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In vivo mRNA display enables large-scale proteomics by next generation sequencing

Significance The need for mass-spectrometric determination of protein identity severely limits the throughput of many whole-cell proteomic analyses. We have developed an approach to physically connect in vivo-expressed proteins to their encoding messenger RNAs (mRNAs). This genotype–phenotype linkage enables us to carry out a variety of whole-proteome assays and determine the identity of each protein using nucleic acid sequencing. We show that the resulting in vivo mRNA display technology enables determination of protein subcellular localization and protein–protein interactions, with high sensitivity and specificity, using simple purification followed by high-throughput sequencing. In vivo mRNA display should enable more efficient interrogation of proteome behavior in a variety of applications that are currently limited by the cost and throughput of mass-spectrometric analysis.


Plasmid Construction
The backbone for all in vivo mRNA display plasmids and respective controls is pSH100 (URA3 selection marker; Addgene #45930) (1). MS2 coat protein (MCP) was PCR amplified from pSH100 (1), while stem-loop sequences from pDZ415 (1) were ordered as Blocks from IDT; defective MCP (MCP*) mutations were introduced via overlap PCR. For cloning, PCR insert fragments for MCP variants and a Gateway cloning ccdB cassette were amplified using Q5 polymerase (NEB Μ0491). The backbone was digested using restriction enzymes (Dataset S1), combined with PCR inserts for Gibson Assembly according to the manufacturer (NEB E2611), and transformed into One Shot ccdB Survival Cells (Invitrogen A10460). The resulting Destination vectors (plPOIVMD156,160,155) allow for Gateway cloning of ORFs flanked by Gateway attL sites in to the display constructs. The in vivo mRNA display constructs in this study are under the control of a MET25 promoter (induced in methionine dropout media). Super folder GFP (2) and mCherry (1) were amplified with flanking attB sites and Entry Vectors were generated via a BP reaction. Individual yeast ORFs were amplified from the ORFeome collection purchased from Dharmacon (3) using the available flanking attB sites. Plasmids expressing SAM2-and ARC40-GFP fusion baits were constructed using Gibson assembly based on the pSH62 (4) backbone (HIS3 selection marker; plIVMD495, 496). DH5α competent cell were used for all bacterial transformations (NEB C2989K). See Dataset S1A-B for a complete list of plasmids in this study, as well as primers and restriction enzymes used for construction.

Yeast Strains
The BY4742 S288c MATα laboratory deletion strain was used as the starting strain for all strains harboring in vivo mRNA display constructs. All plasmids were transformed using the LiAc-PEG-ssDNA method (5), and selected in 2% glucose -URA dropout media. EY0986 MATa deletion strains expressing genomically integrated SAM2-and ARC40-GFP fusions (6) were purchased from Thermo Fischer (Catalog no. 95701; HIS3 selection marker). Plasmids expressing SAM2and ARC40-GFP fusion baits were transformed into BY4741 S288c MATa laboratory deletion strain and selected on 2% glucose -HIS dropout media. For diploid strains, MATa and MATα haploids were mated in YPD at 30 ⁰C with vigorous shaking for 1-2 hours and selected on 2% glucose -URA, -HIS dropout media. Dropout media supplement powders from ForMedium and US Biological were used interchangeably. Single strain selections were plated on appropriate 2% hard agar SC dropout plates. See Dataset S1C for a list of all strains used in this study.
In vivo mRNA display Library Generation E.coli strains from the yeast ORFeome plasmid collection were outgrown, pooled and pelleted.
Pooled plasmid was extracted from the pellets using a Qiagen Maxi-prep kit (#12963). Yeast ORFs were PCR amplified using the attB1 and attB2 flanking sequences. A two-step recombination reaction (first a BP recombination into pDONR221, followed by an LR reaction) was used to transfer the sequence into the Gateway cloning site of the in vivo mRNA display Destination Vector (plIVMD156). BP and LR reactions were transformed in DH5α cells (NEB C2989K) and colonies were selected in semi-liquid soft agarose gel (7) (0.3% Lonza Seaprep #50302) LB media with the appropriate antibiotics (kanamycin and ampicillin for BP and LR reactions respectively). More than 1 million colonies were collected for the BP reaction (~200x coverage) and over 125,000 colonies (~25x coverage) for LR reactions. The final in vivo mRNA display library was transformed into BY4742 using the LiAc-PEG-ssDNA method (5), and selected in 2% glucose SC-URA semi-liquid soft agarose gel (7) (0.3% Lonza Seaprep #50302). Over 500,000 colonies were collected, outgrown for 6 hours in SC-URA and stored at -80 ⁰C in 15% glycerol media. Mated diploid libraries were selected in 2% glucose SC-URA-HIS semi soft agar  For in vivo mRNA display yeast libraries, biological replicates were independently revived from frozen stock and outgrown in semi-liquid soft agarose gel as colonies to avoid any growth biases.
All resulting colonies were pooled and outgrown in liquid SC dropout media as described above.

Excess Coat Protein
Unless otherwise noted, we provided an excess of MS2 coat protein for all high throughput experiments in order to titrate any non-specific interactions. To this end, we mixed into each sample an excess culture of yeast cells that express an MCP fusion that does not display its own mRNA. Additionally, the MCP is not isolated specifically in any given protein assay and its mRNA is not processed during first strand and second strand synthesis. Moreover, lysis and all purification steps were performed at 4 ⁰C since increased temperatures also compromise precision, possibly due to partner exchange ( Fig. S10-11).

Non-Specific Functional Controls for in vivo mRNA display
We included a set of in vivo mRNA display constructs in every library that function as internal negative and positive controls for a given protein purification assay. Their mRNA frequencies provide a background with respect to which we normalize the frequencies of each ORF (Fig. S8-S10). For that, we chose a small set of reporter genes and peptides that should not participate in any biological interactions inside the cell. These control ORFs included GFP (2), mCherry (1), BFP (Addgene #44839), acGFP (pBI-CMV2; Clontech), Firefly Luciferase, Renilla Luciferase (psiCHECK-2; Promega) as well as short peptides derived from these reporter genes. All ORFs were cloned in vectors with an N-terminal MCP, a downstream SL and various purification tags (MYC, FLAG, 6xHIS, HA-tag) such that a subset of them works as a non-specific control set for every protein purification. Moreover, during anti-GFP magnetic bead purifications, the GFP construct works as a positive control for the assay. Additionally, we designed a smaller set of 7 control proteins that harbored an MCP but no SL such that their mRNA progenitors function as non-displaying controls. These proteins are variants of mCherry with 6 additional bases on the 5' and 3' ends of the ORF, so that they can be identified during sequencing. Control strains were added at various concentrations to represent a wide range of construct frequencies and allow us to assess any representation biases and determine read depth cut offs. Overall, the controls represented 2-5% of the total processed cell culture. See Dataset S1E for a complete list of strains and stoichiometry of the control mix.
Additionally, these constructs allow us to assess RNA integrity and the efficiency of our assay post purification but prior to sample preparation for sequencing by means of Quantitative PCR (see relevant Qunatitative PCR Method section). sec disruption pulse at 6m/s followed by 5 minutes of rest in contact with ice cold ethanol packs in between disruptions). Glass beads were removed by a 1 min centrifugation at 7,000 g, and lysate was transferred to a new tube and further cleared by a 30 second spin at 11,000 g. We set aside roughly 100 μl of the resulting sample, which is referred to as the lysate. Additionally, proteins were eluted in 300mM of imidazole and re-purified using a fresh aliquot of His-Tag Isolation Dynabeads (Invitrogen 10103D). To that end, 100 μl of eluted sample was mixed with 1000 μl of Wash Buffer and incubated on a roller, and washed 4 times.

Whole cell lysate preparation
See Dataset S1D for a list of purification assays each library was submitted to.

Crude Mitochondrial Isolation
We processed frozen library pellets (scIVMD580) equivalent to 20 OD600 units of cells per replicate. There was no excess coat protein culture added to the yeast libraries for these purifications. We performed a crude mitochondrial isolation using a commercially available kit from Sigma (MITOISO3). The frozen library cell pellets were resuspended in 2 ml of ice cold Buffer A (Sigma B3311 with 0.02 U/µl SUPERase RNase Inhibitor) and incubated for 15 min at 30 ⁰C with gentle shaking. Next, they were centrifuged at 1,500 g for 5 min, resuspended in 1ml of Buffer B (Sigma B3186 with 0.02 U/µl SUPERase RNase Inhibitor), and supplemented with 40 units of Lyticase Solution (Sigma L2524). Spheroplasts were formed by incubating at 30 ⁰C with gentle shaking for roughly 10 min (until OD600 decreases to 30% of the initial value). The reaction was stopped by centrifuging at 1,200 g for 5 min at 4 ⁰C. Spheroplasts were homogenized in 1 ml of Storage Buffer (Sigma S9689 with 0.2 U/µl SUPERase RNase Inhibitor) with 10 strokes using a pre-chilled sterile Dounce homogenizer at 4 ⁰C (Sigma T2690; P1110). To remove nuclei, samples were centrifuged at 600 g for 10 min at 4 ⁰C. The supernatant was transferred to a new Eppendorf tube and centrifuged at 6,500 g for 10 min at 4 ⁰C. The supernatant was saved for further processing (75 μl for RNA extraction). Storage buffer was added to the pellet and the sample was centrifuged at 6,500 g for an additional 10 min at 4 ⁰C. Supernatant was discarded and the final pellet was saved for further processing.

RNA extraction
We extracted RNA from all protein samples (50 μl of whole cell extract; up to 100μl of purified protein bound on beads; 75 μl of 6,500 g supernatant from crude mitochondrial isolation; or the complete 6,500 g pellet) using TRIzol (Invitrogen 15596026). We added 750 μl of TRIzol reagent to each sample, vortexed and incubated at RT for 5 min. We added 150 μl of chloroform (Sigma C2432), vortexed and incubated for 2 min at RT. Samples were centrifuged at 12,000 g for 15 min at 4 ⁰C. The aqueous phase was transferred to a new tube and mixed with an equal volume of 100% ethanol. The mixture was applied to a spin column, washed and concentrated according to the manufacturer's recommendations (Zymo Research, RNA Clean & Concentrator R1015).
RNA from lysates was eluted in 50 μl, while RNA from purified protein samples was eluted in 15 μl of RNase free water with 0.2 U/µl SUPERase RNase Inhibitor.

cDNA synthesis
For whole cell extract samples, we used 8 μg of purified total RNA as input. For purified protein samples, we used the whole sample. We treated extracted RNA with dsDNase (Thermo Scientific

Quantitative PCR
We assessed extracted RNA for quality and in vivo mRNA display efficiency using qPCR.
Quantitive PCR (PerfeCTa SYBR Green FastMix, QuantaBio 95073-012; on an Applied Biosystems QuantStudio5 384-well instrument) was used to determine the relative abundance of mCherry and GFP transcript in each sample. Protein purification experiments were designed such that either GFP or mCherry is co-purified in the experiment (specific positive control) and the other is washed away (non-specific reference). We calculated a ΔCt value for each sample and a -ΔΔCt between purified sample and input lysate. Therefore the Log2 Fold Enrichment is: For random hexamer RT (Fig.1D), the relative abundance to ACT1 was quantified as a reference.
For each sample, technical duplicate replicate measurements were made, and if they were inconsistent, they were repeated for quadruplicates resulting in the reporting of average values and standard deviations as error bars (Fig. S1, S10, S11, S12). For Fig. 1D-E, purification experiments were conducted in biological replicates, and averages of biological replicates are reported as bars and replicate values as grey dots. See Dataset S1A for qPCR primers. The smaller reaction was split in two technical replicates and cycled on a qPCR machine.
Amplification was observed to determine the number of cycles needed or the amplification to reach the exponential phase (or roughly 30% of the maximum signal) and the number of cycles were noted (11

In vivo mRNA display Sequencing Data Analysis
After de-multiplexing Illumina indexes, we used Cutadapt (12) to trim low-quality sequences and trim universal 3' adapter sequences corresponding to the Y-Linker primers. Cutadapt was also used to de-multiplex internal custom indexes, and remove universal 5' adapter sequences.
Surviving reads of sufficient length (>20 nt) were mapped to the 5' and 3' ends of all yeast ORFs using Bowtie (13). If the sum of all reads mapping to the 5' end (or the 3' end) of each ORF is ( ), we can calculate an average log frequency, for the 5' fragments, the 3' fragments and the average: where represents the frequency for ORF , and ∑ represents the total sum of reads for all the 5' (or 3') end reads . If is the set of constructs in the non-specific functional control library, we calculate a log normalized frequency, ℎ , for each ORF with respect to the control set: The Display Score for each ORF between two matched samples is calculated as the difference of the log normalized frequencies between the samples. For example, the Display Scores for ORF for a protein purification experiment are: where ℎ is the log normalized frequency in the purified protein sample and ℎ is the log normalized frequency in the input whole cell extract. An ORF is considered to be present in an experiment only if it had more than 8 reads in either the input or the purified sample (for Fig. 2D-G a threshold of 4 reads was set based on the distribution of reads). An ORF is considered present in an assay with replicates, if it is present in half or more of the 3' and 5' samples of all the replicates. The Display score represents an enrichment ( > 0) or depletion ( < 0) of the reads of ORF in the purified sample compared to the lysate with respect to the non-specific functional controls. The distribution of the non-specific functional controls can be used to calculate a z Score for the Display Score: where is the standard deviation of the Display Scores of the non-specific functional controls, and = 0 by definition. Z Scores for biological replicate experiments were averaged using the Stouffer rule. Display Score p-values for biological replicates (Fig. 2D-G, Fig.   3 and Fig. 4) were calculated by comparing the distribution of and measurements for every ORF to the distribution of Display Scores for all the non-specific functional controls using a Mann-Whitney U test.

Western Blots
We treated 10μl of Lysate and 40% of the purified protein bound beads in 2xSDS Sample Buffer

Mass Spectrometry
Immunoprecipitation and in-gel digestion: SAM2-and ARC40-GFP were purified as described above from in vivo display libraries using anti-GFP magnetic beads. Samples were processed in biological duplicate. Protein-bound beads were washed three times with ultrapure water. Samples Fisher's exact test for the combined counts of every protein in the SAM2 and the ARC40 samples.

GO Term Analysis for Crude Mitochondrial Isolation
We considered mutually exclusive GO Term categories. For cytosolic, cytoplasmic and nuclear proteins, we filtered out the genes common to other organelle and membrane fractions category compared to the significant Display Scores present in the assay (Fig. 3). We calculated calculate enrichment of genes in organelle and membrane categories with respect to cytosolic (GO:0005829) proteins.

Supplementary Datasets
Supplementary Dataset S1 (Dataset S1A) Catalogue of primers used for in vivo mRNA display processing and sequencing, primers for plasmid construction, and primers for Quantitative PCR analysis.
(Dataset S1B) Catalogue of plasmids used in this study.
(Dataset S1C) Catalogue of strains used in this study.
(Dataset S1D) Catalogue of in vivo mRNA display libraries generated for this study.
(Dataset S1E) Non-specific functional controls for in vivo mRNA display.

Supplementary Dataset S2
Assessment of in vivo mRNA display precision NGS sequencing results. Log2 Reads for Lysates and Purified Samples, Log Normalized frequencies, Display Scores and Display z Scores per each ORF. ORFs specific and non-specific to each purification are noted. Data corresponds to While the presence of a SL allows for in vivo mRNA display in the MCP constructs, it has no effect for MCP*. (C) In vivo mRNA display is independent of purification tags used. Constructs (mCherry and GFP) with no stem-loop and a single hairpin purified with anti-HIS, -MYC, -FLAG, -RFP, and -GFP magnetic beads. (D) Similar to C, but Log Fold Enrichments were calculated with respect to a housekeeping gene in each purified sample and normalized to the lysate. qPCR averages are shown as bars and SD of technical replicates as errors.       Percentage of protein and mRNA in the flow-through and purified fractions with respect to the levels in the input sample. We tested a single step purification for two HIS-tagged (A, B) and one FLAG-tagged (C) construct using reduced salt concentration to avoid excessive loss of RNA and protein. Percentages of protein and RNA levels were calculated with respect to the total input whole cell extract for each strain (using fluorescence and qPCR respectively). Tagged protein constructs were purified in similar amounts with specificity independent of the presence of a stem loop (left-most column). To assess the percentage of cognate mRNA co-purified during the isolation process, we quantified RNA levels of specific and background RNA for constructs with and without stem loops in their 3'UTR. For all three panels, the MCP fusion constructs with no stem-loop co-purified similar levels of construct specific and ACT1 mRNA. In contrast, for the MCP constructs with a stem-loop, purification of the protein resulted in enriched construct specific mRNA with respect to both the reference and the construct with no stem loop. In addition, for the MCP constructs with a stem-loop, construct specific mRNA was depleted in the first flow-through (unbound fraction) with respect to the references.
(A) anti-HIS purification of MCP-mCherry fusion construct with no stem-loop (HIS tag), one stemloop (HIS tag) and a no HIS tag construct. ~30% of isolated protein co-purified ~25% of mRNA carrying a stem loop. However, ~10% of background RNA with no SL is also co-purified, resulting in an excess ~15% that can be specifically attributed to stem loop binding. Therefore, we estimate that ~30% of isolated protein specifically co-purified ~15% of its cognate mRNA. This percentage of RNA amounts to ~%50 of the RNA that proportionally corresponds to the purified protein.
(B) anti-HIS purification of MCP-GFP fusion constructs with no stem-loop (HIS tag), one stemloop (HIS tag) and no HIS tag constructs. ~36% of isolated protein co-purified ~25% of mRNA carrying a stem loop. However, ~10% of background RNA with no SL is also co-purified, resulting in an excess ~15% that can be specifically attributed to stem loop binding. Therefore, we estimate that ~36% of isolated protein specifically co-purified ~15% of its cognate mRNA. This percentage of RNA amounts to ~%40 of the RNA that proportionally corresponds to the purified protein.
(C) anti-FLAG purification of MCP-GFP fusion constructs with no hairpin (FLAG tag), one stemloop (FLAG tag) and no FLAG tag constructs. ~4045% of isolated protein co-purified ~10% of mRNA carrying a stem loop. However, background RNA with no SL is co-purified in small amounts (<0.05%), resulting in a specific co-purification. Therefore, we estimate that ~4045% of isolated protein specifically co-purified ~10% of its cognate mRNA. This percentage of RNA amounts to ~%20 of the RNA that proportionally corresponds to the purified protein.
Overall, we estimate that isolated protein co-purifies roughly 20-50% of its corresponding mRNA with specificity. At the same time, while ~80% of the displayed protein is missing from the flowthrough, the construct specific RNA is depleted in an excess of 20-40% compared to both a no SL control and a housekeeping reference gene. The protein and RNA that is not present in the first flow-through will either be purified or removed during the wash steps. Protein levels were assayed by means of fluorescence using a plate reader (Synergy MX, BioTek). Here, protein constructs were bound using the respective magnetic beads and washed with a reduced salt Wash Buffer (150mM NaCl). RNA was precipitated from every sample using TRIzol in order to avoid inconsistent losses on the spin columns used otherwise in this manuscript. RNA levels were Log Fold Enrichment of specific over non-specific mRNA for purifications from mixed populations. Constructs with no hairpin loop are mixed with functional in vivo mRNA display constructs. In vivo display efficiency is lower in the presence of another functional construct (compare M4 to M3 and M8 to M7). Additionally, when we purify no HL constructs in the presence of functional constructs (M2 and M6), the non-specific mRNA is enriched which is an additional concern. qPCR averages are shown as bars and SD of technical replicates as errors.
Figure S11. Excess Coat Protein or Stem-loop for increased display enrichment.
In vivo mRNA display GFP constructs were mixed with defective coat protein mCherry constructs and the Log Fold Enrichment of specific over non-specific mRNA was quantified for samples purified with anti-GFP magnetic beads. qPCR averages are shown as bars and SD of technical replicates as errors. Figure S12. Increasing temperature decreases precision of in vivo mRNA display assay.
In vivo mRNA display GFP and mCherry constructs were mixed together and the Log Fold Enrichment of specific over non-specific mRNA was quantified for samples purified with anti-RFP (M1, M2) and anti-GFP (M3, M4) magnetic beads. For M1 and M3, samples were kept at 4ºC throughout purification. For M2 and M4, samples were incubated at 30ºC for 30 min post lysis. qPCR averages are shown as bars and SD of technical replicates as errors.  Scatter plot for average log normalized reads for the lysate (x-axis) against the purified samples (y-axis) for the purified yeast library (Fig. 2D-G). Reads for each sample were normalized by the mean of non-specific functional controls (grey crosses). GFP is an specific functional positive control (green cross). The area between a rolling 10 th and 90 th percentile is shaded with the respective color. Purified ORFs are enriched in the purified sample compared to the non-specific functional controls.  Percentage of in vivo mRNA display yeast library proteins with significant Display Scores per GO term compartment category. Figure S18. in vivo mRNA display yeast library proteins span biological processes.
Percentage of in vivo mRNA display proteins with significant Display Scores per GO term biological process category. Percentage of in vivo mRNA display proteins with significant Display Scores per GO term molecular function category. Figure S21. Receiver Operating Characteristic and Precision Recall curves for the crude mitochondrial isolation in Fig. 3.
Members of the library were classified according to their respective Display Score and compared to the GO Term compartment categories. ROC and PR curves for individual replicates are shown. Figure S22. Distribution of reads for in vivo mRNA display yeast library SAM2 purification.
Distribution of log (reads+1) for every SAM2 purification in Fig.3C. Total number of reads noted in thousands for each sample. Figure S23. Distribution of reads for in vivo mRNA display yeast library ARC40 purification.
Distribution of log (reads+1) for every ARC40 purification in Fig.4B. Total number of reads noted in thousands for each sample.  Shown in red are proteins that contain a N-terminal Signal peptide (UniProt annotation), or a Transit peptide (UniProt annotation), or membrane proteins (GO term: 16020). Cytoplasmic and nuclear fractions are reported in grey for reference. Membrane proteins are enriched at an overall higher percentage than proteins carrying peptides responsible for transport, which are usually cleaved from the mature protein and could interfere with the function of the MS2 N-terminal fusion (hypergeometric test for p-values).