The author thanks the National Institutes of Health for the past funding and thanks Michael R. Green for synthesizing oligonucleotides.

NOTE: Figure 1 provides an overview of phosphorothioate mutagenesis and summarizes key steps in the process.
1. Generation of a Library of Mutants &#8212; Doping DNA Template with Non-wild Type Nucleotides
<ol>
	<li>Synthesize T7 Primer (5&#8217;-GTAATACGACTCACTATAG-3&#8217;) by chemical synthesis on a DNA synthesizer.</li>
	<li>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&#8217;-GTTCACTACACTXGAXAXAXAXCAXCXAXAXGXTGCCCTATAGTGAGTCGTATTAC-3&#8217;. 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-mRNA14,15,16,17, 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.</li>
</ol>
2. Synthesis of RNA
<ol>
	<li>Synthesize RNA in a 20 &#181;L transcription reaction, as previously described18.
	<ol>
		<li>Mix T7 transcription buffer and 1 &#181;M T7 oligonucleotide, 1 &#181;M 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/&#181;L T7 RNA polymerase.</li>
		<li>Add, in two separate microcentrifuge tubes, 0.167 mM &#945;-thio ATP or 0.05 mM &#945;-thio UTP to incorporate phosphorothioates (see schematics in Figure 2) into RNAs. 
		NOTE: For alternative protocols, add 0.2 mM &#945;-thio CTP or 0.2 mM &#945;-thio GTP to an appropriate transcription reaction containing a doped oligonucleotide to test the two remaining nucleotide substitutions.</li>
		<li>Incubate the RNA synthesis reaction mixture for 2 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Add 2.5 &#181;L heat-labile alkaline phosphatase and 2.5 &#181;L 10xphosphatase buffer to the RNA sample. Incubate this 25 &#181;L reaction for 10&#8211;30 min at 37 &#176;C to remove 5&#8217; phosphates.</li>
	<li>Inactivate the alkaline phosphatase enzyme by heating at 80 &#176;C for 2&#8211;5 min.</li>
	<li>Radiolabel the 5&#8217; end of dephosphorylated RNA (5 pmole) using 1 &#181;L T4 polynucleotide kinase and 1 &#181;L &#947;-32P ATP in a 10 &#181;L reaction volume. Incubate the reaction mix for 30&#8211;60 min at 37 &#176;C.</li>
	<li>Inactivate the T4 polynucleotide kinase enzyme by heating at 65 &#176;C for 20&#8211;30 min.</li>
	<li>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.</li>
	<li>Centrifuge the gel slurry, discard the gel and collect the buffer solution.</li>
	<li>Extract the solution twice with equal volume of phenol-chloroform and once with chloroform and collect the aqueous phase.</li>
	<li>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 &#176;C for 1 h.</li>
	<li>Centrifuge the sample for 5&#8211;10 min in a high speed microcentrifuge at 16,873 x g. Remove the buffer/ethanol solution carefully without disturbing the RNA pellet.</li>
	<li>Wash the pellet with 70% ethanol and centrifuge for 2&#8211;5 min. Remove ethanol carefully. Dry the pellet in air.</li>
	<li>Resuspend the pellet in 20&#8211;50 &#181;L diethyl pyrocarbonate (DEPC)-treated water. Store at -20 &#176;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.</li>
</ol>
3. Protein Binding Reaction and Separation of Bound RNA
<ol>
	<li>Express the recombinant protein in and purify from E. coli.</li>
	<li>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 &#176;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&#8211;1.0 mM protease inhibitor phenylmethane sulfonyl fluoride (PMSF) is optional.</li>
	<li>Perform RNA-binding reaction (20&#8211;100 &#181;L) in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50 mM KCl, 0.5 units/&#181;L RNase inhibitor, 0.09 &#181;g/&#181;L acetylated bovine serum albumin, 1 mM EDTA, 0.15 &#181;g/&#181;L tRNA, 5&#8217;-end radiolabeled RNA, and 6 &#181;L of appropriate concentration (a concentration at which ~50% of RNA binds to protein) of the protein. 
	NOTE: This binding buffer works for three RNA&#8211;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 Kd represents the most sensitive part of the RNA-binding curve.</li>
	<li>Incubate the protein binding reaction for 20&#8211;30 min at 25 &#176;C (or on ice).</li>
	<li>Separate the protein-bound RNA fraction from the unbound fraction using one of two approaches:
	<ol>
		<li>Nitrocellulose filter binding
		<ol>
			<li>Apply the binding reaction (20&#8211;100 &#181;L) 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.</li>
			<li>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&#8211;500 &#181;L containing 10&#8211;20 &#181;g PK) to immerse the filter pieces, and elute RNA from the filter for 2&#8211;3 h or overnight.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Gel mobility shift
		<ol>
			<li>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.</li>
			<li>Pre-run the gel for 15 min at 250 V in a cold room (4 &#176;C).</li>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Analysis of Iodine-cleaved Phosphorothioate Products for Detection of Mutant Nucleotide Positions
<ol>
	<li>Add 1 mM iodine in a 20 &#181;L DEPC-treated water containing up to 10 &#181;g 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 &#8212; 7% (v/v) iodoethanol, heating at 95 &#176;C for 3 min &#8212; can be found in Gish &#38; Eckstein 198819.</li>
	<li>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&#8211;20% denaturing polyacrylamide gel.</li>
	<li>Expose the polyacrylamide gel to an X-ray film.</li>
	<li>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.</li>
</ol>

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Drosophila as a paradigm.</a> Bioessays. 14 (8), 513-518 (1992).</li><li>Salz, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex,+stem+cells+and+tumors+in+the+Drosophila+ovary.">Sex, stem cells and tumors in the Drosophila ovary.</a> Fly (Austin). 7 (1), 3-7 (2013).</li><li>Gawande, B., Robida, M. D., Rahn, A., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Sex-lethal+protein+mediates+polyadenylation+switching+in+the+female+germline.">Drosophila Sex-lethal protein mediates polyadenylation switching in the female germline.</a> Embo J. 25 (6), 1263-1272 (2006).</li><li>Robida, M. 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The author declares no competing financial interests.

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...

Gene regulation plays an important role in biology. Genes can be regulated at the level of transcription, pre-mRNA splicing, 3&#39; 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 &#8212; 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.

<table><tbody><tr><td>Uridine 5&amp;rsquo; a-thio triphosphate</td><td>NEN (Boston, Massachusetts)</td><td>NLP-017</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo; a-thio triphosphate</td><td>NEN (Boston, Massachusetts)</td><td>NLP-016</td><td></td></tr><tr><td>Vacuum manifold</td><td>Fisher Scientific</td><td>XX1002500</td><td>Millipore&amp;nbsp;25&amp;nbsp;mm Glass Microanalysis Vacuum Filter</td></tr><tr><td>Vacuum manifold</td><td>Millipore</td><td>XX2702552</td><td>1225 Sampling Vacuum Manifold</td></tr><tr><td>Nitrocellulose</td><td>Millipore</td><td>HAWP</td><td></td></tr><tr><td>Nitrocellulose</td><td>Schleicher &amp;amp; Schuell</td><td>PROTRAN</td><td></td></tr><tr><td>Dephosphorlyation Kit</td><td>NEB</td><td>M0508</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>M0201S</td><td></td></tr><tr><td>Proteinase K</td><td>NEB</td><td>P8107S</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>NEB</td><td>M0251S</td><td></td></tr><tr><td>RNasin</td><td>Promega</td><td></td><td>RNase inhibitor</td></tr><tr><td>Glass Plates</td><td>Standard</td><td>Standard</td><td></td></tr><tr><td>Gel Electrophoresis equipment</td><td>Standard</td><td>Standard</td><td></td></tr><tr><td>X-ray films</td><td>Standard</td><td>Standard</td><td></td></tr><tr><td>Polyacrylamide gel solutions</td><td>Standard</td><td>Standard</td><td></td></tr></tbody></table>

a novel saturation mutagenesis approach: single step characterization of regulatory protein binding sites in rna using phosphorothioates

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.

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...

Watch this Scientific Journal Video about A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates at JoVE.com

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

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 ...

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6.5K Views.  University of Colorado at Boulder. Eiwitten die specifieke RNA opeenvolgingen binden spelen een kritieke rol in genexpressie. Gedetailleerde karakterisering van deze bandplaatsen is van cruciaal belang voor ons begrip van de genexpressie. Hier, wordt een-voor-stapmodus aanpak voor verzadiging mutagenese van eiwit-bandplaatsen in RNA beschreven. Deze aanpak geldt voor alle websites van de binding aan eiwitten in RNA.

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

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Biochemistry

An Assay for Quantifying Protein-RNA Binding in Bacteria

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.

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 ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biology

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

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

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