Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach^*

Plasmid Construction for tagged-IRES and tagged-Cap

Dicistronic reporter plasmids pRstF and pRstF-5′UTR (20) were used to generate tagged-Cap and tagged-IRES expression constructs, respectively. To generate a monocistronic reporter plasmid, the NheI and EcoRI sites were used to remove the upstream Renilla luciferase open reading frame and bisect and destroy the subsequent stem-loop. The circular plasmid was regenerated by blunt end ligation. The monocistronic reporters were then linearized (XbaI site) between the Firefly luciferase stop codon and poly(A) signal sequence and a MS2 stem-loop fragment containing four tandem stem-loops was inserted by blunt end ligation (MS2 stem-loop template, SP73-βglobin-(MS2)⁴, was a gift from Klemens Hertel).

Plasmid Construction for MS2-HB

The MS2 coat protein sequence was amplified from pCT119-N55K (gift from David Peabody, University of New Mexico) using a three-piece ligation strategy. To generate a tandem-linked dimer of open reading frames, the first MS2 coat protein in the dimer was generated by PCR amplification of the MS2 coat protein plasmid sequence using a sense primer (5′-AATCTGAGCGGCCGCGCATGGCTTCTAACTTTACTCA-3′) containing a NotI site (italicized) upstream of MS2 sequence and an antisense primer containing a BglI site downstream of the MS2 sequence (5′-ATTCAGCCGTAGAGGCCGGAGTTTGCTGCGATT-3′). The second MS2 coat protein was amplified using a sense primer (5′-ACTCAGGCCTCTACGGCGCAATGGCTTCTAACTTTACTCA-3′) containing a BglI site upstream of the MS2 sequence end and an antisense primer (5′-CCTTAATTAAGGAGTTTGCTGCGATT-3′) containing a PacI site downstream of the MS2 sequence. BglI restriction enzyme sites at the 3′ and 5′ end of each PCR product allowed for blunt end ligation and creation of a tail-to-head tandem placement of two MS2 coat protein open reading frames. The two PCR products were digested with the indicated restriction enzymes and cloned into the HTBH (a derivative of HB tag) tag vector (pQCXIP backbone (18, 19)) linearized with NotI and PacI.

Establishment of Stable Cell Lines (293^MS2-HB)

Two hundred and ninety-three stable cell lines expressing MS2-HB (293^MS2-HB) were generated by retrovirus infection as described (19). Briefly, 293GP2 packaging cells were plated at a density of 7 × 10⁶ cells/10 cm-diameter tissue culture dish and transfected with pQCXIP-MS2-HB retroviral vector using a calcium phosphate protocol. Following 8 h, the medium was replaced with fresh Dulbecco's modified Eagle's medium (DMEM). Thirty-six hours post-transfection, the conditioned medium containing the retroviruses was collected every 8 h for 60 h. Two hundred and ninety-three cells were infected by incubation with equal volumes of fresh DMEM and retrovirus conditioned medium and 4 μg/ml of polybrene. Following 4–6 h, cells were washed and a second infection was performed. Thirty hours postinfection, cells were seeded at 2 × 10⁶ cells/10-cm plate and cultured in DMEM containing the selection antibiotic puromycin (3 μg/ml). Following ∼5 days of selection, cells were seeded at a density of 2 × 10² to 2 × 10³ cells per 10 cm-diameter tissue culture dish. Individual clones were picked from the plates and expanded to generate stable cell clones expressing MS2-HB.

Cell Culture and Metabolic Stable Isotope Labeling Using SILAC (21)

The stable cell line expressing MS2-HB, 293^MS2-HB, was grown in SILAC DMEM (Thermo Scientific, #89985) supplemented with 28 μg/ml ¹²C₆¹⁴N₄-arginine, 73 μg/ml ¹²C₆¹⁴N₂-lysine (Sigma) (light medium) or ¹³C₆¹⁵N₄-arginine and ¹³C₆¹⁵N₂-lysine (heavy medium) purchased from Cambridge Isotope Laboratories (Andover, MA), 10% fetal bovine serum, 1% penicillin/streptomycin, 3 μg/ml puromycin (stable cell selection), and 5 μm biotin (Sigma). Cell lines were grown for more than seven cell doublings in the labeling media to ensure complete incorporation. The cells were then grown to confluence prior to cell lysis.

Cell Transfection, Harvest, and Lysis for Affinity Purification

Two hundred and ninety-three^MS2-HB cells were cultured in 150-mm plates and transfected with the respective tagged-RNAs as described (21–23). Forty-eight hours post-transfection, cells were washed with ice-cold phosphate-buffered saline (PBS), cultures were then immersed in 7 ml 1× PBS, and UV cross-linked using 2400 Stratalinker to irradiate one time for 400 mJ/cm². Cells were harvested by scraping and collected by centrifugation. Cell pellets were lysed using either the native lysis buffer L (100 mm NaCl, 50 mm Tris, 5 mm MgCl₂, 10% glycerol, 0.5% NP-50, RNasIN (Optizyme Ribonuclease Inhibitor, Fisher, 5000×), 1 mm PMSF, Protease Inhibitor (Sigma, 1000×), 50 mm NaF, 0.1 mm Na₄VO₄, 5 mm EDTA, 5 mm EGTA, 0.5 mm β-mercaptoethanol)) or denaturing lysis buffer A-8 (8 m Urea, 300 mm NaCl, 50 mm NaH₂PO₄/NaHPO₄, 0.5% Nonidet P-40) (lysis volumes were chosen to achieve 15–20 mg/ml total protein). Lysate was sonicated using the Branson Sonifier 450 (Setting Duty 50%, Output 4) three times for 15-s intervals. Lysate was centrifuged to remove large debris and clarified by filtration through 1.6 μm GD/X Glass Microfiber filters (Whatman). All steps were performed on ice, with ice-cold reagents.

Affinity Purification

The purification procedure is similar to a previously reported procedure (19, 22). Briefly, Dynal streptavidin M-280 magnetic beads (Invitrogen) were prepared by washing three times with the same buffer used for cell lysis. Amount of beads used varied with amount of total protein in the lysate (∼1 μl bead slurry/20 μg lysate). For native purification, we employed the MAP-SILAC method (mix after purification) to isolate RNA-protein complexes from light and heavy labeled lysates separately to prevent interaction exchange during purification (21). Each lysate was applied to beads in a 1.5-ml tube and rotated at 4 °C for 5 min. An ice-cold magnet was applied to the side of the tube to isolate the beads along with the captured RNP complexes. The flow-through lysate was removed and the beads were washed on ice 2× with ∼50× bed volume of the same buffer used for cell lysis.

For on-bead trypsin digestion, beads were washed again in ice-cold 25 mm NH₄CO₃ (∼30× bed volume) and resuspended with 5–10 ng/μl Promega (Madison, WI) trypsin (diluted in 25 mm NH₄CO₃). Digestion of the sample proceeded at 37 °C for 8–12 h. For denaturing conditions, equal amounts of lysate were mixed from the light and heavy conditions, followed by addition of the magnetic beads (PAM-SILAC, purification after mixing) (21). The steps thereafter were the same as in native purification. Optional TEV digestion and elution: For native purification, TEV (Tobacco Etch Virus Protease; Invitrogen) digestion can be used to release MS2-HB and captured RNA/protein complexes. Following the wash step with lysis buffer, complexes were washed again with 30× bed volume of ice-cold TEB buffer (50 mM Tris-HCl pH 8.0, 10% glycerol, 0.5 mM EDTA, 1% Triton X-100, 1 mm DTT), then incubated with two bed volumes of TEV (0.25 U/μl, diluted with TEB) for 1 h at room temperature. A magnet was applied to collect the elution and followed with trypsin digestion.

Mass Spectrometric Analysis-Liquid Chromatography (LC) MS/MS

Tryptic digests were first separated by strong cation exchange chromatography using a polysulfoethyl A column (2.1 mm i.d. × 10 cm long) (Nest Group) at a flow rate of 200 μl/min using AKTA Basic 10 (GE Healthcare) (21). Peptide elution was achieved by a salt gradient (0–350 mm KCl), and fractions were manually collected based on UV absorbance at 215 nm. The collected fractions were desalted using Vivapure C18 microspin columns (Vivascience, Aubagne, France) prior to LC MS/MS. LC-MS/MS was carried out by nanoflow reversed-phase liquid chromatography (RPLC) (Eksigent, Dublin, CA) coupled online to a linear ion trap (LTQ)-Orbitrap XL mass spectrometer (Thermo-Electron Corp) (24). The LC analysis was performed using a capillary column (100 μm i.d. × 150 mm long) packed with Inertsil ODS-3 resin (GL. Sciences, CA); the peptides were eluted using a linear gradient of 2% to 35% B in 85 min at a flow rate of 350 μl/min (solvent A, 100% H₂O-0.1% formic acid; solvent B, 100% acetonitrile-0.1% formic acid). A cycle of one full Fourier transform scan mass spectrum (350–1800 m/z, resolution of 60,000 at m/z 400) followed by 10 data-dependent MS/MS acquired in the linear ion trap with normalized collision energy (setting of 35%). Target ions already selected for MS/MS were dynamically excluded for 30 s.

Database Searching for Protein Identification and Quantification

The MS data was extracted and analyzed as described (24). Monoisotopic masses of parent ions and corresponding fragment ions, parent ion charge states, and ion intensities from LC-MS/MS spectra were extracted using in-house software based on Raw_Extract script from Xcalibur v2.4. Following automated data extraction, the resultant peak lists for each LC-MS/MS experiment were submitted to the development version (5.3.0) of Protein Prospector (UCSF) for database searching using a concatenated Swissprot database (857302 sequence entries) composed of a SwissProt database (3/24/2009) and its randomized version.. Homo sapiens was selected as the restricted species. Trypsin was set as the enzyme with a maximum of two missed cleavage sites. The mass tolerances for parent and fragment ions were set as 20 ppm and 0.8 Da respectively. Chemical modifications such as protein N-terminal acetylation, methionine oxidation, N-terminal pyroglutamine, and deamidation of asparagine were selected as variable modifications. For SILAC experiments, ¹³C₆¹⁵N₄-Arg and ¹³C₆¹⁵N₂-Lys were also chosen as variable modifications. The Search Compare program in Protein Prospector was used for summarization, validation, and comparison of results. A false positive (% FP) rate of ≤0.2% was used for peptide identification calculated in Search Compare (24). At this false positive rate, false protein hits from the decoy database were not observed. General protein identification was based on at least two peptides. If a protein was identified by multiple peptides from more than two preparations, it was considered as a hit in a preparation identified by only one peptide. For SILAC experiments, the Search Compare program was also used to calculate the relative abundance ratios of Arg/Lys-containing peptides based on ion intensities of monoisotopic peaks observed in the LC MS spectra. The SILAC ratios were further validated by checking all of the raw spectra within the Search Compare program. The ratio outliers (with >30% standard deviations) were excluded for further calculation. If the peptide peaks were mixed with other peptide peaks, they were excluded for quantification. The SILAC ratios reported here were average values, and the accuracy and significance of the measurements were evaluated using standard deviations. The ratios obtained from replicate experiments were examined manually. The resulting SILAC ratios were normalized to the MS2-HB protein in each sample.

Western Blot Analysis

Cell lysates and purified samples were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting. Blots were probed (1:1000) with antibodies purchased from Thermo Fisher Scientific (Streptavidin-HRP), Cell Signaling (eIF4A-1, #2013; eIF-2A, #9722, LEF-1, #4777; Lamin A/C, #2286) (Beverly, MA) and Abcam (PSF, #ab38148; hnRNP H/F, #ab10689; FUS, #ab23439; NonO, #ab45359; YB-1, #12148) (Cambridge, MA) and Zymed Laboratories Inc. (β-tubulin, #32–2600) (San Francisco, CA). PCBP2 antisera was a gift from Bert Semler.

Transient Transfections and Reporter Assays

For luciferase reporter assays 2 × 10⁵ cells were seeded 18–24 h prior to transfection. FuGENE (Roche) transfection reagent was used according to the manufacturer's protocol to transfect 500 ng of the respective Firefly luciferase reporter vector and 100 ng of a control CMV β-galactosidase reporter vector. Cells were washed with 1× PBS-EDTA and lysed with 1× passive lysis buffer (Promega) 24 h post DNA transfection. Cell lysates were assayed for luciferase activities with luciferin substrate (Sigma) using a SIRIUS luminometer (Berthold Detection Systems, Oak Ridge, TN). β-galactosidase activities were determined using the Galacton-Plus substrate (Applied Biosystems, Foster City, CA); these activities served as an internal control for transfection efficiency. Firefly luciferase/β-galactosidase ratios were calculated to determine normalized luciferase values for each sample. Lysate for dicistronic reporter assays were prepared similarly and analyzed using the Dual Luciferase Reporter Assay System (Promega, Seattle, WA) according to the manufacturer's protocol.

siRNA Knock-down

Monolayers of cells were seeded (2 × 10⁵ cells) 24 h prior to transfection of siRNA (Qiagen, Valencia, CA, USA) using the Qiagen Hiperfect Transfection reagent. Two siRNA transfections (3 nm siRNA) were performed over a 48 h period. Following 48 h, fresh media was added and cells were transfected with dicistronic reporter vectors using FuGene 6 transfection reagent according to the manufacturer's protocol. Cells were harvested following 24 h for Western blot and luciferase activity analysis.

RNA Isolation, Northern Blot Analysis

Total RNA was isolated from cells and streptavidin bead purification using TriZol reagent (Invitrogen) according to the manufacturer's instructions. RNA was denatured in Northern Max Formaldehyde Loading Dye (Ambion, Austin, TX) at 65 °C for 5 min and resolved on a 1.2% formaldehyde (37%) agarose gel at 5 V/cm (100V) for 2 h. RNA was transferred to a Brightstar-Plus (Ambion) membrane by vacuum transfer (Bio-Rad Vacuum Blotter) with 10× SSC/10 nm NaOH for 2 h. Following transfer, membranes were briefly rinsed with ddH₂O, UV-irradiated, stained with 0.04% methylene blue dye (in 0.5 m NaOAc) for 1 min, and rinsed with ddH₂O to visualize the ribosomal 28S and 18S RNAs. Membranes were then prehybridized at 60° in ExpressHyb Hybridization Solution (BD Biosciences) with 0.2 mg/ml salmon sperm DNA (Stratagene, La Jolla, CA) for 2 h in a hybridization chamber. The membranes were hybridized in hybridization solution with 20 μg/ml yeast tRNA (Invitrogen) and random prime [³²P]-labeled probes (3 × 10⁶ cpm/ml) against the Firefly luciferase (FLuc) coding region sequence at 60 °C, overnight. To generate a Fluc-specific probe, a ∼800 bp DNA fragment of the Fluc coding region served as the DNA template to generate radiolabeled probes using the MegaPrime DNA Labeling System (GE Healthcare). Following probe hybridization, membranes were washed twice with SSC/0.1% SDS, 60 °C and subsequently washed twice in SSC/0.5% SDS at 65 °C. Bands were visualized using a phosphorimager and Quantity One version 4.3.0 software (Bio-Rad).

Semi-quantitative RT-PCR

First-strand cDNA synthesis was performed using oligo(dT) primers and iScript cDNA synthesis kit as directed by the manufacturer (Bio-Rad). Quantitation of target transcripts was performed by real-time PCR using SYBR Green qPCR master mix (SABiosciences) with an ABI Prism HT7000 cycler (Applied Biosystems). Primer sets were as follows: Firefly luciferase sense 5′-ACGCAGGTGTCGCAGGTCTTC-3′, antisense 5′-TTCGGTACTTCGTCCACAAACACA-3′ and internal control UBA52 sense 5′-AGACAAGGAGGGTATCC-3′, antisense 5′-TGAAGGCGAGCATAGC-3′.

Immunofluorescence

To visualize MS2 coat protein localization 24 h post-transfection of the respective reporter plasmids, 293^MS2-HB cells were cross-linked with 3.7% formaldehyde, blocked with 5% normal goat serum (Vector Labs, Burlingame, CA) for 1 h at room temperature and hybridized with α-MS2 (1:500; Tetracore) at 4 °C overnight. Cells were washed with 1× PBS and incubated with phalloidin (F-actin staining) and secondary goat α-rabbit antibody conjugated to Alexa Fluor 594 (1:1000; Molecular Probes/Invitrogen) for 1 h at room temperature. Subsequently, cells were washed, stained with Dapi (nucleus), and mounted on glass slides. Images were captured using a Zeiss LSM 510 Meta confocal microscope and analyzed with LSM5 Image Browser (Zeiss).

PANTHER and STRING Analysis (25, 26)

PANTHER (Protein analysis through evolutionary relationships) Classification System is a database of annotated gene and gene functions based on their evolutionary relationships. This database was used to determine the types and general function of the proteins purified (see Fig. 3A). Gene IDs were submitted to the database and sorted by GO molecular function. Minor modifications were made to the PANTHER categorization to combine all nucleic acid associated protein category into a single nucleic acid binding category. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database displays predicted protein-protein interactions based on various sources (genomic context, high-throughput experiments, co-expression, and literature). To illustrate the possible interactions between the purified factors, gene IDs for the enriched subset were submitted for analysis.

Design and Expression of MS2-HB and Stem-loop Tagged-RNA

To capture a comprehensive set of IRES regulatory factors, we aimed to develop a new method that integrates an RNA/Protein tagging system with quantitative mass spectrometry. Our approach utilizes the bacteriophage RNA binding protein MS2 to capture RNAs (Fig. 1A) (15, 17, 27). MS2 dimers bind specifically and with high affinity to a RNA stem-loop target sequence (11, 15, 16, 28, 29), which enables MS2 to capture target RNAs in vitro from cell extract systems including target RNA assembled in spliceosome or exon junction complexes, or even to visualize mRNA target localization in living cells (12, 15, 17, 30–34). To create an in vivo target for MS2 in living cells, we constructed a LEF1 IRES expression plasmid in which the 1.2 kb IRES region was cloned upstream of Firefly luciferase coding sequences (Fig. 1A). To tag the mRNA produced from this plasmid, four MS2-targeted stem-loops (tagged-IRES) were cloned at the 3′ end of the luciferase open reading frame (Fig. 1A). Similarly, a stem-loop tagged-Cap construct lacking the IRES element was engineered and used to analyze canonical cap-dependent translation. In addition, the tagged-Cap served as the control RNA for quantitative identification of specific IRES interacting proteins during SILAC-based mass spectrometry analysis. Each mRNA sequence was cloned into a modified pRL-SV40 luciferase reporter plasmid with the RNA polymerase II SV40 promoter/enhancer directing transcription (35). Other features include an intron flanked by a set of splice sites placed ∼200 nucleotides downstream of the transcription start site, and a polyadenylation signal downstream of the luciferase stop codon. Splicing and polyadenylation signals ensure that the expressed mRNAs follow basic steps of RNA processing, so that each transcribed mRNA should be capped at the 5′ end with a 7-methylguanosine cap, marked at the site of splicing, and finished with a polyadenylated 3′ end.

In order to effectively isolate MS2-associated RNA-protein complexes, a eukaryotic expression vector for MS2 was constructed in which the MS2 protein was tagged at its C-terminal end with a HTBH tag (19), a derivative of the HB tag (18). The HB tag is a novel and versatile tandem affinity tag that can be used for purification of protein complexes under native or fully denaturing conditions (18, 19). The HTBH tag consists of two 6×-histidine clusters separated by a TEV cleavage sequence and an in vivo biotinylation site (19) (Fig. 1A). The high affinity of biotin for streptavidin (K_d = 10⁻¹⁵ m) enables efficient isolation of endogenously biotinylated HB-tagged proteins and allows for stringent washing conditions to eliminate nonspecific background binding (18). To significantly increase the affinity and target specificity for the RNA stem-loop sequence (16, 17), an HB-tagged MS2 mutant (N55K) dimer was generated by linking two copies of the N55K open reading frame followed by the HB sequence. Others have shown that a linked dimer has enhanced stem-loop recognition and suppressed nonspecific interactions with other proteins (16, 17). As shown in Fig. 1B, co-expression of the MS2-HB dimer with the stem-loop tagged RNA was not detrimental to Firefly luciferase production by either the IRES or Cap transcripts, instead, it may have provided mRNA stability and a modest increase in expression. To minimize overexpression, a 293 stable cell line expressing MS2-HB (i.e. 293^MS2-HB) was generated by retroviral infection. The localization of MS2-HB was detected in both the nucleus and cytoplasm of 293^MS2-HB cells, and was not significantly altered upon co-expression of tagged-IRES or tagged-Cap RNA (Fig. 1C).

MS2-HB Based Purification of Stem-Loop Tagged RNAs

Because RNAs are labile in cellular extracts, rapid isolation of RNAs from cell lysates is preferred. To this end, we first tested a single-step purification procedure using streptavidin magnetic beads to isolate MS2-HB associated RNA-protein complexes under native conditions. Because of its high capacity and ease of use, streptavidin magnetic beads allow rapid and efficient capture of MS2-HB in 5 min from 293^MS2-HB cell lysates (>95%) (Fig. 1D). To assess purification background, we analyzed purified MS2-HB samples in the absence or presence of a target RNA. As a result, RNA-independent binding to the streptavidin beads and/or MS2-HB ranged between 17–26% (supplemental Fig. S1), indicating that the purification procedure has good specificity for MS2-HB associated RNAs. In order to evaluate the integrity of stem-loop tagged-RNAs in purified samples and their capture efficiency, Northern blot analysis and semi-quantitative RT-PCR was performed. As shown in Fig. 1E, full-length tagged-IRES mRNA (∼3kb) was specifically copurified with MS2-HB, whereas no IRES RNA was isolated in the absence of MS2-HB (Fig. 1E, compare “bound” lanes). In addition, semi-quantitative RT-PCR analysis indicated that our method has captured ∼1% of both tagged-RNAs from whole cell extracts (Fig. 1F, and data not shown). Together, the results have demonstrated that MS2-HB is effective for rapid isolation of functional stem-loop tagged RNAs.

MS2-BioTRAP Strategy for Quantitative Profiling of RNA-Protein Complexes

In order to isolate and identify specific RNA-protein complexes, we have developed an integrated proteomic approach coupling MS2-BioTRAP with SILAC-based quantitative mass spectrometry (21, 36). The general workflow is displayed in Fig. 2. As shown, one population of 293^MS2-HB stable cells was grown in light-labeled (¹²C¹⁴N-Arg/Lys) media and another population was grown in heavy-labeled (¹³C¹⁵N-Arg/Lys) media (Fig. 2). Each culture was propagated extensively (at least seven doublings) to permit sufficient incorporation of stable isotope labeled amino acids. To identify IRES specific interacting proteins, the stem-loop tagged IRES target RNA was transiently expressed in the light culture, whereas the stem-loop tagged Cap control RNA was transiently expressed in the heavy culture (Fig. 2). To obtain authentic RNA-protein complexes, UV irradiation was applied to living cells to freeze RNA-protein interactions prior to cell lysis. In this work, two purification strategies were employed as illustrated in Fig. 2 (Path I and II), in an effort to generate a comprehensive in vivo RNA-protein interaction profile.

The first strategy (Fig. 2, path I) involves the isolation of MS2-HB associated RNA-protein complexes under native conditions, in which the MAP-SILAC (mix after purification) method was employed to prevent the exchange of dynamically interacting proteins between their light and heavy labeled forms as previously reported for native purification in a standard SILAC protocol (21, 37). Using this method, cells in the light and heavy cultures were lysed separately. RNA-protein complexes were subsequently purified from the tagged-IRES (light) and tagged-Cap (heavy) containing lysates by binding to streptavidin magnetic beads. The resulting purified samples were then mixed equally for trypsin digestion on the beads. This strategy presumably allows the capture of cross-linked RNA-protein as well as noncross-linked protein-protein interactions; however, nonspecific background might be high. Alternatively, the bound proteins can be directly eluted with TEV cleavage (see Experimental Procedures).

In comparison, the second strategy (Fig. 2, path II) employs the purification of MS2-HB associated RNA-protein complexes under fully denaturing conditions (e.g. 8 m urea), permitting the capture of only cross-linked RNA-protein interactions. With this strategy, the PAM-SILAC (purification after mixing) method was used because it is best suited for isolating cross-linked complexes under denaturing conditions (37). In this strategy, equal amounts of cell lysates from the two compared samples were mixed first and then HB-tag based purification was carried out. Although the HB tag allows two-step purification, we only performed one-step purification by binding to streptavidin beads. Single-step isolation enabled a fair comparison of the results from both strategies. As for the PAM purification under native conditions, denatured proteins bound to streptavidin beads were directly digested with trypsin on-bead for subsequent analysis.

Trypsin digests from both strategies were analyzed by LC MS/MS for protein identification and quantification. With SILAC-based quantitative mass spectrometry, the relative abundance of a given protein in the two compared samples can be determined by the relative abundance ratios (i.e. SILAC ratios) of its arginine/lysine containing peptides (i.e. light-labeled versus heavy labeled) measured by mass spectrometry. If proteins specifically interact with the tagged-IRES RNA, relative abundance ratios (i.e. SILAC ratios) of their light labeled tryptic peptides (from the tagged-IRES sample) to the corresponding heavy labeled peptides (from the tagged-Cap sample) should be different from 1 (17). However, if proteins bind at equal levels to both tagged-RNAs, relative abundance ratios of their tryptic peptides should be close to 1. Thus a large set of RNA binding factors that bind to the tagged-IRES versus the tagged-Cap control RNA can be quantitatively determined.

Quantitative Profiling of IRES RNA Interacting Proteins

With the strategies described above, we have carried out two native and two denaturing purifications for four independent pair-wise comparisons. As a result, the SILAC ratio of MS2-HB was determined as 1.2 ± 0.2 in all four experiments, demonstrating that MS2-HB is isolated uniformly using our purification strategies. Uniform isolation of MS2-HB thus enabled normalization of SILAC ratios of proteins in each experiment to SILAC ratios of MS2-HB for parallel comparison among multiple samples. Based on a threshold previously established by immunoblotting (38), proteins with normalized SILAC ratios greater than 1.5 were determined as specific, enriched interactors of the tagged IRES RNA.

From the four experiments, a comprehensive list of proteins was identified. This list is composed of only those proteins that were consistently detected in the two replicate experiments for each purification strategy. Proteins identified by a single peptide were not included in a list unless they were identified in both replicate purifications. We reproducibly captured a total of 535 proteins from the native purification and 326 proteins from the denaturing purification (Fig. 3A; supplemental Table S1 and S2). The majority of the proteins (75%) captured under denaturing conditions were also captured under native conditions. The large overlap suggested that the more stringent denaturing condition retained relevant factors whereas removing indirectly interacting (components of complexes) or nonspecific binding factors that are isolated by the less stringent native purification. Categorization of proteins in functional categories using PANTHER classification tools defined the overall spectrum of proteins isolated in each purification condition (Fig. 3A) (26). Overall, the classification profiles were similar for native and denaturing conditions, with nucleic acid binding proteins the most prominent category, and the category with the greatest difference between native and denaturing conditions.

Further analysis of nucleic acid binding proteins revealed the greatest number of common and IRES-enriched regulatory factors (Fig. 3B). Panther analysis identified five subcategories of nucleic acid binding proteins isolated in each condition: “mRNA processing,” “other RNA binding proteins,” “translation,” “hnRNPs,” and “DNA related factors.” The diverse functions represented by these subcategories suggest that RNA was captured from multiple subcellular compartments with the target RNA undergoing different processes from transcription to translation (Fig. 3B; supplemental Table S3 and S4). Immunofluorescent detection of MS2-HB in both the nuclear and cellular space correlated with this result (Fig. 1C). To understand the interaction specificity of the identified proteins with RNAs, we have categorized them into two groups: (1) IRES-enriched proteins (SILAC ratios >1.5), indicating that these proteins either preferentially interact with the IRES, or they are relatively more abundant in the IRES-RNA complex (Fig. 3B) and (2) commonly purified proteins (SILAC ratios ≤1.5), meaning that they bind equally well to the tagged-IRES and tagged-CAP matrix (38).

Common Interacting Proteins

Common interacting factors were predominantly translation and mRNA processing subunits of complexes predicted to be equivalently associated with both target-RNAs. These include components of poly(A) binding complexes, splicing complexes, and translation complexes (Fig. 3B). Some of these complexes were isolated more intact than others, and not surprisingly, were better represented in the native condition. For example, 53 of 81 known ribosomal subunits were isolated as common interacting proteins under native conditions as was all subunits of translation elongation factors EF-1 and EF-2. In the denaturing condition, 27 ribosomal subunits, but no translation elongation factors were purified as common interactors. Likewise, at least one subunit from each of the translation initiation factors (eIF 1, 2, 3, 4, 5, and 6), core components of the core exon junction complex (eIF4A-III, WIBG), and several of its outer shell and auxiliary factors were isolated as common interactors in the native condition (UAP56, Aly/REF, RBM8A, RNPS1, and ACIN1). With the denaturing purification, several translation initiation subunits and ribosomal subunits were enriched with the tagged-IRES (see below). Splicing and processing factors were also purified as common interactors, including subunits and auxiliary factors for each of the snRNPs (U1, U2, U4, U5, and U6), and poly(A) binding proteins PABP1, PABP2. Other complexes expected to interact equally well with tagged-IRES and tagged-CAP mRNAs were not detected, such as the nuclear and cytoplasmic cap binding complexes and proteins associated with sites of RNA decapping, degradation, and/or storage (stress granules or P-bodies). The observation that many of the commonly associated proteins are involved in splicing and translation and not mRNA degradation suggests that MS2-HB captured pools of newly processed RNA in the nucleus as well as actively translating RNA-protein complexes in the cytoplasm.

HnRNP and DNA binding proteins from other nucleic acid binding protein categories were also identified as common interacting proteins in both native and denaturing conditions. The hnRNP category represents a group of abundant RNA binding proteins that shuttle between the nucleus and cytoplasm. Even though many hnRNPs have been characterized as IRES interacting proteins, the majority of the hnRNPs we isolated were common to both tagged-IRES and tagged-Cap RNAs (17 hnRNPs isolated in the native- and 12 hnRNPs isolated in the denaturing-conditions). Likewise, proteins in the DNA binding protein category were isolated in all conditions and were the largest subcategory of nucleic acid binding protein commonly bound to RNAs in the native purification. Much of this binding is most likely to be nonspecific and/or indirect because denaturing purification eliminated most of these interactions. Taken together, the types of common interacting proteins detected in our analysis suggest that MS2-HB captured pools of newly processed RNA in the nucleus as well as actively translating RNA-protein complexes in the cytoplasm.

IRES Interacting Proteins

Compared with the number of proteins common to both tagged-RNAs, a much smaller number of nucleic acid binding proteins were enriched on the tagged-IRES (Fig. 3B). These proteins are components of chromatin, mRNA transcription, processing, export, and translation. Some of these proteins are multifunctional nuclear proteins found in several complexes associated with various RNA processes (SFPQ/PSF and NONO/p54nrb) or even with IRES activities (PCBP2 and ELAVL1/HuR) (1, 7, 8, 39, 40). Others are uncharacterized RNA binding proteins and helicases not previously associated with eukaryotic or viral IRESs (EWS and FUS) (41, 42). Interestingly, several proteins involved in general translation were highly enriched with the IRES target. These include translation factors (eIF4A-1, eIF-2A, and eIF3g) and ribosomal subunits (RPS7, RPS19, and RPL26) with known canonical as well as noncanonical functions (43–47). That selected subunits of the translation machinery might be specifically enriched with an IRES template has significance for how IRES and cap translation might differ (see Discussion). Under stringent denaturing conditions, 36 IRES-enriched nucleic acid binding proteins were isolated (Fig. 3B) and subjected for further analysis using STRING to generate an interaction network based on known and predicted protein interactions from multiple databases (www.string-db.org). The STRING results show that the IRES-enriched proteins are highly interactive and that the central core of this interaction network is comprised of RNA binding proteins, some of which are known ITAFs (ELAV1/HuR and SFPQ/PSF) (Fig. 4) (1, 25). In summary, use of the MS2-BioTRAP system with complex extracts from whole cells appears to provide a global snapshot of putative LEF1 IRES regulatory factors that assemble onto mRNA.

Comparison of IRES Binding Specificity by SILAC Analysis

In this work, we have used protein SILAC ratios (>1.5) as the quantitative measure to determine specific IRES interacting proteins purified under either native or denaturing conditions. During the analysis, we noticed that the SILAC ratios of the specific IRES interactors indicated a moderate level of enrichment in our purifications. This may be attributed to the nature of proteins associated with RNA as many of them are abundant proteins. In addition, it is possible that the purification methods retained sufficiently high levels of indirect or background binding proteins such that SILAC ratios were masked. For example, a protein that binds specifically to the IRES and nonspecifically to other features of the RNA or the bead matrix will be detected as a protein with a modestly enriched SILAC ratio. To assess how many enriched IRES binding proteins identified in the stringent denaturing or gentle native purification condition also engage in background binding, we performed an additional SILAC analysis. This analysis was performed in the absence of any RNA target to reveal background binding of proteins to MS2-HB protein and streptavidin beads. Under these conditions, any proteins purified were expected to bind equivalently and be detected as peptides with SILAC ratios close to 1.0. We compared the list of proteins isolated as background with the list of IRES-enriched nucleic acid binding proteins (excluding proteins identified by a single peptide; supplemental Table S6 and S7). One out of the 35 IRES-enriched proteins identified in the native purifications could be purified without the RNA target. This suggests that the majority of proteins isolated with a tagged-RNA target are specific interactors. It should be noted however, that the average SILAC ratio of background binding proteins was 1.42 ± 0.29, a range of values that comes close to the range of SILAC ratios for IRES-enriched proteins (1.84 ± 0.32). Under denaturing conditions, 16 of 36 IRES proteins were detected in the background binding experiment. Although this represents a significant degree of overlap in protein identification, the SILAC ratios of the 16 shared proteins were significantly lower in the background binding experiment (1.06 ± 0.16) than their enriched ratios in the presence of tagged-IRES (Fig. 5C). Therefore, these 16 factors are true IRES binding candidates. These results demonstrate that although our procedure will isolate “sticky” or abundant proteins that interact nonspecifically with the bead matrix, SILAC ratios can reliably distinguish true interactions—even for abundant proteins that exhibit both modes of binding (supplemental Table S6). In fact, when we consider the overall spread of average SILAC ratio for IRES-enriched proteins versus their background counterparts, we find that the ratios are separated by three standard deviations (1.86 ± 0.50 versus 1.06 ± 0.16) (Fig. 3C). Graphical representation of the SILAC ratios in the native and denaturing purifications highlight how the denaturing condition reduces background and ensures a more reliable SILAC ratio (Fig. 3C). We conclude that either purification condition reliably detects RNA-associated factors above background, and that the SILAC ratios derived from the denaturing purification condition enable a more reliable distinction between enriched and common sets of proteins.

Validation of Selected IRES Binding Factors

For a validation test of the results from the native and denaturing isolations, eight proteins were chosen for RNA-protein complex purification and Western blot analysis (Fig. 5A). Target factors were selected based on reproducible enrichment in the denaturing purifications (SFPQ/PSF, hnRNP H, hnRNP F, and FUS), and/or for their known or potential connections to other IRESs and each other (SFPQ/PSF and NONO/p54nrb, paraspeckle factors; FUS, TEF family member; hnRNP H, hnRNP F, and PCBP2, hnRNP proteins) (39, 41, 48). We also included two translation factors identified as IRES-enriched in the native purification (eIF-2A, eIF4A-1) to test the validity of enrichment under less stringent conditions. UV cross-linking and native purification were performed for both tagged-IRES and tagged-Cap RNA and the relative levels of capture analyzed by Western blot. Preferential enrichment with the IRES target was detected for all eight factors regardless of whether they were identified as enriched in native or denaturing purifications. Interestingly, the extent of IRES enrichment detected by immunoblotting ranged from 1.5- to greater than 30-fold where eIF4A-1, an RNA helicase component of the cytoplasmic cap binding complex, showed the greatest level of enrichment (34-fold).

To test for a functional link between validated proteins and IRES activity, we interfered with ITAF function using either siRNA knockdown methods or a small molecule inhibitor (Fig. 5B). For siRNA knockdown, a dicistronic vector, which assesses cap-dependent translation of the first open reading frame (Renilla Luciferase) and IRES-dependent translation of the second open reading frame (Firefly Luciferase), was used to compare effects on both modes of translation. We targeted proteins that were enriched on the IRES as well a factor common to both tagged-IRES and tagged-Cap (YB-1, SILAC ratio <1.5). YB-1 is a known eukaryotic ITAF for a heterologous IRES in the cMYC messenger RNA (7). Reduced levels of YB-1 by siRNA (85% knockdown efficiency) did not significantly affect either cap- or IRES-dependent translation (Fig. 5B) compared with the control scrambled siRNA. Knockdown of some of the more abundant validated proteins was difficult to achieve (NONO/p54nrb, EWS), but was successful for FUS and SFPQ (Fig. 5B). Transient transfection of the dicistronic vector into cells with lower levels of FUS (30% knockdown efficiency) did not have an effect on IRES-dependent translation, but increased expression of cap-dependent Renilla Luciferase almost twofold. Knockdown efficiency of SFPQ was better, with proteins levels reduced to 50% that of the control siRNA knockdown. Under these conditions translation of both cap- and IRES-dependent open reading frames was increased, with the strongest effects observed for the cap-dependent open reading frame. SFPQ is a multifunctional protein, but its role as a paraspeckle protein might have been detected as knockdown could have relieved nuclear retention of the large, highly folded dicistronic luciferase reporter. These results show that, as opposed to a nonenriched protein, which has no effect on the dicistronic reporter mRNA (YB-1), knockdown of two of the IRES-enriched factors show specific effects on expression.

Finally, we tested for the functional relevance of the strongly associating eIF4A-1 RNA helicase (Fig. 5C). We used the small molecular inhibitor Hippuristanol, which binds specifically and directly to eIF4A-1, to inhibit RNA binding. Hippuristanol has previously been shown to interfere with the function of several viral IRESs and IRES activity in cMYC mRNA (43, 44). We used a low dose of Hippuristanol that has no effect on cap-dependent translation (100 nm) and is eightfold lower than the IC50 dose for inhibition of HeLa cell growth (43). We observed a selective, significant inhibition of the tagged-IRES reporter, reducing gene expression by as much as 50% (Fig. 5C). No significant inhibition of the tagged CAP reporter was detected (Fig. 5C). In addition, no effect was detected for expression from the cotransfected control plasmid—a cap-dependent production of β-galactosidase. Thus, the IRES is much more sensitive to inhibition of eIF4A-1 RNA binding activities than conventional cap-dependent translation mechanisms, an observation that may apply to other cellular IRESs (44). To test whether this sensitivity extends to full-length, endogenous LEF1 mRNA, we treated 293 cells with 100 nm Hippuristanol for 12 h and harvested cells for Western blot analysis of endogenous LEF-1 protein (Fig. 5D). We observed a threefold decrease in LEF-1 protein levels, suggesting that as for the tagged-IRES reporter, LEF-1 translation is highly dependent on eIF4A-1 activity (Fig 5D).