In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.
In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.
RNA binding protein (RBP)-based post-transcriptional regulation, specifically characterization of the interaction between RBPs and RNA, has been studied extensively in recent decades. There are multiple examples of translational down-regulation in bacteria originating from RBPs inhibiting, or directly competing with, ribosome binding1,2,3. In the field of synthetic biology, RBP-RNA interactions are emerging as a significant tool for the design of transcription-based genetic circuits4,5. Therefore, there is an increase in demand for characterization of such RBP-RNA interactions in a cellular context.
The most common methods for studying protein-RNA interactions are the electrophoretic mobility shift assay (EMSA)6, which is limited to in vitro settings, and various pull-down assays7, including the CLIP method8,9. While such methods enable the discovery of de novo RNA binding sites, they suffer from drawbacks such as labor-intensive protocols and expensive deep sequencing reactions and may require a specific antibody for RBP pull-down. Due to the susceptible nature of RNA to its environment, many factors can affect RBP-RNA interactions, emphasizing the importance of interrogating RBP-RNA binding in the cellular context. For example, we and others have demonstrated significant differences between RNA structures in vivo and in vitro10,11.
Based on the approach of a previous study12, we recently demonstrated10 that when placing pre-designed binding sites for the capsid RBPs from the bacteriophages GA13, MS214, PP715, and Qβ16 in the translation initiation region of a reporter mRNA, reporter expression is strongly repressed. We present a relatively simple and quantitative method, based on this repression phenomenon, to measure the affinity between RBPs and their corresponding RNA binding sites in vivo.
1. System Preparation
2. Experiment Setup
NOTE: The protocol presented here was performed using a liquid-handling robotic system in combination with an incubator and a plate reader. Each measurement was carried out for 24 inducer concentrations, with two duplicates for each strain + inducer combination. Using this robotic system, data for 16 strains per day with 24 inducer concentrations was collected. However, if such a device is unavailable, or if fewer experiments are necessary, these can easily be done by hand using an 8-channel multi-pipette and adapting the protocol accordingly. For example, preliminary results for four strains per day with 12 inducer concentrations and four time-points were acquired in this manner.
3. Preliminary Results Analysis
4. Dose Response Function Fitting Routine and KRBP Extraction
The presented method utilizes the competition between an RBP and the ribosome for binding to the mRNA molecule (Figure 1). This competition is reflected by decreasing mCherry levels as a function of increased production of RBP-mCerulean, due to increasing concentrations of inducer. In the case of increasing mCerulean fluorescence, with no significant changes in mCherry, a lack of RBP binding is deduced. Representative results for both a positive and a negative strain are depicted in Figure 2. In Figure 2A, the OD, mCherry, and mCerulean channels are presented as a function of time and inducer over a range of four hours, with T0 = 1 h and Tfinal = 3.5 h. In Figure 2B, averaged mCerulean fluorescence (top) and mCherry rate of production (bottom) are presented as a function of inducer concentration, for the two example strains. As can be seen, the results for a positive strain display a clear down-regulatory effect in the mCherry rate of production (Figure 2B,C), which translates into a significant non-zero value of KRBP (Figure 2D). For the positive strain, the fitting procedure yielded the following values: KRBP = 394.6 a.u., Kunbound = 275.6, n = 2.1, C = 11.2 a.u., and R2 = 0.93. After normalization by the maximal mCerulean fluorescence, the KRBP value was 0.24. For the negative strain, a lack of distinct response was observed (Figure 2C), and no KRBP value was extracted (Figure 2D).
In Figure 3, we present the results of this assay for two phage coat RBPs, PP7 and MS2, on several mutated binding sites, at different locations within the initiation region of the mCherry mRNA. The results are roughly classified into three kinds of responses (Figure 3A): strains exhibiting a down-regulatory effect at a low mCerulean level, reflecting a low KRBP value (high binding affinity); strains exhibiting down-regulatory effect at either intermediate or high mCerulean levels, reflecting a high KRBP value (intermediate or low affinity); and strains exhibiting no distinct response to rising levels of mCerulean, reflecting a higher KRBP value than the maximum RBP concentration in the cell (no detectible binding affinity). Figure 3B presents the minimal KRBP value computed for every RBP−binding-site combination based on all combinations of the two RBPs and ten binding-sites at different positions. The binding sites include a negative control (no binding site), non-matching binding sites, and a positive control ― the native binding site for each RBP (PP7-wt for PP7 coat protein [PCP], and MS2-wt for MS2 coat protein [MCP]). The results match the predictions, as both RBPs present a high affinity for their positive controls, and a non-detectible binding affinity for the negative controls. Additionally, previous studies using these two RBPs27,28 have observed that they are orthogonal, which is clearly conveyed in the heatmap presented: both MCP and PCP do not bind the native site of the other RBP. Furthermore, the mutated binding sites present varying results, where some binding sites displayed a similar level of affinity as that of the native site, such as PP7-mut-1, PP7-mut-2, and MS2-mut-3, while others displayed a significantly lower affinity, such as PP7-mut-3 and MS2-mut-2. Thus, the assay presented a quantitative in vivo measurement of the binding affinity of RBPs, yielding results that are comparable to those of past experiments with these RBPs.
Since the assay is based on repression of the mCherry gene, a viable mCherry signal is required. Therefore, when designing the binding site cassette, there are two design rules to keep in mind. First, the open reading frame (ORF) of the mCherry should be kept. Since the binding-site length can vary, inserting it into the gene can cause a shift of one or two bases from the original mCherry ORF. Therefore, if needed (Figure 4A), insert one or two bases immediately downstream to the binding site. For example, a binding site that is 20-base long, with a δ of two bases, will yield an addition of 22 bases to the mCherry gene. To keep the ORF, we need to add two bases, for a total of 24 bases. The second design rule is to avoid insertions of stop codons into the mCherry ORF. Some binding sites, as the MS2-mut-2 (Figure 4B, inset), contain stop codons when positioned in one or more of the three possible ORFs. Such an example is illustrated in Figure 4A, where the binding site contained a stop codon that is in-frame with the mCherry ORF only when no bases are added. As can be seen in the dose-response curve for that position (Figure 4B), mCherry production rate was undetectable, thus the binding affinity could not be measured.
A closer look at Figure 4B demonstrates the effect of the spacing δ on mCherry production. For instance, for δ = 4, basal production rate was a factor of six more than those for δ = 5, ensuring a higher fold-repression effect. For δ = 14, however, the basal production levels were too low to observe a down-regulatory effect.
Figure 1: Overview of system design and cloning steps. Illustration of the cassette design for the binding site plasmid (left) and RBP-mCerulean plasmid (right). The next step is consecutive transformations of both plasmids into competent E. coli cells, with RBP plasmids first. Double-transformants are then tested for their mCherry expression levels in increasing inducer concentrations; if the RBP binds to the binding site, mCherry levels decline as a function of mCerulean (gray bubble). Please click here to view a larger version of this figure.
Figure 2: Analysis scheme. (A) Three-dimensional (3D) plots depicting raw OD levels (top), mCerulean fluorescence (middle), and mCherry fluorescence (bottom) as a function of time and inducer concentration, for a positive strain. (B) Top: mCerulean steady-state expression levels for each inducer concentration is computed by dividing each fluorescence level by the respective OD and averaging over all values in the 2−3 h exponential growth time window for both the positive (left) and negative (right) strains. Bottom: mCherry production rate computed according to Eq. 3 for time-points 2−3 h after induction. (C) mCherry production rate plotted as a function of mean mCerulean fluorescence averaged over two biological duplicates for two strains. Error bars are standard deviation of both mCherry production rate and averaged mCerulean fluorescence acquired from at least two replicates. (D) Fit for KRBP using the fitting formula in Eq. 4 shown for the positive strain (left), exhibiting a specific binding response. For the negative strain (right), no KRBP value was extracted. Data is shown in duplicate. This figure has been adapted with permission from Katz et al.10. Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
Figure 3: Representative final results. (A) Normalized dose-response curves for thirty different strains based on two RBPs and ten binding sites at different locations. Three types of responses are observed: high affinity, low affinity, and no affinity. (B) Quantitative KRBP results for two RBPs (MCP and PCP) with five different binding site cassettes (listed). All RBP−binding-site strains were measured in duplicate. This figure has been adapted with permission from Katz et al.10. Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
Figure 4: Example design and results for MCP with a mutant binding site. (A) Design illustration of the binding site cassettes in four different locations. Cassette including the ribosome binding site, start codon for the mCherry, δ spacer bases, the binding site tested, one or two bases to maintain the ORF, and the rest of the mCherry gene. Red stars indicate a stop codon. (B) Dose-response curves for MCP with a mutant binding site at four different locations. Inset: the sequence of the tested mutated binding site. Results presented are for duplicates of each strain. Please click here to view a larger version of this figure.
Name | Binidng site location, A in AUG = 1 | Binding site sequence (RBS for controls) | Site: ATG to second mCherry codon GTG Controls: RBS to second mCherry codon GTG |
Source |
MS2_wt_d5 | 5 | acatgaggattacccatgt | atgcacatgaggattacccatgtcgtg | Gen9 Inc. |
MS2_wt_d6 | 6 | acatgaggattacccatgt | atggcacatgaggattacccatgtgtg | Gen9 Inc. |
MS2_wt_d8 | 8 | acatgaggattacccatgt | atggcgcacatgaggattacccatgt cgtg |
Gen9 Inc. |
MS2_wt_d9 | 9 | acatgaggattacccatgt | atggcgccacatgaggattacccatg tgtg |
Gen9 Inc. |
MS2_U(-5)C_d8 | 8 | acatgaggatcacccatgt | atgcacatgaggatcacccatgtgg tg |
Gen9 Inc. |
MS2_U(-5)C_d9 | 9 | acatgaggatcacccatgt | atggcacatgaggatcacccatgtg tg |
Gen9 Inc. |
MS2_U(-5)C_d8 | 8 | acatgaggatgacccatgt | atgcacatgaggatgacccatgtgg tg |
Gen9 Inc. |
MS2_U(-5)G_d9 | 9 | acatgaggatgacccatgt | atggcacatgaggatgacccatgtg tg |
Gen9 Inc. |
MS2_struct_d9 | 9 | cacaagaggttcacttatg | atggccacaagaggttcacttatgg tg |
Gen9 Inc. |
MS2_struct_d8 | 8 | cacaagaggttcacttatg | atgccacaagaggttcacttatggg tg |
Gen9 Inc. |
PP7wt_d5' | 5 | taaggagtttatatggaaaccctta | atgctaaggagtttatatggaaacc cttacgtg |
Gen9 Inc. |
PP7wt_d6' | 6 | taaggagtttatatggaaaccctta | atgaataaggagtttatatggaaac ccttagtg |
Twist Bioscience |
PP7wt_d8' | 8 | taaggagtttatatggaaaccctta | atgaacataaggagtttatatggaa acccttacgtg |
Twist Bioscience |
PP7wt_d9' | 9 | taaggagtttatatggaaaccctta | atgaacaataaggagtttatatgga aacccttagtg |
Twist Bioscience |
PP7_USLSBm_d6 | 6 | taaccgctttatatggaaagggtta | atggctaaccgctttatatggaaag ggttagtg |
Gen9 Inc. |
PP7_USLSBm_d15 | 15 | taaccgctttatatggaaagggtta | atgggcgccggcgctaaccgcttta tatggaaagggttagtg |
Gen9 Inc. |
PP7_nB_d5 | 5 | taagggtttatatggaaaccctta | atgctaagggtttatatggaaaccc ttagcgtg |
Gen9 Inc. |
PP7_nB_d6 | 6 | taagggtttatatggaaaccctta | atggctaagggtttatatggaaacc cttatgtg |
Gen9 Inc. |
PP7_USs_d5 | 5 | taaggagttatatggaaccctta | atgctaaggagttatatggaaccct tagtg |
Gen9 Inc. |
PP7_USs_d6 | 6 | taaggagttatatggaaccctta | atggctaaggagttatatggaaccc ttagcgtg |
Gen9 Inc. |
No_BS_d1 | – | – | ttaaagaggagaaaggtacccatgg tg |
Gen9 Inc. |
No_BS_d4 | – | – | ttaaagaggagaaaggtacccatgg gcgtg |
Gen9 Inc. |
No_BS_d10 | – | – | ttaaagaggagaaaggtacccatgg gcgccggcgtg |
Gen9 Inc. |
Sequencing primer for binding site cassettes | gcatttttatccataagattagcgg | IDT | ||
Sequencing primer for RBP cassettes | gcggcgctgggtctcatctaataa | IDT |
Table 1: Binding sites and sequencing primers. Sequences for the binding sites and binding site cassettes used in this study, as well as the primers for the sequencing reactions detailed in the protocol (steps 1.2.5.1 and 1.3.3).
RBP name in this work | source organism name, protein | source organism gene | source organism refseq | wt aa seq | changes from wt (and references) | aa seq used in this work | nt seq used in this work |
MCP | Escherichia virus MS2 | cp | NC_001417.2 | MASNFTQFVLV DNGGTGDVTV APSNFANGVA EWISSNSRSQ AYKVTCSVRQ SSAQNRKYTI KVEVPKVATQT VGGVELPVA AWRSYLNMEL TIPIFATNSD CELIVKAMQG LLKDGNPIPS AIAANSGIY |
delF-G [1] V29I [1] taken from addgene plasmid 27121 |
MASNFTQFVLV DNGGTGDVTV APSNFANGIA EWISSNSRSQ AYKVTCSVRQ SSAQNRKYTI KVEVPKG AWRSYLNMEL TIPIFATNSD CELIVKAMQG LLKDGNPIPS AIAANSGIY |
ATGGCTTCTA ACTTTACTCA GTTCGTTCTC GTCGACAATG GCGGAACTGG CGACGTGACT GTCGCCCCAA GCAACTTCGC TAACGGGATC GCTGAATGGA TCAGCTCTAA CTCGCGTTCA CAGGCTTACA AAGTAACCTG TAGCGTTCGT CAGAGCTCTG CGCAGAATCG CAAATACACC ATCAAAGTCG AGGTGCCTAA AGGCGCCTGG CGTTCGTACT TAAATATGGA ACTAACCATT CCAATTTTCG CCACGAATTC CGACTGCGAG CTTATTGTTA AGGCAATGCA AGGTCTCCTA AAAGATGGAA ACCCGATTCC CTCAGCAATC GCAGCAAACT CCGGCATCTAC |
PCP | Pseudomonas phage PP7 | cp | NC_001628.1 | MSKTIVLSVGEA TRTLTEIQST ADRQIFEEKV GPLVGRLRLT ASLRQNGAKT AYRVNLKLDQ ADVVDCSTSVC GELPKVRYTQ VWSHDVTIVA NSTEASRKSL YDLTKSLVAT SQVEDLVVNL VPLGR |
delF-G [2] taken from addgene plasmid 40650 |
MLASKTIVLSVG EATRTLTEIQ STADRQIFEE KVGPLVGRLR LTASLRQNGA KTAYRVNLKL DQADVVDSG LPKVRYTQVW SHDVTIVANS TEASRKSLYD LTKSLVATSQ VEDLVVNLVP LGR |
ATGCTAGCCTC CAAAACCATC GTTCTTTCGG TCGGCGAGGC TACTCGCACT CTGACTGAGA TCCAGTCCAC CGCAGACCGT CAGATCTTCG AAGAGAAGGT CGGGCCTCTG GTGGGTCGGC TGCGCCTCAC GGCTTCGCTC CGTCAAAACG GAGCCAAGAC CGCGTATCGC GTCAACCTAA AACTGGATCA GGCGGACGTC GTTGATTCCG GACTTCCGAA AGTGCGCTAC ACTCAGGTAT GGTCGCACGA CGTGACAATC GTTGCGAATA GCACCGAGGC CTCGCGCAAA TCGTTGTACG ATTTGACCAA GTCCCTCGTC GCGACCTCGC AGGTCGAAGA TCTTGTCGTC AACCTTGTGC CGCTGGGCCGT |
References: | |||||||
1.Peabody, D.S., Ely, K.R. Control of translational repression by protein-protein interactions. Nucleic Acids Research. 20 (7), 1649–1655 (1992). | |||||||
2. Chao, J.A., Patskovsky, Y., Almo, S.C., Singer, R.H. Structural basis for the coevolution of a viral RNA–protein complex. Nature Structural & Molecular Biology. 15 (1), 103–105, doi: 10.1038/nsmb1327 (2008) |
Table 2: RBP sequences. Amino acid and nucleotide sequences of the coat proteins used in this study.
The method described in this article facilitates quantitative in vivo measurement of RBP-RNA binding affinity in E. coli cells. The protocol is relatively easy and can be conducted without the use of sophisticated machinery, and data analysis is straightforward. Moreover, the results are produced immediately, without the relatively long wait-time associated with next generation sequencing (NGS) results.
One limitation to this method is that it works only in bacterial cells. However, a previous study12 has demonstrated a repression effect using a similar approach for the L7AE RBP in mammalian cells. An additional limitation of the method is that the insertion of the binding site in the mCherry initiation region may repress basal mCherry levels. Structural complexity or high stability of the binding site can interfere with ribosomal initiation even in the absence of RBP, resulting in decreased mCherry basal levels. If basal levels are too low, the additional repression brought on by increasing concentrations of RBP will not be observable. In such a case, it is best to design the binding site cassette with the binding site still in the initiation region, but on the verge of the transition from initiation region to elongation region (δ in the range of 12−15 bp10,29). We have shown that for such δ values a repression effect can still be observed. To increase the chances that the assay will work, regardless of structural complexity, we advise performing the assay on at least three different positions for a given binding site.
The main disadvantage of the method in comparison to in vitro methods, such as EMSA, is that the RBP-RNA binding affinity is not measured in absolute units of RBP concentration, but rather in terms of fusion-RBP fluorescence. This disadvantage is a direct result of the in vivo setting, which limits our ability to read out the actual concentrations of RBP. This disadvantage is offset by the benefits of measuring in the in vivo setting. For example, we have found differences in binding affinities when comparing results from our in vivo assay to previous in vitro and in situ assays. These differences may stem from discrepancies in the structure of the mRNA molecules in vivo that emerge from their presence inside cells10,11,30,31. Such structural differences may lead to changes in the stability of the folded states in vivo which, in turn, either stabilize or de-stabilize RBP binding.
Since the method is relatively simple and inexpensive, we advise running multiple controls alongside the actual experiment. Running a negative control, i.e., a sequence that has no affinity to the RBP yet has similar structural features, can help avoid false positives stemming from non-specific interactions with the mRNA. In the representative results shown, the two negative controls were the mCherry gene alone (no binding site), and the native binding site of the other RBP (i.e., PP7-wt for MCP and MS2-wt for PCP). Moreover, we propose incorporating a positive control (such as an RBP and its native binding site). Such a control will help in quantifying the binding affinity by presenting a reference point, and in avoiding false-negatives stemming from low fold-repression.
Finally, for those who wish to obtain a structural perspective of RBP-RNA binding, we propose carrying out a selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)11,32,33 experiment. SHAPE-Seq is an NGS approach combined with chemical probing of RNA, which can be used to estimate secondary structure of RNA as well as RNA interactions with other molecules, such as proteins. In our previous work we conducted a SHAPE-Seq experiment on a representative strain in both in vivo conditions34 and in vitro with purified recombinant protein10,35. In our case, the results revealed that RBP-binding affected a much wider segment of RNA than previously reported for these RBPs in vitro36.
The authors have nothing to disclose.
This project received funding from the I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation (Grant No. 152/11), Marie Curie Reintegration Grant No. PCIG11-GA- 2012-321675, and from the European Union's Horizon 2020 Research and Innovation Program under grant agreement no. 664918 – MRG-Grammar.
Ampicillin sodium salt | SIGMA | A9518 | |
Magnesium sulfate (MgSO4) | ALFA AESAR | 33337 | |
48 plates | Axygen | P-5ML-48-C-S | |
8- lane plates | Axygen | RESMW8I | |
96-well plates | Axygen | P-DW-20-C | |
96-well plates for plate reader | Perkin Elmer | 6005029 | |
ApaLI | NEB | R0507 | |
Binding site sequences | Gen9 Inc. and Twist Bioscience | see Table 1 | |
E. coli TOP10 cells | Invitrogen | C404006 | |
Eagl-HF | NEB | R3505 | |
glycerol | BIO LAB | 071205 | |
incubator | TECAN | liconic incubator | |
Kanamycin solfate | SIGMA | K4000 | |
KpnI- HF | NEB | R0142 | |
ligase | NEB | B0202S | |
liquid-handling robotic system | TECAN | EVO 100, MCA 96-channel | |
Matlab analysis software | Mathworks | ||
multi- pipette 8 lanes | Axygen | BR703710 | |
N-butanoyl-L-homoserine lactone (C4-HSL) | cayman | K40982552 019 | |
PBS buffer | Biological Industries | 020235A | |
platereader | TECAN | Infinite F200 PRO | |
Q5 HotStart Polymerase | NEB | M0493 | |
RBP seqeunces | Addgene | 27121 & 40650 | see Table 2 |
SODIUM CHLORIDE (NaCL) | BIO LAB | 190305 | |
SV Gel and PCR Clean-Up System | Promega | A9281 | |
Tryptone | BD | 211705 |