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.
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.
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 cells1,2,3,4. In addition, it regulates polyadenylation site switching by binding to uridine-rich polyadenylation enhancer sequences in the enhancer of rudimentary (e(r)) transcript5,6. SXL likely regulates additional targets in the female germline that remain to be identified1,7,8,9,10,11,12,13.
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 (Kd or equilibrium dissociation constant) for the protein of interest; Kd 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.
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
2. Synthesis of RNA
3. Protein Binding Reaction and Separation of Bound RNA
4. Analysis of Iodine-cleaved Phosphorothioate Products for Detection of Mutant Nucleotide Positions
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 also be templates with multiple non-wild type nucleotides in a molecule, precluding analysis of the effect of single-nucleotide substitutions. Thus, as a rule of thumb, use a ratio of non-wild type to wild-type nucleotides of 1/n, where n is the number of positions to be analyzed. Here, we analyzed 10 positions simultaneously and used a mixture containing 90% of the wild-type nucleotide and 10% of the non-wild type nucleotide for doping the DNA template. Separate transcription reactions are done, in which each transcription reaction is performed with one of the four phosphorothioates. Thus, each reaction monitors a specific nucleotide at the doped positions.
Principle of partitioning due to interference:
In the mutagenesis approach presented here, RNAs have on an average fewer than one phosphorothioate residue per molecule. During the process of binding, RNA molecules partition between the protein-bound fraction and the unbound fraction. Since a particular nucleotide is linked to a phosphorothioate linkage, cleavage of the phosphorothioate backbone linkage is a readout of the presence of a specific nucleotide at that position. It is expected that at any given position, when a nucleotide is changed it may have either no effect on binding or it may inhibit binding, partially or completely. If presence of a specific nucleotide has no effect on binding it will partition equally between the protein-bound and unbound fractions. However, if a specific nucleotide at a given position interferes with protein binding it will be preferentially excluded from the protein-bound fraction. The degree of interference can be quantitatively monitored for each of the positions in the same reaction following gel electrophoresis. This concept is illustrated in a schematic (Figure 3). In total RNA fraction (lane T), band intensity is approximately equal for all doped positions (bands 1, 3–7). In protein-bound RNA fraction (lane B), at positions 1, 4, and 7, the nucleotide has no effect on binding. However, at positions 3 and 6, it interferes with binding and thus is excluded from the bound fraction. At position 5, interference is partial. Thus, comparisons of four paired lanes, T and B for each nucleotide, allow for analysis of all four nucleotides. It should be noted that wild-type nucleotide for a given position reflects the effect, if any, of sulfur in the RNA backbone on protein binding.
The accompanying autoradiograph (Figure 4) shows two pairs (α-thio A and α-thio U) of lanes from a denaturing gel (T for Total pool and B for Bound fraction). Several observations can be made by comparing the intensities of bands at each position between each pair of lanes (T and B). First, the majority of the signal is at the top of the gel, i.e., uncleaved product, for both α-thio A lane and α-thio U lane. Second, for α-thio A pair lanes, several bands (iodine cleavage products) are identical between the bound and the total RNA fractions for example, bands above 1; relevant bands within the binding site for the α-thio A lane are numbered for reference. Third, bands at the bottom of the gel are more closely spaced (e.g., bands below 6) than is usual for a sequencing gel. Fourth, several bands are present in the total RNA fraction but absent or significantly reduced in the bound RNA fraction (e.g., bands 1, 2, 3, and 5). Fifth, for the α-thio U lane pair, while most bands are comparable between the total and bound lanes, some bands are relatively less intense in the bound fraction (e.g., bands 7 and 8).
These observations provide evidence for successful development of this new method and lead to the following conclusions: First, for both α-thio A pair lanes and α-thio U pair lanes, because most of the RNA is uncleaved, it provides evidence that only a tiny fraction of the RNA contains modified or phosphorothioate residues. This is important because it ascertains that RNA molecules contain no more than one phosphorothioate residue. Second, identical or similar intensities for several bands outside of the binding site between the two lanes, for both α-thio A and α-thio U, demonstrate that loading is comparable for both lanes, allowing easy comparison between lanes. Third, more closely spaced bands are achieved, for α-thio A and α-thio U, by the wedge-shape of the gel (thinner at the top and thicker at the bottom), thus allowing analysis of longer sequence reads and offering higher resolution. Typically, shorter fragments are more widely spaced in a gel with uniform thickness. Fourth, disappearance or reduced intensity of certain bands in the bound fraction indicates that the non-wild-type nucleotide is preferentially excluded from the bound fraction (α-thio A). Relative intensities of different bands within the bound lane also offer quantitative information about the extent of interference from non-wild type residues at different locations. In other words, the mutant or non-wild type nucleotides at specific positions interfere with protein binding. Finally, testing the effect of backbone sulfur substitution (phosphorothioate) at one of the non-bridging oxygens (α-thio U), shows that three positions show minor effects from backbone sulfurs (e.g., 6, 7 and the one below 7). Our combined results show that several positions within the binding site show preferential disappearance or reduced intensities of specific bands in the bound fraction. This is due to base rather than backbone substitution, indicating protein interactions with specific bases in the binding site (α-thio A). Meanwhile, incorporation of α-thio C shows an interference pattern comparable to α-thio A. However, only 3–4 α-thio G substitutions show small but detectable interference centered around, for example, positions numbered 2 and 3 (data not shown). This method has been used to reveal differences in how the splicing repressor SXL and the general splicing factor U2 snRNP Auxiliary Factor (U2AF65) bind to an identical pre-mRNA splicing signal sequence (polypyrimidine-tract) in distinct manner15.
Figure 1: Flow chart of key steps in phosphorothioate mutagenesis (PTM). Please click here to view a larger version of this figure.
Figure 2: Schematic of a phosphorothioate linkage between two nucleotides, which can be chemically cleaved by iodine. Sulfur replaces one of the non-bridging phosphate oxygens. Please click here to view a larger version of this figure.
Figure 3: Principle of partitioning due to interference. Hypothetical RNA contains representative α-thio nucleotides at six positions, including a protein-binding site. Following protein binding, RNA is cleaved at sites of phosphorothioate incorporation by iodine. Positions 1–7 within and around the binding site are arbitrarily numbered for reference in the text. Position 2 or missing band represents no phosphorothioate incorporation or non-doped nucleotide. Lane T is total RNA and lane B is protein-bound RNA. Please click here to view a larger version of this figure.
Figure 4: Phosphorothioate mutagenesis (PTM) approach allows saturation mutagenesis of a polypyrimidine-tract/3' splice site present in the transformer pre-mRNA. Lane T is total RNA and lane B is protein-bound RNA. α-thio A represents RNA synthesized in the presence of α-thio ATP and identifies doped positions with α-thio adenosines in the sequence. Three strong bands correspond to adenosines in the sequence at those positions. α-thio U represents RNA synthesized in the presence of α-thio UTP and identifies all uridines, including doped positions, in the sequence. Vertical line on the left marks the SXL binding site. Positions 1–8 within the binding site are numbered for reference purposes and for ease of description in the results section. Please click here to view a larger version of this figure.
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 sequences. Second, a sequence can be randomized and the pool used for multiple cycles of binding and polymerase chain reaction (PCR) amplification. These sequences that bind protein will have to be cloned and sequenced to identify and validate a binding site from the consensus sequence. Nonetheless, this iterative binding and amplification option involves multiple steps and is laborious in identifying residues that are important within the binding site. Moreover, repeated sequence amplifications inherent to PCR may introduce sequence bias. Third, sequences selected from the random pool can be sequenced using a faster option of high throughput sequencing, although it is relatively expensive. Therefore, to overcome these limitations of multiple steps, laborious and/or expensive methods, we devised a faster, inexpensive, and most importantly a single-step method, described here, to accomplish saturation mutagenesis of a binding site (PTM).
The key steps in this approach include identification or tagging of the non-wild-type nucleotide(s). We used phosphorothioate nucleotides, in which one of the oxygens in the phosphate backbone is substituted with sulfur, as described (Figure 2). The advantage is that iodine can be used to chemically cleave the backbone of the phosphorothioate nucleotide. Thus, iodine cleavage of the RNA substrate containing a phosphorothioate backbone generates a sequencing-type ladder, where the site of cleavage is a proxy or tag for the presence of one of the four bases at that position. A comparison of the unbound and total fractions identifies residues that are preferentially excluded from the bound fraction and thus are important for protein binding. In contrast, residues that are not important for binding remain equally distributed between the two fractions. This approach can be used to define binding sites for any RNA-binding protein.
In summary, this one-step saturation mutagenesis approach, combining doping, phosphorothioates, and iodine cleavage, offers a powerful means for the characterization of any binding site in RNA. However, this approach requires that the binding site is already known prior to mutagenesis. Furthermore, protein–binding conditions need to optimized for each protein. The following precautions need to be emphasized. Precautions for RNA handling must be exercised, such as wearing gloves, preparation of solutions and buffers in DEPC-treated water, autoclaving pipette tips and tubes, and dedicating equipment for RNA use only to avoid RNAses. Similarly, care involving radioactive shields, radioactive monitoring while using radioactive material is essential, and use of exhaust hood while using hazardous chemicals are necessary.
The authors have nothing to disclose.
The author thanks the National Institutes of Health for the past funding and thanks Michael R. Green for synthesizing oligonucleotides.
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 |