specifically bound to the rG4 formed in bswR mRNA; the ligand-enhanced fluorescence spectra showed an enhanced fluorescence for the wild type under K+ compared to Li+ conditions. The rG4 mutant was used as a control. (D) ThT specifically bound to rG4 in the bswR mRNA, producing an enhanced fluorescence signal for the wild type under K+ conditions compared to Li+ conditions. The rG4 mutant was used as a control. (E) The folded rG4 structure positively regulated the expression of bswR in vivo. (F) rG4 positively regulated the protein expression of BswR. (G) Swarming motility measurement between overexpressed bswR-WT and bswR-ΔrG4 strains. (H) Biofilm production detection between overexpressed bswR-WT and bswR-ΔrG4 strains. All the experiments were performed in at least three repetitions. *, P < 0.5; **, P < 0.01; ***, P < 0.001. Results represent means ± SD. (I) The schematic diagram of inserting bswR-rG4-WT into the 5′ UTR (−20 from ATG) of bswR-rG4-WT and bswR-ΔrG4-WT, respectively. (J) Immunoblot probed for the translation products of the bswR-5′-UTR (−20)-WT-rG4 and bswR-5′-UTR (−20)-Δ-rG4 constructs with wild-type bswR-rG4 inserted into the 5′ UTR of BswR. All the experiments were performed in at least three repetitions. ***, P < 0.01. Results represent means ± SD.
Supplemental Material
FIG S1
RNase A-treated RNA samples led to complete loss of the fluorescent signal. As a negative control, there was no loss of signal upon DNase I treatment. (A) P. aeruginosa. (B) P. syringae. (C) E. coli. (D) Acinetobacter. (E) K. pneumoniae. (F) V. parahaemolyticus. (G) S. Typhimurium. (H) S. aureus. (I) Enterococcus. (J) Bacillus cereus. All the RNA samples were divided into 6 groups (RNA + QUMA-1, RNA + RNase A + QUMA-1, RNA + DNase I + QUMA-1, RNA, Tris-KCl buffer + QUMA-1, and Tris LiCl buffer + QUMA-1), respectively. Each group had three repetitions, and results represent means ± SD. The RNA samples were first treated by RNase A or DNase I in a Tris-KCl buffer, and the control samples also had the equal volume of Tris-KCl buffer added in a total of 100 μl of reaction mixture. After the treatment, 0.5 μM (final concentration) QUMA-1 was added, mixed well, and then incubated at room temperature for 2 min. The fluorescence intensity at 670 nm was detected when the sample was excited at 555 nm in a Synergy 2 plate reader (BioTek). Download FIG S1, TIF file, 0.8 MB.
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FIG S2
The rRNA samples showed no fluorescent signal. (A and B) rRNA of P. aeruginosa and E. coli was detected by QUMA-1 ligand-enhanced fluorescence assay. (C) The relative fluorescence intensity (per ng) of total RNA was higher than the total rRNA in both P. aeruginosa and E. coli. (D to G) The four synthetic E. coli 23S rRNA oligonucleotides (RlmF, 5′-CCCCAAACCGACAC-3′ and 5′-AGGUGCUCAGGU-3′; RlmJ, 5′-AACUCGCUGUG-3′ and 5′-AAGAUGCAGUGUACC-3′) showed no significant difference between KCl and LiCl conditions in the QUMA-1 ligand-enhanced fluorescence assay. Each group had three repetitions. Download FIG S2, TIF file, 0.7 MB.
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TABLE S1
(A) Whole-genome location analysis of rG4 sites from rG4-seq in E. coli. (B) Whole-genome location analysis of rG4 sites from rG4-seq in P. aeruginosa. (C) Comparisons between this study and that by Guo and Bartel (Science 353:aaf5371, 2016, https://doi.org/10.1126/science.aaf5371). Download Table S1, PDF file, 1.7 MB.
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FIG S3
The guanine (G) bases present in the third position in codons were replaced with adenine in the hemL and bswR rG4 region. Sequencing peaks of hemL-WT (A), hemL-ΔrG4 (B), bswR-WT (C), and bswR-ΔrG4 (D). Download FIG S3, TIF file, 0.4 MB.
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FIG S4
The mutated codons did not change the protein expression level compared with the wild-type hemL-WT and bswR-WT lux reporters. (A) Three mutations in the alanine codon (CCG) in hemL-WT (non-rG4 region) by replacing the G with A (A is highlighted in yellow in the table). (B) Three mutations in the glutamine codon (CAG) in hemL-WT (non-rG4 region) by replacing the G with A (A is highlighted in yellow in the table). (C) Three mutations in the proline codon (GCG) in hemL-WT (non-rG4 region) by replacing the G with A (A is highlighted in yellow in the table). (D) Three mutations in the glutamine codon (CAG) in bswR-WT (non-rG4 region) by replacing the last G with A (A is highlighted in yellow in the table). (E) Three mutations in the leucine codon (CTG) in bswR-WT (non-rG4 region) by replacing the G with A (A is highlighted in yellow in the table). All the experiments were performed in at least three repetitions, ***, P < 0.01. Results represent means ± SD. Download FIG S4, TIF file, 0.7 MB.
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FIG S5
CD assay showed presence of rG4 in hemL and bswR mRNA. (A and C) The CD spectrum of the wild-type oligonucleotide (from hemL or bswR) was monovalent ion dependent and showed a negative peak at ∼240 nm and positive peak at ∼263 nm under K+ conditions but not under Li+ conditions, suggesting the formation of parallel topology. (B and D) The CD spectrum of rG4-mutant oligonucleotide (from hemL or bswR) was monovalent ion independent and showed no sign of rG4 formation under both lithium and potassium ion conditions, due to the positive peak at 270 nm and a negative peak at 244 nm. Download FIG S5, TIF file, 0.3 MB.
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FIG S6
The folded rG4 structure affected the transcriptional level of hemL while it did not affect the bswR mRNA level. (A) The folded rG4 structure positively regulated the transcriptional level of hemL. All the strains were cultured at 37°C and 220 rpm overnight in 2 ml LB supplemented with 60 μg/ml carbenicillin and then inoculated into 2 ml fresh LB (1:100 dilution) until OD600 reached 0.6. All the experiments were performed in at least three repetitions. **, P < 0.01. The results represent means ± SD. (B) The folded rG4 structure did not affect the mRNA level of bswR. All the strains were cultured at 37°C and 220 rpm overnight in 2 ml LB supplemented with 150 μg/ml carbenicillin and inoculated into 2 ml fresh LB (1:100 dilution) until OD600 reached 0.6. All the experiments were performed in at least three repetitions. The results represent means ± SD. NS, no significant difference. Download FIG S6, TIF file, 0.2 MB.
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FIG S7
The rG4 formed in the 3′ UTR of hemL and bswR did not affect their protein expression level. (A) The wild-type HemL-rG4 sequence was moved to the 3′ UTR of hemL-WT-FLAG or hemL-ΔrG4-FLAG. (B) Western blotting detected the protein expression of hemL-WT-FLAG and hemL-ΔrG4-FLAG containing the wild-type hemL-rG4. The total lysate of each strain was loaded with equal quantity. All the experiments were performed in at least three repetitions. (C) The wild-type BswR-rG4 sequence was moved to the 3′ UTR of bswR-WT-FLAG or bswR-ΔrG4-FLAG. (D) Western blotting detected the protein expression of bswR-WT-FLAG and bswR-ΔrG4-FLAG containing the wild-type bswR-rG4. The total lysate of each strain was loaded with an equal quantity. All the experiments were performed in at least three repetitions. Download FIG S7, TIF file, 0.3 MB.
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TABLE S2
Strains, plasmids, and primers used in this study. Download Table S2, PDF file, 0.7 MB.
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TEXT S1
Supplemental references. Download Text S1, PDF file, 0.2 MB.
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