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 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.
RNA-binding proteins (RBPs) have emerged as crucial players in transcriptional and post-transcriptional gene regulation, since they are involved in processes such as mRNA splicing, RNA cellular localization, translation, modification, and degradation1,2,3. Such interactions between the two macromolecules are highly coordinated, precisely balanced, and essential for the formation of functional and processing hubs. Variations or dysregulations within these hubs have the potential of disrupting the finely regulated protein-RNA networks and are increasingly associated with a variety of human diseases, including cancer4,5 and neurodegenerative disorders6,7,8. The interactions between RNA molecules and their protein binding partners can be either stable and easy to validate experimentally, or highly dynamic, transient, and more difficult to characterize.
In recent years, intensive efforts have been undertaken to understand these interactions. Among the most established methods, protein pull-down assays (PDs) are probably the most appreciated and commonly used approaches to unravel the main players constituting ribonucleoprotein (RNP) complexes and other protein-RNA interaction networks3,9,10. PDs include a broad umbrella of informative techniques, such as the immunoprecipitation of either the RNA (RIP)11,12 or the protein (CLIP)13,14 of interest. Some of these RNA-PD protocols employ a known RNA as bait for proteins15, most frequently by taking advantage of high affinity tags such as biotin. In this instance, the interaction partners of a biotinylated RNA can be detected by anchoring the RNA on streptavidin-coated beads, enabling efficient isolation of the RNPs. The main limitations of these approaches are usually the design of the biotinylated probes and the testing of their ability to bind target proteins. For this purpose, it could be useful to rely on published CLIP data of the protein of interest, if available, since they reveal, with high precision, the short RNA regions that correspond to peaks of interactions with the target protein13,16. These same regions could be used to develop probes for PDs. An alternative method to design such RNA baits might be the systematic evolution of ligands by exponential enrichment (SELEX)17, which enable the design of aptamers through in vitro selection, starting from a comprehensive randomized library and via a series of PCR-driven optimization cycles. However, SELEX is complex and time consuming, and the final results are highly dependent on the initial library. To select the RNA bait to use in the protocol presented here, yet another approach was exploited, consisting of using an RNA bait designed de novo by means of the computational power of the algorithm catRAPID, which predicts the preferential binding of a given protein toward certain RNA sequences18,19,20.
The protocol introduced here is a version of an RNA-PD optimized to elute specific protein partners in near-to-physiological conditions, without the use of detergent, denaturing agents, or high temperatures. It relies on nano-superparamagnetic beads covalently coated with highly purified streptavidin and the use of a specific in silico designed biotinylated RNA as a bait. This protocol provides a rapid and efficient method to isolate the binding partners of biotinylated RNA molecules in native conditions, offering the potential for a wide range of downstream applications. To test this protocol, a 10-nucleotide single stranded RNA aptamer sequence, previously designed to bind the protein TAR DNA-binding protein 43 (TDP-43) with high affinity and specificity, was used20. Starting from HEK 293T cell lysates, the interactors of the biotinylated RNA aptamer were identified by means of mass-spectrometry analysis performed on samples detached from the RNA bait using a hypertonic buffer. This analysis confirmed the successful identification and quantification of TDP-43 as preferred binder.
This protocol enables the successful identification of protein interactors using only a short, in vitro synthesized RNA oligonucleotide. Moreover, the use of in silico designed RNA aptamers as PD probes21,22 guarantees specificity for the targets at significantly reduced costs.
1. General methods and material
2. Mammalian cell line preparation
3. Total protein harvest
4. Protein concentration determination
5. Bead preparation
6. Bead loading
7. Protein binding on beads
NOTE: From now on, when possible, perform the steps at 4 °C.
8. Washing of nonspecific binders
9. Elution of specific binders
10. Identification of protein binders by mass spectrometry
11. Results validation by western blot
To verify the validity of the proposed protocol, the PD experiments presented here were performed with a biotinylated RNA aptamer designed in silico to specifically bind TDP-4320. This RNA binds its protein target with high binding affinity (Kd = 90 nM)20. Here, this RNA, of sequence 5'-CGGUGUUGCU-3', is referred to with the name "+RNA". As a negative control, the reverse complementary sequence of +RNA, which is here called "-RNA", was used. Its sequence is 5'-AGCAACACCG-3'. -RNA shows a significantly lower binding affinity toward TDP-43 (Kd = 1.5 µM)19. For the purpose of the protocol described here, these RNA oligonucleotides have been purchased conjugated to a biotin molecule, to allow binding to the streptavidin beads. +RNA was purchased with a biotin-TEG at its 3' end, which includes a 15-atom triethylene glycol spacer between the biotin and the phosphate group of the nucleic acid; -RNA instead had a biotin at its 5' end, conjugated to the nucleic acid via an amino-C6 linker. However, if the design of the RNA bait is robust, and as long as there is no structural or chemical interference between the linker and the RNA, other positions for the biotin conjugation and other linker lengths could be employed.
Knowing the identity of the main protein to be found bound to the +RNA probe after the PD enabled the validation of the protocol by identification of TDP-43 in the eluate, using both mass spectrometry (MS) and western blot (WB) (Figure 1).
MS analysis was carried out on four PD replicates performed using either +RNA or -RNA (Figure 2). The identification of the interactomes of +RNA and -RNA is beyond the scope of this protocol, however some results that validate the accuracy of the protocol are reported. Of note, plotting the significantly enriched proteins in a volcano plot revealed that the total protein content and the enriched proteins eluted from +RNA was significantly higher that what was recovered from -RNA (Figure 2). This means that, despite having the same length and structural content (linear), +RNA can establish a higher number of specific interactions, which are retained up to the elution step with high salt. It is likely that -RNA instead establishes a higher number of nonspecific contacts that are disrupted during the washing steps. As expected, TDP-43 was identified as a unique interactor of +RNA20; the average label-free quantification (LFQ) for the four PD replicates performed with +RNA is 31.96 ± 0.56, while the protein is not identified among the interactors of -RNA. In addition, among all unique interactors of +RNA, TDP-43 was found to be the most abundantly enriched protein.
To further validate the protocol, the in-house algorithm catRAPID18,19 was used to computationally predict which other proteins would specifically bind either +RNA or -RNA. In particular, interaction scores for +RNA and -RNA with the proteins composing the human proteome were computed using the catRAPID 'interaction propensity' feature, as defined in our previous work27. Among the proteins scored with high confidence, HNRNPH3 was predicted to bind selectively +RNA (+RNA interaction score = 1.01; -RNA interaction score = 0.21) and PCBP2 to interact specifically with -RNA (+RNA interaction score = -0.5; -RNA interaction score = 0.31) (Figure 3A). In addition, the protein RBM41 was predicted to be promiscuous for both RNA oligonucleotides (+RNA interaction score = 0.4; -RNA interaction score = 0.39) (Figure 3A). The MS analysis indeed confirmed the presence of HNRNPH3 and PCBP2 in the PD of +RNA and -RNA respectively, while RBM41 was found interacting with both (Figure 3B).
WB was used to detect the presence of TDP-43 to further confirm the results and during protocol optimization (Figure 4). In the procedure described here, different samples were collected at different stages. The input sample (IN) consisted of the total proteins diluted in lysis buffer. The flowthrough (FT) was obtained after an overnight incubation of the total proteins with the streptavidin beads pre-coated with the biotinylated RNA, representing the fraction of proteins that did not bind the RNA. Finally, the eluate (EL) contained all the proteins that recognized specifically the RNA under investigation, since between the FT and the EL steps three washing steps with 150 mM salt and 0.1% triton-X should have removed the weakest interactions.
For each replicate, the same amount (5% v/v) of IN, FT, and EL was run in parallel on an SDS-PAGE and stained with an anti-TDP-43 antibody (Figure 4). In the case of +RNA, the band of TDP-43 was observed in IN and in EL, indicating that the protein, present from the start in the total protein extract, is retained by +RNA during the washing steps and is only eluted at the end with a high salt buffer. TDP-43 was also present in IN for -RNA, however the band corresponding to the protein is also visible in FT, indicating that this RNA does not bind TDP-43. The absence of the TDP-43 band in EL confirms this result.
During the optimization of the protocol, the elution of the proteins specifically bound to the RNA sequences was probed both with an elution buffer containing 1 M NaCl (EB1) and with an elution buffer complete with 2 M NaCl (EB2) (Figure 4). Eluates obtained with either EB were compared on an SDS-PAGE and blotted with the anti-TDP-43 antibody. The images obtained were then analyzed with ImageJ28 to quantify any difference in TDP-43 amount eluted with the two buffers. Overall, no significant difference was observed, and we concluded that, within these assays, 1 M salt is sufficient to disrupt even the strongest protein-RNA interactions.
Overall, the results reported here for MS and WBs demonstrate that this protocol is efficient in capturing the protein interactors of a given RNA in a specific manner, and that it enables the elution in buffers compatible with downstream analysis.
Figure 1: Sketch of the experimental pipeline used in the proposed protocol. (A) The biotinylated RNA oligonucleotide is prepared in lysis buffer at the appropriate concentration. (B) Magnetic streptavidin beads are washed, blocked with yeast tRNA, and loaded with the biotinylated RNA. (C) Total protein extract derived from cultured mammalian cell lines are added to the beads-RNA mixture. (D) Multiple washes are performed to remove nonspecific interactions. (E) The specific protein interactors are detached from the RNA with a hypertonic solution. (F) The identity of the interactors is revealed by mass spectrometry, and specific cases are validated by western blot. Please click here to view a larger version of this figure.
Figure 2: Analytical strategy for label-free MS-based protein quantification. (A) Eluted proteins are precipitated in cold acetone overnight. Proteins are then denatured, and an in-solution digestion is performed. Proteolytic peptides are concentrated and desalted. (B) Peptides are analyzed via LC-MS/MS using a "shotgun approach". (C) Raw data processing and analysis is performed using MaxQuant and Perseus software, respectively. (D) Statistically significant enriched proteins are displayed in a volcano plot. Please click here to view a larger version of this figure.
Figure 3: Correlation between predicted interaction propensities and experimentally determined interactions of +RNA and -RNA. (A) catRAPID interaction scores relative to HNRNPH3, PCBP2, and RBM41, indicating preferential binding of HNRNPH3 for +RNA and of PCBP2 for -RNA, while RBM41 is predicted to indiscriminately bind both RNA sequences. (B) Label-free quantification averages determined by mass-spectrometry analysis from the pull-downs performed with +RNA and -RNA. The analysis confirms that HNRNPH3 solely binds +RNA, PCBP2 solely binds -RNA, and RBM41 binds both equally. Please click here to view a larger version of this figure.
Figure 4: Western blot validation of the presence/absence of TDP-43 among the interactors of chosen RNA sequences. The WB membrane has been treated with anti-TDP-43 antibody. IN = input; FT = flow-through; EL (EB1) = elution with elution buffer 1; EL (EB2) = elution with elution buffer 2; the sign "+" indicates samples derived from the pull-down performed with +RNA; the sign "-" indicates samples derived from the pull-down performed with -RNA; lane 1 contains a protein ladder. TDP-43 is indicated by an arrow. The WB indicates that TDP-43 is found among +RNA interactors but not among -RNA interactors. Please click here to view a larger version of this figure.
Buffer Name | Composition | |||||
10x Tranfer buffer | 250 mM tris, 1.92 M glycine, 1% SDS, 20% methanol. Dilute 10 folds prior use | PD | ||||
20X MES SDS running buffer | 1 M MES, 1 M tris, 2% SDS , 20 mM EDTA. Adjust pH to 7.3. Dilute 20 folds prior use | |||||
4x Sample loading buffer | 0.25 M Tris base, 0.28 M SDS, 40% glycerol, 20% 2-mercapto-ethanol, 4 mg/ml bromphenol blue | |||||
Elution buffer 1 | 20 mM phosphate pH 7.5, 1 M NaCl, 0.5 mM EDTA, 0.1 % Triton X-100, 1 mM DTT (to be added after quantification) | |||||
Elution buffer 2 | 20 mM phosphate pH 7.5, 2 M NaCl, 0.5 mM EDTA, 0.1 % Triton X-100, 1 mM DTT (to be added after quantification) | |||||
Lysis buffer | 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.1 % Triton X-100, 1 mM DTT and protease inhibitors | |||||
Tris-buffered saline with Tween-20 | 1 M Tris-HCl pH 7.4, 3 M NaCl, 2.0% Tween-20 | |||||
Wash buffer 1 | 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.1 % Triton TM X-100, 1 mM DTT and protease inhibitors | |||||
Wash buffer 2 | 25 mM Hepes pH 8, 150 mM NaCl, 0.5 mM EDTA, 0.1 % Triton X-100, 1 mM DTT and protease inhibitors | |||||
Buffer A | 0.1% formic acid | MS | ||||
Buffer B | 60% acetonitrile, 0.1% formic acid | |||||
Denaturation buffer | 8M urea, 50 mM Tris-HCl |
Table 1: PD and MS buffers. Names and composition of the buffers used for either the pull-down experiments (PD) or for the mass-spectrometry analysis (MS).
This work reports the optimization of a PD protocol performed with biotinylated RNA oligonucleotides to capture their protein interactors. The protocol described here is simple to perform, requires little material, and produces highly reliable results. Importantly, the most novel aspects of this protocol consist of the use of an RNA bait designed fully in silico and specific for the protein target, and the elution of all proteins bound to the RNA bait by directly disrupting their interactions with a high-salt solution, rather than by dissociating the streptavidin from the biotin with detergent and high temperature treatment.
This protocol takes advantage of the strength of the bond between biotin and streptavidin29,30. According to the chosen streptavidin beads, loading of the biotinylated RNA must be tested and quantified before proceeding. Also, the RNA tri-dimensional folding might affect the loading efficiency on the beads, since it might limit the exposure of the biotin to the streptavidin. Blocking the beads with non-biotinylated tRNA improves the cleanliness of the results by limiting nonspecific interactions with the beads. The loading buffer and the elution buffer must be chosen depending on the downstream applications. Here, very mild conditions, suitable to the majority of the applications and developed to preserve potential protein complexes, were proposed. This method is however highly adaptable; the user can choose any cell line and any RNA size, and could decide to repeat the protocol after folding/unfolding of the RNA to determine the effect of the structure on the binding properties.
Another original aspect of this protocol is the use of in silico prediction tools to ensure the correctness of the results20. Knowing in advance which proteins should be identified as interactors of the RNA of interest gives the unprecedented advantage of validating the technical aspects of the protocol. For example, using a simple WB analysis, it is possible to verify the presence of a known protein target in the samples derived from the different steps of the protocol before proceeding with the MS analysis, which requires specialized instrumentation and is more costly. In addition, a method to use catRAPID20, an in-house protein-RNA prediction algorithm, to design de novo RNA specific for a target protein was recently reported. Until recently, the only available pipeline to design DNA/RNA aptamers for a target protein was the SELEX (systematic evolution of ligands by exponential enrichment) approach31. The in silico method enables for a much faster and cost-effective design of RNA aptamers.
The main limitations of this method are associated with the need of working in nuclease-free buffers and tools. Moreover, if it is considered necessary to confirm in vitro the binding between a de novo-designed RNA and a target protein prior PD, the protein needs to be produced and purified and the binding determined with biophysical approaches. This is a limitation that is shared with the production of monoclonal antibodies.
Despite these minor issues, reliable methods to map RNA-protein interactions, such as the one presented here, can bring scientists closer to unveil macromolecular networks and complex main actors of many physiological and pathological mechanisms, such as the ones involved in cancer, cardiomyopathies, diabetes, microbial infections, and genetic and neurodegenerative disorders.
The authors have nothing to disclose.
The authors would like to thank Prof. Tartaglia's and Dr. Cuomo's research group for the support offered. E.Z. received funding from the MINDED fellowship of the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 754490.
6-well tissue culture plates | VWR | 10861-554 | CELLS |
Cell scrapers | BIOSIGMA | 10153 | CELLS |
Dulbecco′s Modified Eagle′s Medium (DMEM) | Thermo Fisher Scientfic | 11995065 | CELLS |
Fetal Bovine Serum, qualified, heat inactivated, Brazil | Thermo Fisher Scientfic | 10500064 | CELLS |
Phosphate Buffer Saline (PBS, Waltham, MA) | Thermo Fisher Scientfic | 14190169 | CELLS |
Trypsin (0.25%), phenol red | Thermo Fisher Scientfic | 15050065 | CELLS |
Anti-rabbit IgG horseradish peroxidase (HRP) | Cellsignal | 7070 | PD |
Biotinylated RNA | Eurofins | Custom RNA oligonucleotides | PD |
Bovine serum albumin | Sigma-Aldrich | A9418 | PD |
Clarity Western ECL Substrate, 500 ml | Biorad | 1705061 | PD |
cOmplete, EDTA-free Protease Inhibitor Cocktail | Merck – Sigma Aldrich | 5056489001 | PD |
NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well | Invitrogen | NP0321BOX | PD |
Recombinant anti-TDP43 antibody | Abcam | ab109535 | PD |
Ribonucleic acid, transfer from baker's yeast (S. cerevisiae) | Merck – Sigma Aldrich | R5636-1ML | PD |
Streptavidin Mag Sepharose | Merck – Sigma Aldrich | GE28-9857-99 | PD |
Trans-Blot Turbo RTA Mini 0.2 µm PVDF Transfer Kit | Biorad | 1704272 | PD |
Acetone | Thermo Fisher Scientfic | 022928.K2 | MS |
C18 cartridge | Thermo Fisher Scientfic | 13-110-018 | MS |
Dithiothreitol (DTT) | Thermo Fisher Scientfic | 20290 | MS |
EASY-Spray HPLC Columns | Thermo Scientific | ES902 | MS |
iodoacetamide (IAA) | Sigma Aldrich S.r.l. | I6125 | MS |
Lys-C/Trypsin | Promega | V5073 | MS |
Trifluoroacetic acid (TFA) | Thermo Fisher Scientfic | 28904 | MS |
Urea | Thermo Fisher Scientfic | J75826.A7 | MS |
Equipment | |||
ChemiDoc imaging system | Bio-Rad | CELLS | |
Dyna Mag -2 , Magnetic rack | Invitrogen | CELLS | |
Forma Series 3 water jacketed C02 incubator | Thermo Scientific | PD | |
PROTEAN II xi cell , power supply for PAGE applications | Bio-Rad | PD | |
Rotating wheel, rotator SB3 | Stuart | PD | |
Water bath set at 37 °C | VWR | PD | |
XCell SureLock Mini-Cell electrophoresis system | ThermoFisher Scientific | MS | |
Easy-nLC 1200 UHPLC | Thermo Scientific | MS | |
Q exactive Mass Spectrometer | Thermo Scientific | MS | |
Software | Version | ||
MaxQuant | 2.0.3.0 | MS | |
Perseus | 1.6.14.0 | MS |