A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Summary

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Abstract

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Introduction

Gene regulation plays an important role in biology. Genes can be regulated at the level of transcription, pre-mRNA splicing, 3' end formation, RNA export, translation, mRNA localization, decay, post-translational modification/stability, etc. Both DNA- and RNA-binding proteins play key roles in gene regulation. While molecular genetic analyses have identified numerous regulatory proteins, only a small subset of them have been characterized fully for their cellular functions or binding sites in vivo. Phylogenetic sequence analysis and mutagenesis offer complementary approaches to characterize DNA- or RNA-protein interactions.

RNA-binding proteins are important in developmental processes, including sexual differentiation. The Drosophila protein Sex-lethal (SXL) or the master sex-switch protein is absent in males, but present in females. It recognizes uridine-rich sequences or pyrimidine-tracts adjacent to specific splice sites in downstream pre-mRNA targets (transformer, Sex-lethal, and male-specific lethal2) in somatic cells^1,2,3,4. In addition, it regulates polyadenylation site switching by binding to uridine-rich polyadenylation enhancer sequences in the enhancer of rudimentary (e(r)) transcript^5,6. SXL likely regulates additional targets in the female germline that remain to be identified^{1,7,8,9,10,11,12,13}.

Typically, characterization of a binding site involves mutagenesis, for example, by deletion or substitution of single or multiple nucleotides. Each mutant binding site, relative to the wild-type RNA sequence, is then analyzed using a series of protein concentrations to determine its binding affinity (K_d or equilibrium dissociation constant) for the protein of interest; K_d is the protein concentration required to obtain 50% RNA binding. This labor-intensive process of detailed mutagenesis involves generation and analysis of numerous mutants — three non-wild type nucleotides for each position in the binding site. Thus, there is a need for an alternative approach for faster, simpler, and inexpensive saturation mutagenesis of protein binding sites in RNA.

Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Protocol

NOTE: Figure 1 provides an overview of phosphorothioate mutagenesis and summarizes key steps in the process.

1. Generation of a Library of Mutants — Doping DNA Template with Non-wild Type Nucleotides

1. Synthesize T7 Primer (5’-GTAATACGACTCACTATAG-3’) by chemical synthesis on a DNA synthesizer.
2. Synthesize a doped oligonucleotide (complementary strand) by chemical synthesis on a DNA synthesizer corresponding to the protein binding site. Use an appropriate mixture of phosphoramidites during chemical synthesis for each site of doping (X below) with a ratio of 90% A as the wild-type nucleotide and 10% T as the non-wild type nucleotide (see Representative Results for more detail on this ratio).
   Note: Here, the sequence of the doped oligonucleotide is 5’-GTTCACTACACTXGAXAXAXAXCAXCXAXAXGXTGCCCTATAGTGAGTCGTATTAC-3’. The underlined sequence is the reverse complement of SXL protein binding site, plus additional nucleotides outside the binding site that provide useful controls confirming that not every change in the RNA affects binding, as well as being loading controls for comparison and normalization of nucleotides within the binding site. The SXL-binding site sequence UUUUUGUUGUUUUUUUU, which is present in transformer pre-mRNA^14,15,16,17, was used to devise the proposed methodology. The sequence in italics is complementary to the T7 primer sequence and is the T7 promoter for in vitro transcription.

2. Synthesis of RNA

1. Synthesize RNA in a 20 µL transcription reaction, as previously described¹⁸.
   1. Mix T7 transcription buffer and 1 µM T7 oligonucleotide, 1 µM doped oligonucleotide, 10 mM dithiothreitol (DTT), 2 mM GTP, 1 mM each ATP, CTP, and UTP (guanosine, adenosine, cytidine, and uridine triphosphate), and 2 U/µL T7 RNA polymerase.
   2. Add, in two separate microcentrifuge tubes, 0.167 mM α-thio ATP or 0.05 mM α-thio UTP to incorporate phosphorothioates (see schematics in Figure 2) into RNAs.
      NOTE: For alternative protocols, add 0.2 mM α-thio CTP or 0.2 mM α-thio GTP to an appropriate transcription reaction containing a doped oligonucleotide to test the two remaining nucleotide substitutions.
   3. Incubate the RNA synthesis reaction mixture for 2 h at 37 °C.
2. Add 2.5 µL heat-labile alkaline phosphatase and 2.5 µL 10xphosphatase buffer to the RNA sample. Incubate this 25 µL reaction for 10–30 min at 37 °C to remove 5’ phosphates.
3. Inactivate the alkaline phosphatase enzyme by heating at 80 °C for 2–5 min.
4. Radiolabel the 5’ end of dephosphorylated RNA (5 pmole) using 1 µL T4 polynucleotide kinase and 1 µL γ-³²P ATP in a 10 µL reaction volume. Incubate the reaction mix for 30–60 min at 37 °C.
5. Inactivate the T4 polynucleotide kinase enzyme by heating at 65 °C for 20–30 min.
6. Gel purify the RNA by electrophoresis in a 10% denaturing polyacrylamide gel. Locate RNA on the gel by autoradiography. Excise the gel slice containing radiolabeled RNA, crush in a microcentrifuge tube by pressing against the walls with a pipette tip, and soak in proteinase K (PK) buffer (100 mM Tris, pH 7.5, 12.5 mM EDTA, 150 mM NaCl, 1% Sodium dodecyl sulfate). Rotate the tube at room temperature from 2 h to overnight.
7. Centrifuge the gel slurry, discard the gel and collect the buffer solution.
8. Extract the solution twice with equal volume of phenol-chloroform and once with chloroform and collect the aqueous phase.
9. Add to the aqueous phase 0.1 volume of 3M Sodium Acetate, pH 5.2, carrier tRNA or glycogen, and 2.5 volume of ethanol. Keep the sample at -80 °C for 1 h.
10. Centrifuge the sample for 5–10 min in a high speed microcentrifuge at 16,873 x g. Remove the buffer/ethanol solution carefully without disturbing the RNA pellet.
11. Wash the pellet with 70% ethanol and centrifuge for 2–5 min. Remove ethanol carefully. Dry the pellet in air.
12. Resuspend the pellet in 20–50 µL diethyl pyrocarbonate (DEPC)-treated water. Store at -20 °C until use.
    NOTE: Perform all steps with radiolabeled RNA using appropriate precautions and a plexiglass shield to protect from radioactivity. Presently, spin columns are more commonly used and more convenient to remove unincorporated radioactivity, as an alternative to gel purification of RNA.

3. Protein Binding Reaction and Separation of Bound RNA

1. Express the recombinant protein in and purify from E. coli.
2. Estimate recombinant protein concentration by spectrophotometry or by separating in an SDS-polyacrylamide gel next to a known protein standard, bovine serum albumin (BSA). Visualize the protein of interest and the BSA standard by staining the gel with Coomassie Brilliant Blue R-250. Quantitate the protein in comparison to known quantities of BSA dilutions on the same gel.
   NOTE: Store recombinant protein at -80 °C until use and dilute in 20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 8.0, 1 mM dithiothreitol (DTT), 0.2 mM Ethylenediaminetetraacetic acid (EDTA), 0.05% NP-40, 20% glycerol. Use of 0.5–1.0 mM protease inhibitor phenylmethane sulfonyl fluoride (PMSF) is optional.
3. Perform RNA-binding reaction (20–100 µL) in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50 mM KCl, 0.5 units/µL RNase inhibitor, 0.09 µg/µL acetylated bovine serum albumin, 1 mM EDTA, 0.15 µg/µL tRNA, 5’-end radiolabeled RNA, and 6 µL of appropriate concentration (a concentration at which ~50% of RNA binds to protein) of the protein.
   NOTE: This binding buffer works for three RNA–binding proteins (SXL, U2AF⁶⁵, and PTB), but must be standardized for a protein of interest. Estimate protein concentration empirically, using various dilutions for a given protein preparation, that is required to obtain approximately 50% RNA binding. This condition or K_d represents the most sensitive part of the RNA-binding curve.
4. Incubate the protein binding reaction for 20–30 min at 25 °C (or on ice).
5. Separate the protein-bound RNA fraction from the unbound fraction using one of two approaches:
   1. Nitrocellulose filter binding
      1. Apply the binding reaction (20–100 µL) onto a nitrocellulose filter connected to a vacuum manifold at room temperature.
         NOTE: Only the RNA-protein complex is retained on the filter and unbound RNA flows through the filter. The filter binding approach allows higher recovery of the bound RNA and is faster and simpler, compared to the gel mobility shift assay. By placing a DEAE membrane underneath the nitrocellulose filter it is also possible to collect the unbound RNA fraction or free RNA for comparison with the bound fraction.
      2. Cut the portion of the nitrocellulose filter containing the retained radioactive RNA into smaller pieces to fit into a microcentrifuge tube, soak in sufficient PK buffer (300–500 µL containing 10–20 µg PK) to immerse the filter pieces, and elute RNA from the filter for 2–3 h or overnight.
      3. Extract with phenol-chloroform, chloroform, and collect aqueous phase. Add sodium acetate and ethanol and, after incubation in freezer, centrifuge, wash, dry and resuspend RNA in DEPC-treated water. Follow these steps as detailed in steps 2.10 to 2.14 above.
   2. Gel mobility shift
      1. Prepare and polymerize a 5% native polyacrylamide gel (60:1 Acrylamide:bis-acrylamide) in 0.5x TBE (standard Tris-Borate-EDTA buffer) before starting the binding reaction, as an alternative to the filter binding method.
      2. Pre-run the gel for 15 min at 250 V in a cold room (4 °C).
      3. Load each binding reaction (above) into separate wells of the above pre-run gel.
         NOTE: The protein storage buffer provides sufficient glycerol for sample loading into wells.
      4. Separate the protein-bound RNA by gel electrophoresis in a cold room at 250 V for 1 to 2 h, depending on the RNA size and specific protein.
      5. Locate the position of the RNA-protein complex on the gel by autoradiography. Excise the gel slice containing the RNA-protein complex. Elute the RNA from the gel slice by crushing the gel slice and soaking in the PK buffer.
      6. Extract with phenol-chloroform, chloroform, and collect aqueous phase. add Sodium acetate and ethanol and, after incubation in freezer, centrifuge, wash, air dry, and resuspend RNA in DEPC-treated water. Follow these steps as detailed in steps 2.10 to 2.14 above.

4. Analysis of Iodine-cleaved Phosphorothioate Products for Detection of Mutant Nucleotide Positions

1. Add 1 mM iodine in a 20 µL DEPC-treated water containing up to 10 µg carrier tRNA to cleave RNAs (bound and total RNAs) at the sites of phosphorothioate incorporation. Incubate at room temperature for 5 min.
   NOTE: Further details on iodine cleavage with slightly different conditions — 7% (v/v) iodoethanol, heating at 95 °C for 3 min — can be found in Gish & Eckstein 1988¹⁹.
2. Precipitate cleaved RNA by adding sodium acetate/ethanol, as described above. Resuspend in a loading dye for denaturing gels. Heat and load the sample to separate RNA fragments by electrophoresis in a 15–20% denaturing polyacrylamide gel.
3. Expose the polyacrylamide gel to an X-ray film.
4. Detect bands in the bound fraction versus total pool using autoradiography.
   NOTE: Perform all steps with iodine in an exhaust hood. For RNA separation shown here, a wedge-shaped gel is employed, which is achieved by doubling the thickness of the both spacers at the bottom of the gel plates by inserting an extra inch-long spacer at the bottom of the gel. This shape allows more uniform or closer spacing between shorter RNA fragments. Alternatively, a phosphorimager can be used rather than X-ray films for detection and quantitation of radioactivity.

Results

Principle of saturation mutagenesis using doping:

For an appropriate molar ratio of wild-type and other nucleotides, use an equal mixture of all four nucleotides if only one position is to be analyzed. However, if multiple positions are analyzed simultaneously, the ratio of non-wild type to wild- type nucleotides must be adjusted, i.e., reduced. Otherwise, in addition to single substitutions, which is desired, there will a...

Discussion

Mutagenesis has long been used to characterize protein binding sites. First, a series of mutants can be constructed and individually tested in binding assays to analyze their effects on binding affinity. While a standard mutagenesis approach offers a way to analyze several sequences, multiple steps involved in the standard approach, such as constructing mutants and performing a series of binding reactions for each mutant, is laborious and time consuming and may not allow saturation mutagenesis, especially for longer sequ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Uridine 5’ a-thio triphosphate	NEN (Boston, Massachusetts)	NLP-017
Adenosine 5’ a-thio triphosphate	NEN (Boston, Massachusetts)	NLP-016
Vacuum manifold	Fisher Scientific	XX1002500	Millipore 25 mm Glass Microanalysis Vacuum Filter
Vacuum manifold	Millipore	XX2702552	1225 Sampling Vacuum Manifold
Nitrocellulose	Millipore	HAWP
Nitrocellulose	Schleicher & Schuell	PROTRAN
Dephosphorlyation Kit	NEB	M0508
T4 Polynucleotide Kinase	NEB	M0201S
Proteinase K	NEB	P8107S
T7 RNA polymerase	NEB	M0251S
RNasin	Promega		RNase inhibitor
Glass Plates	Standard	Standard
Gel Electrophoresis equipment	Standard	Standard
X-ray films	Standard	Standard
Polyacrylamide gel solutions	Standard	Standard