www.sciencemag.org/content/345/6194/328/suppl/DC1

Supplementary Materials for

Human tRNA synthetase catalytic nulls with diverse functions

Wing-Sze Lo, Elisabeth Gardiner, Zhiwen Xu, Ching-Fun Lau, Feng Wang, Jie Zhou, John D. Mendlein, Leslie A. Nangle, Kyle P. Chiang, Xiang-Lei Yang, Kin-Fai Au, Wing

Hung Wong, Min Guo, Mingjie Zhang, Paul Schimmel*

*Corresponding author. E-mail: schimmel@scripps.edu

Published 18 July 2014, Science 345, 328 (2014)

DOI: 10.1126/science.1252943

This PDF file includes:

Materials and Methods Figs. S1 to S6 Tables S1 to S6

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Materials and Methods Samples and messenger RNA isolation

A total of six RNA samples were submitted to high-throughput RNA deep sequencing. The six human samples included adult brain (polyA+ RNA, Cat# 636102, Clontech), fetal brain (polyA+ RNA, Cat#636106, Clontech), peripheral blood leukocytes (polyA+ RNA, Cat #636170, Clontech), leukemia Jurkat T-cells (Cat# TIB-152, ATCC®), Burkitt’s lymphoma Raji cells (Cat# CCL-86, ATCC®), and monocytic leukemia THP-1 cells (Cat# TIB-202, ATCC®). For differential gene expression studies, a panel cDNA of 19 human tissues and cells was synthesized and tested. The 19 human samples included the four that were submitted for deep sequencing. Additional tissues included human lung (total RNA, Cat# 636524, Clontech), liver (polyA+ RNA, Cat# 636101, Clontech), heart (polyA+ RNA, Cat# 636113, Clontech), spleen (polyA+ RNA, Cat# 636121, Clontech), pancreas (polyA+ RNA, Cat# 636119, Clontech), small intestine (polyA+ RNA, Cat# 636125, Clontech), colon (polyA+ RNA, Cat# 636146, Clontech), kidney (polyA+ RNA, Cat# 636118, Clontech), skeletal muscle (total RNA, Cat# 636534, Clontech), bone marrow (total RNA, Cat# 636591, Clontech), thyroid (polyA+ RNA, Cat# 636128, Clontech), adipose (polyA+ RNA, Cat# 636162, Clontech), fetal lung (polyA+ RNA, Cat#636109, Clontech), and two commercially available cell lines: the neuroblastoma line IMR32 (Cat# CCL-127, ATCC®) and THP-1 (Cat# TIB-202, ATCC®). Total RNA was extracted from the cell line samples and the mouse monocytes using PureLinkTM RNA Mini kit (Cat# 12183018A, Life Technologies). Isolated total RNA was quantified by NanoDrop 1000 spectrometer. Ratio of 260-to-280nm was 2 and 1.94 for the RNA from cell line samples and from mouse monocytes, respectively. Genomic DNA was digested using TURBO DNase (Cat # AM2238, Life Technologies). Messenger RNA was isolated from the total RNA using the FastTrack MAG Maxi mRNA Isolation kit (Cat# K158002, Life Technologies). Enrichment of ARS-gene transcriptome by multiplex PCR

The AARS transcriptome was defined as containing the transcripts of 37 cytoplasmic or mitochondrial AARS genes. To enrich the AARS transcriptome a polymerase chain reaction (PCR)-based method was employed using AARS-gene exon-specific primers. For each exon a forward and a reverse PCR primers was designed. The AARS-exon primers were grouped into 13 forward primer sets and 13 reverse primer sets, e.g. forward primer set #1 contains forward exon-primers for targeting Exon 1, Exon 14 and Exon 27 of all AARS genes; reverse primer set #2 contains reverse exon-primers for targeting Exon 2, Exon 15 and Exon 28 of each AARS. AARS-gene-specific complementary DNA (cDNA) was synthesized from mRNA samples using Superscript III First-Strand Synthesis Kit (Cat# 18080-051, Life Technologies) and AARS-gene exon-flanking reverse primer sets. For each sample, thirteen reverse transcription reactions were set-up and preformed. In each reaction, 100 nM of each primer in the AARS-exon reverse primer set was input. The AARS-gene-specific first-strand cDNA were pooled and diluted to 100 ul, and were used as template in the following multiplex PCR step. With 13 primer sets for both forwards and reverse AARS-exon primers, a total of 52 multiplex PCR were performed with each human sample. Each 10-ul multiplex PCR contained 1 ul of ARS-gene first-strand cDNA, 100 nM of each AARS-exon primer

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in the forward or reverse primer sets, 200 uM of each dNTP, 1X of Advantage II PCR buffer and 1.5X of Advantage 2 polymerase mix (Cat# 639201, Clontech). Multiplex PCR cycling conditions were as follows: 95ºC for 1 min, followed by 35 cycles of 95ºC for 20 sec, 60ºC for 20sec and 68ºC for 1 min, and a final extension at 68ºC for 5 min. The multiplex PCR products then were pooled and purified using Nucleospin Extract II Kit (Cat# 740609.250, Macherey-Nagel). Deep sequencing of the AARS-transcriptome enriched cDNA and identification of alternative splicing and exon-skipping events

The AARS-gene-specific multiplex PCR products were purified and pooled and constructed into cDNA libraries using Multiplexing Sample Preparation Oligonucleotide Kit (Illumina, CA) and sequenced by HiSeq 2000 sequencing system (Illumina, CA). Deep sequencing reads were mapped and counted using rSeq version 4, a modified version of rSeq version 2 by Professor Wing H. Wong’s group (19), for counting number of sequencing reads mapped to alternative spliced exon-exon junctions. Annotated exon splice sites of the 42 AARS genes were obtained from RefGene of NCBI based on human reference genome (NCBI version 36, hg18). Quantitative real-time PCR by ViiATM System

To profile AARS splice variant gene expression across 19 human tissues or cells, quantitative real-time PCR (qPCR) was performed using the ViiATM 7 System (Applied Biosystems). Each 20-ul qPCR consisted of 2 ul of diluted cDNA, 250 nM of each forward and reverse primers and 1X of FastStart Universal Probe Master with ROX (Roche Diagnostics). Reactions were performed on the ABI ViiA 7 Real-Time PCR System (Applied Biosystems). For each sample, technical triplicates were performed for quantifying the housekeeping gene and the splice variant of interest, respectively. Thermal cycling conditions included 2 min at 50ºC, and then 10 min at 95ºC, and followed by 40 cycles of 95ºC for 30 sec and 60ºC for 30 sec. qPCR data was analyzed using ViiA 7 RUO Software (Applied Biosystems). The melting curve for each qPCR was generated at the end of the reaction and examined to make sure no noise peaks were being generated. For house-keeping gene selection, we tested a list of ribosomal protein genes that had been suggested by GeNorm for the most stably-expressed genes in the human tissues and cells cDNA panel. Gene expression of splice variants was normalized in each sample using the expression of housekeeping gene Ribosomal protein L9 (RPL9) and Ribosomal protein S11 (RPS11) that were found to be most stably expressed among the other house-keeping genes in this study. Isolation of polyribosomal RNA in cultured cells

Polysome-bound mRNA was isolated from cultured Jurkat T-cells. In brief, Jurkat T-cells were firstly lysed in RLN-lysis buffer (Qiagen) with 0.5% IGEPAL (Sigma-Aldrich), 40 mM dithiothreitol and 500 U/ml RNAse inhibitor (ABI Biosystem). Nuclei then were removed from the cell lysate by centrifugation at 12,000g for 10 seconds at 4ºC, and supernatant was supplemented with 150 ug/ml cyclohexamide, 650 ug/ml heparin, and 10 mM proteinase inhibitor and centrifuged at 12,000g for 5 min at 4ºC. The supernatant was layered onto linear sucrose gradient (15 - 40% sucrose [w/v], supplement with 10 mM Tris-HCl at pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 10 mM dithiothreitol,

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100 ug/ml cycloheximide, 0.5 mg/ml heparin and centrifuged at 15,500 rpm for 6 hours at 4ºC with brake off. Fractions were collected and RNAs in the fractions were resolved by gel electrophoresis to identify the fractions that contain polysome-bound RNAs. RNAs in the polysome-bound RNA fraction were precipitated with 2M lithium chloride at 4ºC overnight to remove heparin. RNAs were purified with the use of an RNAeasy kit (Qiagen) and eluted in RNase-free water. Detection of splice variant genes in the polysome-bound RNA fraction was carried out by qPCR as described previously. Western blot methodology

Human Jurkat T cells were lysed by 50 mM Tris buffer (pH 8.0) containing 1% Triton X-100 and 5 mM EDTA. After incubation on ice for 30 min, lysed cells were centrifuged at 22,000 ×g 4 °C for 15 min, and the supernatant was collected and analyzed for protein concentration by Bio-rad Protein Assay (Bio-rad, Hercules, CA). Whole cell lysates were loaded onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA) at 50 µg proteins per lane next to the protein marker (Invitrogen, Seeblue Plus2) for electrophoresis and transferred to a nitrocellulose membrane. The membranes were each stained by an antibody directed against a specific AARS as detailed in Table S4. In-vitro translation

The cDNA encoding endogenous TyrRS1-C7 splice variant was subcloned into the pCDNA3.1 vector. The construct was employed as the template in the coupled transcription/translation reaction using TnT® T7 Coupled Reticulocyte Lysate System (Promega). A control reaction was performed using the pcDNA3.1 vector that contains no insert. The in vitro translation products were separated by SDS-PAGE electrophoresis. A small amount of the products was analyzed, by western blot analysis, for expression of the splice variant. The remaining amount was run in parallel and not subjected to western blot analysis. A band of protein at the size corresponding to the splice variant was cut out and subjected to mass spectrometry analysis. Mass Spectrometry detection of AARS splice variants

Proteins were selectively immunoprecipitated from the supernatants of lysed Jurkat cells by binding to IgG agarose beads. For the whole cell lysate preparation, Jurkat cells (107) were incubated at 4oC for 30 min with 0.2 ml whole cell lysis buffer (20 mM HEPES, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 10% glycerol, 1% Triton X-100, 1x protease inhibitor mixture). Immunoprecipitation was done for 1 hour at 4’C with commercially available antibodies to TyrRS, GlnRS and ValRS. Beads were gently centrifuged, washed three times in PBS, and subjected to SDS-PAGE. The gel was stained and destained using a standard Coomassie protocol and the visible band was excised and submitted for analysis. The band was cut out wearing gloves to avoid keratin contamination. The excised band was submitted for analysis in de-ionized water in a sterile Eppendorf tube. The samples were processed by the TSRI Mass Spectrometry Core, using the extraction and drying procedure outlined at: http://msf.ucsf.edu/ingel. html. MS/MS data was obtained using nano-LC-MS/MS and searched against the predicted fragment ions from the trypsin digestion of proteins contained in NCBI-nr database using Mascot.

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Recombinant splice variant protein expression AARS splice variants can be expressed as recombinant proteins using standard

expression techniques in a soluble form and purified to sufficiently high purity to discern their biological properties. Cell based assays were used in conjunction with sufficiently pure, stable, soluble protein to reveal potential biological relevance. 6xHis-tagged AARS polypeptides were expressed in bacteria and purified using affinity and ion exchange chromatography. To remove endotoxins, an AcroPrep Advance filter plate with Mustang Q membrane (Pall, Cat# 8171) was utilized and desalted AARS polypeptides were added to the filter plate and incubated on a shaker for 5-10 minutes. The plate was centrifuged at 1,000 g for 5 - 10 minutes and the flow through fractions containing the AARS polypeptides were collected for endotoxin testing. All purified AARS polypeptides were analyzed by SDS-PAGE, their concentration determined based on A280 and calculated extinction coefficient (ProtParam on ExPASy server). Endotoxin levels were measured by the QCL-1000 Endpoint Chromogenic LAL assay (Lonza, Cat# 50-648U) according to the manufacturer’s instructions. Dynamic Light Scattering was done using a Wyatt Technology DynaPro 99 instrument. The data collected included hydrodynamic radius, polydispersity, predicted average molecular weight, percentage of intensity and percentage of mass. Cell-based In vitro profiling with recombinant splice variant proteins Measurements of cell proliferation

Cell proliferation was assessed by adding a solution of Resazurin to cells after 24 or 48 hours of protein incubation, incubating them for 1-4 hours, and reading the fluorescence or absorbance. The amount of fluorescence or absorbance is proportional to the number of living cells and corresponds to the cells metabolic activity. Samples were measured on Spectramax reader (Molecular Devices, Sunnyvale CA) with a 530 nm excitation and 590 nm emission filter setting. Acetylated LDL uptake

A standard assay for measuring acetylated LDL uptake was employed in HepG2C3a cells. HEPG2C3a cells (ATCC# CRL-10741) were maintained in Eagles Minimal Essential (EMEM) medium supplemented with 10% FBS (HyClone Cat#SH30910.03), 50u/ml penicillin, 50μg/ml streptomycin, (Invitrogen) in 15 ml medium in 75 ml flasks. A 100 µl volume of cells was plated on collagen coated plates (Invitrogen Cat#A11428) overnight in complete medium at a cell density of 50,000 cells/ml. Cells were washed once with PBS (Invitrogen Cat# 10010) and 80 µl of serum free EMEM was added to each well. AARS polypeptides at a final concentration of 250 nM per well were added in a consistent volume in sterile PBS to each well. Cells were serum starved and exposed to the AARS polypeptides for 16 hours. Following a 2-hour incubation at 37 ºC 5% CO2, cells were washed twice with sterile PBS before 100 µl PBS was added to each well. Plates were analyzed for total fluorescent intensity using a bottom read on a Victor X5 fluorescent plate reader (Perkin Elmer) at an excitation wavelength of 485 nm, and an emission wavelength of 535 nm.

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Neutrophil oxidative burst and elastase assays Heparinized whole blood was mixed with sterile dextran (0.6% final concentration)

for 1 hour and allowed to separate into layers. The lower layer contained neutrophil, monocytes and red blood cells. An ammonium chloride lysis step was utilized to remove all RBCs and a 97% pure population of neutrophils with approximately 3% monocyte contamination remained following the lysis step. Formation of the reactive oxidants during potential oxidative burst was monitored by the addition and oxidation of Amplex Red (Life Technologies) in the presence of horseradish peroxidase (Sigma). Elastase was measured using the ENZCHEK® Elastase Assay Kit (Invitrogen Catalog # E-12056) according to manufacturer’s instruction. To assay for elastase activity, 50 μl of 1X Reaction Buffer is pipette into each assay well containing 500,000 neutrophils/ ml in a 30 μl volume. AARS polypeptide, 8 μl of each was added per well, and the sample incubated for 20 minutes at 37ºC. 50 μl of 100 μg/ml DQ elastin working solution was added to each well and mixed. Fluorescence intensity in a fluorescence microplate reader equipped with standard filters (ex 485/ Em 535) fluorescence was measured. NF-κB activation assays

Endogenous ligands, as well as microbial components, are recognized by and can activate TLRs, raising the possibility that these receptors may be critical targets for the development of new therapies for multiple diseases. RAW-BLUE™ cells were used according to manufacturer’s instructions. Cells were plated at a concentration of 50,000 cells/well in a 96 well plate in a total volume of 100 µL, and AARS polypeptides, controls, or AARS polypeptides (+LPS) were added to each well at the concentrations shown in the experiments outlined below. Cells were incubated at 37°C in a 5% CO2 incubator for 18 hours. On experimental day 2, SEAP detection medium (QUANTI-BLUE™) (Invivogen Catalog code: rep-qb1) was prepared following the instructions and 120 μl was added per well to a clear flat-bottom 96-well plate, and cell supernatant is added to prepared plate (20 μl). SEAP levels were determined using a spectrophotometer 30 minutes after addition by reading absorbance at 650 nM. AARS polypeptides were added to the cells at a final concentration of about 250nM per well, 1 hour prior to adding 50 ng/ml LPS. PBS control wells with no LPS or AARS polypeptide alone added were used to find the basal level of TLR stimulation at the time of the measurement. Control wells were pretreated with PBS and known TLR agonists. Cytokine release and soluble cell adhesion assays

Cell types used included Human Fibroblast-Like Synoviocytes-Rheumatoid Arthritis, HFLS-RA, adult cells (Cell Applications Cat # 408RA-05a), Human Astrocytes (HA) from Cell Applications (Cat # 882K-05f) maintained in Cell Applications HA Cell Growth Medium (Cat # 821-500) according to manufacturer’s instructions, Human Adult Skeletal Muscle Cells (HSkMC, Cell Application Cat # 150-05f), HPASMC (Cell Applications Cat # 352-05a) were maintained in HPASMC growth media (Cell Applications Cat # 352-05a) and HLMVEC (Cell Applications, Catalog # 540-05) maintained in Cell Applications Microvascular Endothelial Cell Growth Medium (Cat # 111-500). An 80 µl volume of cells was plated overnight in growth medium at a cell density of 10-50,000 cells/ml depending on cell type. AARS polypeptides at a final concentration of 250 nM per well were added in a consistent volume in sterile PBS to

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each well. Cells were exposed to AARS polypeptides for 48 hours and spent media was removed for cytokine assessment. IL-8 and IL-6 ELISA assays were run according to manufacturer’s instructions (RND Systems, Cat # DY206 and DY-208, DY-210 Duo-set kits). Cell adhesion molecules including soluble VCAM and/or ICAM were measured using a standard ELISA kit from RND Systems (Cat # DY643 and DY720 respectively). Cellular differentiation

HPAd (human pre-adipocytes) (Cell Application Cat # 803sD) were maintained according to vendor instructions. AARS polypeptides at a final concentration of 250 nM per well were added to each assay well. Cells were exposed to AARS polypeptides for 48 hours. Proliferation is assessed on day 15, cells were placed in serum free media. On day 16, differentiation to mature adipocytes is assessed with Nile Red (Invitrogen, concentration of 3 µM final) staining and quantified with a fluorescent plate reader with the appropriate wavelengths. To perform this assay cells were fixed with 10% paraformaldehyde, washed in PBS and permeabilized in PBS containing 0.5% BSA and 0.1% Triton X-100. Cell proliferation is assessed with an intensity measurement on a fluorescent reader with Hoechst dye 33432 at a concentration of 1ug/ml final. Adipogenesis was expressed as intensity of Nile Red signal. Hoechst dye signal is used to assess cellular number. To assess the potential role of AARS polypeptides in this process, a standard assay of skeletal muscle cell differentiation was employed. For this assay, Human Adult Skeletal Muscle Cells (HSkMC, Cell Application Cat # 150-05f) were isolated from healthy human donors from limbal skeletal muscle. Cells were maintained in HSkMC Growth Medium (Cell Applications, Cat # 151-500). For differentiation, cells were maintained in growth media for one passage and then trypsinized, (Cell Applications, Cat # 070-100) washed with ion free phosphate buffered saline (Cat # 14190250, Life Technologies) and replated in basal media (Cat # 150-500, Cell Applications) at 50,000 cells per ml in 96-well clear-bottom black-walled TC treated plates pre-coated with a 50 ug/ml solution rat tail collagen (Cat # 122-20, Cell Applications) at 100 µl per well. Cells were allowed to adhere overnight. AARS polypeptides in PBS, or PBS alone, were added to each well at a final concentration of 250 nM protein (or as otherwise indicated in the examples below). Control wells received the same volume of Differentiation Media (Cat # 151D-250, Cell Applications) at this time. Cells were incubated with protein or differentiation media for 48 hours. At 48 hours, cell culture supernatant is collected from all wells and differentiation media is added at a volume of 150 µl to the entire plate with the exception of control wells that were maintained in growth media only. Supernatant was utilized to assess soluble cell adhesion molecule production as well as cytokine secretion including IL6 and IL8. Cells were monitored under the microscope and media was exchanged for fresh media every 2 days. On Day 5, cells were fixed with 10% paraformaldehyde for 30 minutes. Cells were washed twice with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. Cells were stained with alpha actin skeletal muscle antibody (GeneTex Cat # GTX101362) and Hoechst 33432 (Life Technologies, Cat # H3570). A secondary fluorescent antibody (Life Technologies, Cat # A-11008) was used to visualize the alpha actin immunoreactivity. Digital photos using a fluorescent microscope as well as visual inspections and blinded scoring were done. HALO software (Indica Labs, Albuquerque, NM) was used for quantification of fluorescent intensity in stained wells.

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Human bone marrow mesenchymal stem differentiation and proliferation

To explore the potential for AARS polypeptides to act as therapeutic agents to regulate the differentiation of MSCs in vivo, AARS polypeptides were tested as potential inducers of MSC proliferation and differentiation. hMSC (human marrow stromal cells) (Cell Application Cat # 492-05f) were maintained according to vendor instructions. For culturing, cells were thawed quickly, and transferred immediately into 15 ml of Marrow Stromal cell Growth Medium (Cell Application Cat # 419-500) and plated into a standard sterile tissue culture treated flask. Media was replaced with fresh Marrow Stromal cell Growth Medium every other day until cells were >60% confluent. Cells were grown at 37ºC, 5% CO2. Cells were plated in clear bottom black walled 96 well tissue culture treated assay plates for differentiation at a concentration of 50,000 cells/ml. tRNA synthetase derived proteins at a final concentration of 250 nM per well were added to each assay well. Cells were maintained in growth media for 2 days with the exception of the positive controls, stimulated with osteogenic or chondrogenic differentiation media (Cat # A10072-01 and A10071-01 Life Technologies). Cells were exposed to AARS polypeptides for 48 hours. Soluble VCAM was measured using a standard ELISA kit from RND Systems (Cat # DY643). Differentiation was assessed with alkaline phosphatase staining using ELF-97 stain (Cat# E6601, Life Technologies) at day 10 post first differentiation exchange. HPASMC (Cell Applications Cat # 352-05a) were maintained in HPASMC growth media (Cell Applications Cat # 352-05a) in 15 ml medium in 125 mL flasks for 1 passage before use. Cells were maintained at 37ºC, 5% CO2, in a humidified environment. Differentiation was assessed by smooth muscle actin-alpha staining using an anti-SMA-alpha antibody (GeneTex Cat #GTX101362) and an Alexa 405 conjugated secondary antibody. Proliferation was assessed with Hoechst staining after cell fixation in 10% formaldehyde for 30 minutes. Hoechst dye is read using a bottom reading fluorescent plate reader with an excitation wavelength (Ex) of 405 nm, and an emission wavelength (Em) of 450 nm. Total actin staining is assessed via the use of an Alexa-488 labeled phalloidin stain (Cat# A12379, Life Technologies). Transcription analysis of human skeletal muscle and mesenchymal stromal cells

Transcriptional analysis was done using standard protocols for using an ABI (Applied Biosystems, Item # AM1728). TAQMAN® Gene Expression Cells-to-CT™ Kit was utilized to lyse cells and harvest transcriptome material. A Pre-Amp Mix (Applied Biosystems, Cat# 4391128) was used to synthesize pre-amplification products using the cell lysate. Gene specific primers were designed using a Primer 3 program and purchased from IDT technologies. Gene expressions were quantified using Fluidigm profiling arrays (Item # BMK-M-96.96), Fluidigm loading reagents, and Sso Fast EvaGreen Supermix with Low ROX (BioRad, Cat # 172-5212) on the Fluidigm Biomark System. For data analysis Ct values for all genes of interest were normalized to the averaged Ct values for housekeeping genes from the corresponding sample to obtain ΔCt values (ΔCt = Ct gene – Ct average housekeeping genes). Genes from each sample were normalized to the same gene in untreated control to obtain ΔΔCt values (ΔΔCt =ΔCt control sample - ΔCt treated sample). To obtain fold change values up-regulated genes (i.e. ΔΔCts greater than 0) were subject to the following calculation: Fold Change = 2^ΔΔCt. For down-regulated genes (i.e. ΔΔCts less than 0): Fold Change = -(2^|ΔΔCt|)

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Fig. S1 Functional domain-based illustrations of AARS splice variants for Class I and Class II cytoplasmic AARSs. Most AARS splice variants are catalytic nulls. The catalytic aminoacylation domains are represented in blue for Class I representatives, and dark orange for Class II. “S” and “L” designate UNE-S and UNE-L, respectively. All splice variants shown here were validated by PCR, cloning and sequencing and/or by quantitative real-time PCR.

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Fig. S1

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Fig. S2 Polysome association of TyrRS1-AS06 transcripts in Jurkat cell lysate. Endogenous TyrRS1-AS06 splice variant mRNA, which encodes TyrRS1-C7 (fig. S1), was detected in the polyribosomal fractions but not in free mRNA fractions isolated from Jurkat cells. Fourteen fractions were obtained from across a 10–40% sucrose gradient. With specific PCR primers, the mRNA for TyrRS1-AS06 (blue arrow) was detected in the polyribosome-bound fraction (Fractions 10-14) but not in the free mRNA fraction (Fractions 1-4).

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Fig. S3A

2

B

3

C

Fig. S3 Mass spectrometry detection of AARS splice variants. (A) Endogenous AARS splice variants were enriched by immunoprecipitation with anti-GlnRS1, -ValRS1 or -TyrRS1 specific antibodies and separated by electrophoresis. Gel slices with the sizes of the corresponding splice variants were cut out for mass spectrometry (MS) analysis. As a control for any possible contamination of proteolytic fragments that could be misconstrued as splice variants, a gel was run with an overloaded quantity of the recombinant full length TyrRS1. A large chunk of gel ("the proteolytic tail") was cut underneath the ‘full-length’ gel band for the MS analysis, and no peptide of TyrRS1 was obtained from this gel. (B) Analysis of the public PROTOMAP database (25) revealed a protein the size of the HisRS1-C9 splice variant found in this work, with 3 MS peptides on both sides of the internal splice site of HisRS-C9. (C) A cDNA PCR product was generated from endogenous TyrRS1-C7-encoding mRNA and then used for in vitro translation (IVT) as described in Materials and Methods. (Endogenous TyrRS1-C7 was identified by western blot analysis of whole cell lysates as shown in Fig. 3A.) IVT confirmed that the transcripts could be stably translated into proteins. Shown are the western blot analyses. TyrRS1-C7 with a size of ~50 kDa is indicated by the red arrow. For the IVT control, vector containing no insert was employed as template. The dark band at the top is cross-reacting material from the translation mixture.

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Fig. S4 Expressed AARS recombinant fragments have unique biological activities across a diverse panel of biological assays. Recombinant AARS fragments were tested for biological activities in cell based assays. AARS fragments were identified from deep sequencing, by mass spectrometry as proteolytic fragments that closely resembled those also encoded by specific splice variants. Blue bars indicate relative activity in a given assay. Data is the average of 4-12 data points per protein. Proteins were expressed as both N- and C-tagged with a 6XHis tag. In most cases, the data were the same for both tagged forms. Absence of a bar indicates no activity in a given assay for all of the tested batches. Amino acid numbers of specific proteins are given in table S6.

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Fig. S5 Effect of MetRS1-C5 protein on myotube differentiation of human primary skeletal muscle cells. Human skeletal muscle cells were treated with MetRS1-C5 protein or with PBS for 5 days. Myotubes were immune-stained with anti-alpha-actin antibody, and myotube area was measured. Data are means ± SE of n=2.

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Fig. S6. Genes upregulated by MetRS1-C5. Human skeletal muscle cells were treated with or without MetRS1-C5 proteins (N- or C-His tagged) for 48 hours. Gene expressions were compared between MetRS1-C5 treated and untreated. Genes with more than a 10-fold change in expression, and with both tagged MetRS1-C5 proteins showing the same effect, were plotted. LPL, lipoprotein lipase; IGF1, insulin growth factor 1; TERT, telomerase reverse transcriptase; IFNG, interferon gamma; IL10, interleukin 10; HNF4A, hepatocyte nuclear factor 4 alpha; LEP, leptin.

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Table S1. Total alternative splice junctions in the 37 AARS genes that encompass multiple exons

tRNA Synthetase Gene Name*

Exon-skipping events Alternative splice sites

Previously Reported in NCBI† or AceView‡

Newly discovered in this study by RNAseq

Cytoplasmic

AlaRS AARS 1 6 16 ArgRS RARS 1 2 13 AsnRS NARS 1 3 8 AspRS DARS 4 4 15 CysRS CARS 2 6 3 GluProRS EPRS 1 4 22 HisRS HARS 5 4 2 IleRS IARS 1 2 7 LeuRS LARS 1 9 25 MetRS MARS 4 13 4 PheRSa FARSA 0 6 33 PheRSb FARSB 3 7 2 SerRS SARS 2 5 14 ThrRS TARS 1 4 53 TrpRS WARS 4 4 4 TyrRS YARS 3 10 14 ValRS VARS 1 17 1

Shared by both cytoplasmic and mitochondrial

GlnRS QARS 1 18 0 GlyRS GARS 0 3 15

LysRS KARS 4 8 12

Mitochondrial

AlaRS AARS2 0 13 2 ArgRS RARS2 3 7 0 AsnRS NARS2 1 10 2 AspRS DARS2 3 6 2 CysRS CARS2 1 12 33 GluRS EARS2 0 3 21 HisRS HARS2 2 6 17 IleRS IARS2 0 6 24 LeuRS LARS2 0 9 25 MetRS MARS2 0 0 2 PheRS FARS2 0 1 6 ProRS PARS2 0 0 5 SerRS SARS2 1 13 22 ThrRS TARS2 7 9 9 TrpRS WARS2 2 2 3 TyrRS YARS2 0 1 0 ValRS VARS2 1 15 9

Total= 61 248 445 Total Exon-skipping Events = 309

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* Among the AARS genes shown here, only those proteins encoded by multiple exons are included. MARS2 and PARS2 are excluded because their coding sequences are in one exon only. † Known NCBI RefSeq splice junctions are obtained from Gene database of NCBI (http://www.ncbi.nlm.nih.gov/gene/). ‡ Detected splice junctions were previously reported in AceView database of NCBI (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html) by other

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Table S2. Frequencies of AARS domains present in AARS in-frame splice variants AARS No. of in-

frame Splice

Variants

Anti-codon Binding Domain

Catalytic Domain

Editing Domain

N-terminal Single Helix

GST EMAPII

WHEP Containing >1 Appended Domain

AspRS 5 3 1 - 5 - - - 5 LysRS 3 2 0 - 3 - - - 3 AsnRS 1 1 0 - 1 - - - 1 ArgRS 0 0 0 - 0 - - - 0 TyrRS 6 3 1 - - - 6 - 6 ValRS 7 6 1 3 - 4 - - 4 CysRS 2 1 1 - - 2 - - 2 GluProRS 1 1 1 - - 1 - 1 1 MetRS 6 5 3 - - 5 - 5 5 GlyRS 0 0 0 - - - - 0 0 HisRS 8 8 0 - - - - 8 8 TrpRS 2 1 0 - - - - 2 2 SerRS 1 1 0 - - - - - 1 PheRSa 3 1 1 - - - - - 3 PheRSb 4 - - - - - - - 4 GlnRS 10 5 4 - - - - - 8 ThrRS 2 2 1 - - - - - 2 LeuRS 6 4 0 5 - - - - 2 IleRS 1 1 0 1 - - - - 1 AlaRS 2 - 0 2 - - - - 2 Total No. splice variants 70 No. splice variants with specific domain 45 14 11 9 12 6 16 60 Total No. of splice variants in AARSs that can potentially have a specific domain

64 66 16 9 16 6 17 70

% of domain present in splice variants 70 21 69 100 75 100 94 85 “-“ indicates that the particular AARS lacks the specific domain.

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Table S3. Tissue distribution of AARS reference and splice variant transcripts. With specific qPCR primers, 48 AARS splice variant mRNAs and 18 AARS reference mRNAs were profiled and compared across 19 human tissues or cells. Gene expression was represented by fold-change, i.e., expression in a tissue relative to average expression across the 19 tissues/cells. Those with a fold-change of 5-times or more than the median across tissues are highlighted in red. All splice variants were detected in polysome-bound mRNAs of Jurkat cells. Expressions of splice variant transcripts were compared between adult and fetal lung. Those with a fold-change of 10-times or more in adult versus fetal lung are highlighted in green.

AARS Full-length Reference/ Splice Variant

Skipped Exon*

Tota

l Leu

kocy

te

Bon

e M

arro

w

Thyr

oid

Adu

lt Lu

ng

Feta

l Lun

g

Hea

rt

Panc

reas

Sple

en

Live

r

Kid

ney

Col

on

Smal

l Int

estin

e

Adi

pose

Tis

sue

Skel

etal

Mus

cle

Jurk

at T

-cel

ls

Raj

i B-c

ells

THP-

1 M

onoc

yte

THP-

1 M

acro

phag

e

IMR

-32

Adu

lt Lu

ng o

ver

Fe

tal L

ung

AlaRS1

Reference - 0.1 0.5 1.1 0.0 0.0 1.3 0.8 0.4 1.5 1.5 0.7 0.8 1.0 0.8 1.0 1.1 1.1 1.1 0.6 -

AS04 E 12 1.1 0.6 0.8 0.6 0.2 0.8 0.8 1.2 1.0 1.0 1.4 1.2 0.8 1.0 1.6 1.0 0.9 1.0 1.5 3.7

AS06 E 14-15 0.5 1.0 1.6 0.2 0.2 1.4 1.1 1.7 1.0 1.5 1.5 1.5 0.6 0.4 1.3 0.4 0.4 0.7 1.5 1.3

ArgRS1

Reference - 0.5 0.4 0.7 0.8 0.8 1.5 0.5 0.2 1.6 1.5 1.0 1.3 0.8 0.7 2.2 1.0 1.5 4.1 1.4 1.0

ArgRS1-AS01 E 11 116 0.7 0.3 1.3 0.1 1.0 0.4 2.3 0.4 1.5 6.8 2.1 0.8 1.4 16 0.8 0.3 0.5 3.7 12

ArgRS1-AS02 E 10-11 0.4 0.5 0.7 0.0 0.0 1.5 0.6 0.3 1.5 1.3 1.1 1.5 0.8 0.8 3.1 1.0 1.3 3.0 1.4 1.8

AsnRS1 Reference - 0.5 1.0 0.8 0.3 0.4 2.0 0.7 0.4 0.8 2.3 0.9 1.3 0.8 1.2 2.1 1.1 1.2 1.8 1.6 0.8

AsnRS1-AS03 E 5 4.8 2.6 0.4 0.0 0.0 1.0 0.5 2.7 0.6 1.2 0.0 1.3 0.3 0.0 1.6 1.5 1.2 1.0 1.0 1.4

AspRS1

Reference - 0.5 1.0 1.8 0.7 0.7 2.7 0.9 0.6 1.4 1.1 0.9 1.3 1.1 0.7 2.7 1.5 1.7 0.7 0.9 1.0

AspRS1-AS07 E 2-3 0.5 1.0 1.3 0.5 1.8 0.9 0.7 0.7 1.4 3.2 0.9 1.4 1.9 0.0 8.9 0.8 0.9 1.4 1.5 0.3

AspRS1-AS08 E 6-7 2.8 1.7 0.4 1.0 1.2 1.8 0.6 5.4 0.5 1.0 0.8 0.9 1.6 0.1 7.5 2.4 0.7 0.8 1.1 0.8

AspRS1-AS09 E 11 63 0.8 2.5 2.3 0.5 1.0 0.6 6.2 1.4 1.1 1.4 0.9 1.1 0.9 2.8 0.5 0.7 0.3 0.6 4.2

CysRS1 Reference - 0.9 0.3 0.2 1.0 0.5 0.6 1.4 0.1 3.4 0.9 1.0 1.0 1.5 0.2 2.2 2.6 1.5 2.9 3.0 1.9

CysRS1-AS04 E 4-5 58 2.8 0.6 0.5 0.0 2.1 0.9 17 1.0 2.1 2.8 1.1 2.1 0.0 0.9 1.0 0.0 0.0 0.7 14

GlnRS1 Reference - 1.0 1.3 1.5 0.3 0.2 2.8 0.8 0.5 1.9 1.9 1.8 1.7 1.0 1.6 0.9 1.0 0.9 1.0 0.6 1.2

GlnRS1-AS10 E 23 1.0 2.1 0.6 0.1 0.1 1.4 0.7 0.6 0.7 0.9 2.4 2.0 1.1 0.4 5.1 2.0 3.9 6.3 0.9 1.2

GluProRS1

Reference - 0.3 0.9 0.8 0.6 0.5 1.9 0.9 0.2 0.8 1.3 1.0 1.2 0.8 1.6 1.9 2.3 4.6 4.0 2.2 1.3

GluProRS1-AS01 E 7 3.2 1.6 0.6 0.1 0.1 4.5 2.0 2.1 0.8 2.8 2.6 3.1 1.0 0.3 2.9 0.3 0.4 0.5 0.8 1.1

GluProRS1-AS02 E 30 1.4 29 0.3 0.7 1.0 0.7 0.8 0.9 5.3 2.7 2.9 0.2 0.2 1.2 2.9 0.0 2.5 9.6 0.7 0.7

* Exon numbering is according to NCBI database

2

AARS Full-length Reference/ Splice Variant

Skipped Exon*

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GlyRS1 Reference - 0.3 1.0 0.5 0.7 0.5 1.1 1.2 0.6 1.1 1.2 1.0 0.8 0.8 0.6 3.2 3.5 1.7 2.2 4.4 1.3

GlyRS1-AS02 E 15 40 0.2 1.1 6.5 1.4 1.4 0.6 1.9 1.0 1.0 0.6 0.2 0.6 0.4 0.4 4.7 0.8 1.2 1.9 4.7

HisRS1

Reference - 0.9 0.6 0.6 2.5 2.1 2.1 0.7 0.3 1.0 1.9 1.3 0.4 1.0 3.0 1.9 1.0 0.6 1.3 1.0 1.2

HisRS1-AS03 E 4-6 3.6 0.6 0.5 1.9 3.4 4.1 0.4 0.8 1.1 1.7 0.5 0.4 0.8 11 20 6.4 0.2 1.0 0.3 0.6

HisRS1-AS07 E 6 5.5 0.5 0.8 3.2 1.0 0.7 0.7 0.9 1.3 2.8 1.1 0.9 1.4 0.2 8.2 1.0 0.2 1.2 1.2 3.2

HisRS1-AS09 Inserted E2b 0.8 0.9 0.8 8.0 4.3 1.1 1.1 0.4 1.0 1.5 1.2 1.2 1.2 0.2 0.7 0.2 0.4 1.3 1.0 1.9

HisRS1-AS10 E 4-5 5.4 1.6 0.0 1.0 2.1 14 0.3 0.4 0.6 0.9 1.0 0.6 0.5 7.1 13 13 0.1 0.5 1.0 0.5

IleRS1 Reference - 0.3 1.0 0.6 0.1 0.1 1.6 1.0 0.4 0.6 1.2 0.9 1.3 0.7 1.4 4.5 1.4 1.8 2.4 3.7 0.8

IleRS1-AS03 E 21-23 4.9 0.9 0.5 0.2 1.3 2.5 0.9 1.0 1.1 1.3 0.9 1.5 1.3 0.9 1.7 0.4 0.2 0.6 3.2 0.1

LeuRS1

Reference - 0.9 0.5 0.3 0.1 0.1 1.0 1.2 0.9 0.5 1.2 0.4 1.7 1.0 0.3 4.3 2.3 2.4 2.1 4.4 0.6

LeuRS1-AS04 E 4 38 0.3 0.2 0.6 0.3 0.4 0.7 8.1 1.2 1.0 0.4 1.0 1.4 0.0 1.9 1.2 3.7 1.9 1.0 2.0

LeuRS1-AS05 E 2-3 1.9 0.0 0.2 1.5 1.1 0.5 1.1 0.0 0.4 0.9 1.0 1.5 0.7 0.1 2.2 3.0 1.3 0.6 4.8 1.4

LysRS1

Reference - 0.8 0.7 0.7 1.1 1.0 2.2 0.5 0.5 1.0 1.2 0.8 0.7 0.7 1.9 2.1 1.4 1.1 1.3 0.6 1.0

LysRS1-AS04 E 5 2.3 0.6 1.0 0.3 0.4 1.3 0.6 1.4 0.5 1.5 1.1 1.0 1.0 0.2 5.1 2.3 0.3 1.2 1.3 0.8

LysRS1-AS05 E 7-10 1.7 0.4 1.6 0.2 0.2 0.6 1.1 2.2 0.8 2.0 0.8 1.0 1.4 0.3 7.1 0.9 0.9 2.8 1.4 0.9

LysRS1-AS06 E 8 9.3 1.2 0.1 3.0 1.4 0.5 0.0 3.8 0.2 0.2 0.7 0.4 0.2 0.4 14 1.0 1.6 3.1 2.4 2.1

LysRS1-AS08 E 9-13 9.6 0.0 0.0 0.8 1.3 0.0 0.0 0.0 0.0 1.2 0.0 1.0 0.8 0.0 0.8 0.5 0.0 1.9 0.0 0.6

LysRS1-AS10 E 4-5 7.5 0.5 1.3 0.8 0.5 1.0 0.8 2.9 0.7 1.1 1.5 0.9 0.9 0.7 5.0 2.0 0.3 1.7 1.6 1.7

MetRS1

Reference - 1.6 1.0 0.8 0.0 0.0 2.0 1.1 0.4 1.0 1.2 1.1 1.0 0.8 0.7 2.3 1.1 0.7 0.6 1.5 -

MetRS1-AS06 E 10 4.0 0.8 0.3 0.1 0.1 2.3 1.8 0.6 0.9 0.9 1.2 1.9 0.6 0.3 2.4 1.0 1.5 1.6 3.5 0.8

MetRS1-AS08 E 14 9.6 1.2 0.9 0.3 1.1 6.6 0.8 0.6 2.2 2.3 0.9 2.3 1.5 1.0 0.6 0.4 0.1 0.4 1.2 0.3

MetRS1-AS09 E 18 1.3 0.8 0.6 0.1 0.4 2.4 1.0 0.0 0.6 0.8 0.5 1.4 0.5 1.8 1.9 1.4 3.9 1.3 3.6 0.2

MetRS1-AS12 E 2-5 1.5 0.0 0.5 0.3 0.2 10 0.3 1.8 0.3 1.8 1.5 0.8 1.0 2.0 19 3.3 0.0 0.0 17 1.2

MetRS1-AS13 E 3-5 163 0.8 0.2 3.4 0.2 2.2 0.6 9.3 0.3 1.0 2.4 0.5 1.5 1.0 1.3 1.4 0.5 0.3 0.8 15

MetRS1-AS14 E 7 40 0.0 0.0 1.1 0.3 3.0 1.0 13 0.0 0.9 2.1 0.8 1.5 1.9 4.2 1.0 0.7 0.0 4.0 3.4

3

AARS Full-length Reference/ Splice Variant

Skipped Exon*

Tota

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PheRSb

Reference - 0.2 0.7 0.7 0.4 0.5 1.8 0.5 0.3 1.1 1.0 1.5 1.7 0.7 1.0 1.9 2.2 1.4 1.4 2.3 0.7

PheRSb-AS04 E 11-13 0.2 2.5 0.4 0.7 4.2 3.8 0.2 0.0 0.2 0.5 1.1 1.2 0.9 1.9 9.2 1.2 0.9 1.0 1.5 0.2

PheRSb-AS07 E 11-15 1.0 0.0 2.6 0.6 1.4 2.5 0.9 5.8 2.0 1.6 1.0 1.8 0.2 18 0.0 0.7 0.6 0.0 4.2 0.4

PheRSb-AS08 E 2-15 0.3 0.0 2.2 0.2 0.6 14 0.6 3.5 1.1 1.8 0.8 1.0 1.0 0.6 1.5 3.8 0.4 1.1 1.8 0.4

PheRSb-AS14 E 3-15 1.2 0.0 0.0 1.6 0.0 2.9 1.3 0.0 2.2 0.0 0.0 0.0 0.8 0.0 2.6 2.1 1.8 0.0 1.7 -

SerRS1

Reference - 0.7 0.7 1.4 2.4 1.9 1.5 1.2 0.5 1.5 1.2 1.3 1.0 1.0 0.9 0.9 0.4 0.7 0.9 1.3 1.3

SerRS1-AS02 E 9 110 2.6 2.9 26 0.7 0.8 0.7 18 0.8 1.0 1.6 0.8 1.3 1.1 0.9 1.7 0.1 0.4 0.7 36

SerRS1-AS03 E 2 1.6 0.5 2.0 1.7 1.3 1.0 0.9 1.2 0.6 1.1 1.0 1.2 1.1 0.5 0.6 0.4 0.2 0.4 5.0 1.3

ThrRS1 Reference - 0.3 1.1 0.7 0.1 0.1 2.0 1.0 0.3 1.9 1.4 1.3 1.0 0.9 0.6 2.4 1.0 1.7 1.7 0.2 0.7

ThrRS1-AS04 E 4 4.1 2.7 0.8 0.1 0.0 1.0 1.3 3.1 0.3 1.5 1.4 0.6 0.9 1.4 1.0 0.6 0.5 0.0 3.1 1.5

ThrRS1-AS05 E 16 14 4.7 1.3 1.2 0.1 0.2 0.8 2.4 1.0 1.0 9.9 0.5 0.2 0.0 3.9 1.6 1.0 0.0 1.0 12

TyrRS1

Reference - 0.9 0.9 0.3 1.8 1.0 1.2 1.3 0.7 1.5 0.9 0.8 0.7 0.8 0.2 2.6 2.2 2.1 2.2 2.2 1.8

TyrRS1-AS02 E 6 1.1 0.2 0.1 0.8 1.2 0.7 1.0 1.7 1.5 0.5 0.5 0.4 0.5 0.1 5.5 6.2 5.0 4.1 10 0.7

TyrRS1-AS06 E 5-6 1.1 0.1 0.5 0.3 0.6 1.1 0.8 1.4 1.0 0.9 0.2 1.0 0.3 1.9 18 3.2 3.2 2.1 2.2 0.5

TyrRS1-AS07 E 2-4 0.3 1.8 0.4 0.0 0.0 2.0 0.2 0.0 0.4 0.3 1.0 0.4 1.8 0.0 6.5 15 2.6 2.2 1.0 -

TyrRS1-AS08 E 3-4 2.9 0.3 0.3 0.0 0.0 1.6 0.5 2.5 0.5 0.5 1.3 0.6 1.6 0.4 2.7 0.6 2.2 1.0 1.5 -

TyrRS1-AS09 E 9 13 0.9 0.4 3.6 0.7 2.1 2.1 5.0 1.0 1.0 1.8 1.4 1.0 0.3 0.7 0.7 1.1 2.0 0.9 5.0

TyrRS1-AS10 E 11 172 0.0 0.0 38 0.9 0.5 0.9 10 0.7 0.5 1.3 0.3 1.0 2.7 3.6 3.1 3.4 0.7 1.1 41

ValRS1

Reference - 0.6 1.0 0.8 0.8 0.4 1.2 0.5 0.6 1.1 1.1 1.0 1.0 0.6 1.0 2.5 1.5 1.6 3.2 2.5 1.7

ValRS1-AS04 E 16 2.3 1.3 0.7 0.0 0.0 3.3 0.8 0.9 0.4 0.3 0.6 1.3 0.4 1.7 7.4 1.9 1.4 1.0 14 1.1

ValRS1-AS12 E 3-16 1.2 0.5 0.4 0.2 0.1 1.4 1.0 1.3 1.0 1.9 0.9 1.5 0.3 1.0 6.8 0.7 0.2 1.1 2.4 2.4

ValRS1-AS16 E 24 3.4 1.6 0.8 0.1 0.1 1.0 0.5 2.3 0.6 0.0 0.6 1.2 0.3 0.5 9.6 1.2 1.1 1.3 6.1 1.0

1

Table S4. Detection of AARS CN Proteins by Western Blot Analysis. In addition to those for the full-length AARSs, bands with the predicted molecular sizes of CNs are listed.

Nam

e

Ori

gin

Cat

alog

#

Ant

igen

Prot

ein

Des

igna

tion

Des

crip

tion

Am

ino

acid

s

MW

(kD

a)

Perc

ent o

f fu

ll le

ngth

Anti-AlaRS1 Santa Cruz SC-165990 aa701-968 AlaRS1 full length 1-968 106.5 100.0 AlaRS1-AS01 C8 293-968 74.3 5.3 Anti-CysRS1 Abnova H00000833-B01P full-length CysRS1 full length 1-831 91.4 100.0 CysRS1-AS06 C5 598-831 26.9 4.7 Anti-LysRS Abcam ab-31532 full-length LysRS1 full length 1-597 65.7 100.0 LysRS1-AS08 N9 1-305 + 518-597 43.5 9.1 LysRS1-AS04 N12 1-129 + 20aa 16.7 3.6 Anti-TyrRS1 TSRI n/a full-length TyrRS1 full length 1-528 58.1 100.0 TyrRS1-AS06 C7 1-170 + 229-528 52.7 8.3 Anti-ValRS1 Abnova PAB3528 N-terminus ValRS1 full length 1-1264 139 100.0 ValRS1-AS16 N15 1-906 + 7aa 101.9 45.4 ValRS1-AS14 N14 1-557 + 2aa 61.9 40.9 ValRS1-AS09 N10 1-525 + 25aa 60.8 41.0 ValRS1-AS08 N5 1-222 + 23aa 26 20.9

1

Table S5. Cells utilized for assays. When possible, cells were sourced directly from primary human tissue. Cells were chosen for diverse cell surface marker and specific biological relevance. Whole blood and neutrophil assays were run from blood sourced at the Normal Blood Donor Service (The Scripps Research Institute, La Jolla, CA). Other cells were sourced directly from ATCC (Manassas, VA) or Invivogen (San Diego, CA). Batch and passage number of all cell types was strictly maintained and most cells were used between passage numbers 2 and 5. (Mo7e cells were the exception, as the passage number for these cells was listed as 10 when the cells were thawed into culture.) All cells were grown and assayed in media obtained from Cell Applications (San Diego, CA) and maintained and passaged per manufacturer’s instructions. Cell Type Catalog # Source HEPG2C3a (human hepatocyte) CRL-10741 ATCC Human Whole Blood EDTA as coagulant Normal Blood Donor Service Human Neutrophils Dextran & ficoll

purified Normal Blood Donor Service

RAW-Blue (modified mouse macrophage) raw-sp Invivogen Human Fibroblast-Like Synoviocytes (HFLS-RA)

408RA-05a Cell Applications: Rheumatoid Arthritis Human Donor (adult)

Human Astrocytes (HA) 882K-05f Cell Applications: Normal Human Donor (fetal) Human Skeletal Muscle (HSkMC) 105-05f Cell Applications: Normal Human Donor (fetal) Human Pulmonary Artery Smooth Muscle (HPASMC)

324-05a Cell Applications: Normal Human Donor (adult)

Human Lung Microvascular Endothelium (HLMVEC)

540-05a Cell Applications: Normal Human Donor (adult)

Human pre-Adipocyte (HpAd) 803-sD Cell Applications: Normal Human Donor (adult) Human Bone Marrow Stromal Cells (HMSC)

492-05f Cell Applications: Normal Human Donor (fetal)

THP-1 (monocytic leukemia) TIB-202 ATCC Mo7e (human megakaryoblastic leukemia) ACC 104 DSMZ (The Leibniz Institute, Germany) HL60 (human promyelocytic leukemia) CCL-240 ATCC RPMI-8226 (human myeloma) CCL-155 ATCC Human Umbilical Cord Endothelial Cells (HUVEC)

200K-05n Cell Applications: Normal Human Donor (neonatal)

1

Table S6. AARS variants can be successfully expressed as recombinant proteins. Biological activities of recombinant AARS fragments were assessed in cell-based assays. Amino acid numbers of specific splice variants are shown here. AARS variant ID Full length AARS amino acids present in variant AlaRS1-C2 758-968 AlaRS1-C6 747-968 AlaRS1-N1 1-401 AlaRS1-N3 1-488 AlaRS1-N6 1-497 + 24 aa ArgRS1-N2 1-71 aa AsnRS1-C2 210-548 AsnRS1-N1 1-322 AsnRS1-N2 1-125 AsnRS1-N3 1-268 + 5 aa AsnRS1-N4 1-111 AsnRS1-N5 1-213 AsnRS1-N6 1-114 + 3 aa AsnRS1-N7 1-267 + 380-548 AspRS1-N3 1-224 AspRS1-N4 1-184 CysRS1-C5 598-831 CysRS1-I2 94-229 CysRS1-I5 555-708 CysRS1-I6 555-748 CysRS1-N6 1-122 + 5 aa GlnRS1-C13 19-208 + 557-793 GlnRS1-C19 1-170 + 344-793 GlnRS1-C25 1-310 + 778-793 GlnRS1-C27 1-556 + 736-793 GlnRS1-C5 580-793 GlnRS1-C9 566-793 GlnRS1-I2 19-253 + 19 aa GlnRS1-I3 19-253 + 38 aa GlnRS1-I7 19-201 GlnRS1-I10 19-267

2

AARS variant ID Full length AARS amino acids present in variant GlnRS1-I11 19-281 GlnRS1-I13 180-281 GlnRS1-I14 19-143 + 6 aa GlnRS1-N15 1-218 GlnRS1-N16 1-238 GluProRS1-C11 992-1512 GluProRS1-C3 950-1512 GluProRS1-C6 1384-1512 GluProRS1-I1 750-1021 GluProRS1-I3 682-1025 GluProRS1-N5 1-212 GlyRS1-C15 612-739 GlyRS1-I3 57-121 HisRS1-C1 405-509 HisRS1-C2 1-60 + 175-509 HisRS1-C3 1-60 + 211+509 IleRS1-C3 942-1266 LysRS1-C2 157-597 LysRS1-C5 469-597 LysRS1-I1 65-214 LysRS1-N2 1-76 aa LysRS1-N4 1-65 aa LysRS1-N5 1-214 LysRS1-N6 1-74 + 22 aa LysRS1-N10 1-74 + 2 aa LysRS2-N10 1-333 + 546-625 LysRS2-N11 1-102 + 2 aa LysRS2-N12 1-48 + 1 aa LysRS2-N2 1-104 LysRS2-N3 1-222 LysRS2-N4 1-93 aa LysRS2-N5 1-242 LysRS2-N6 1-49 + 12 aa LysRS2-N7 1-102 + 22 aa MetRS1-C5 846-900 PheRSa1-N1 1-226 PheRSa1-N2 1-219 PheRSa1-N3 1-128 + 13 aa

3

AARS variant ID Full length AARS amino acids present in variant PheRSa1-N9 1-152 PheRSb1-C1 373-589 PheRSb1-I1 89-304 PheRSb1-N3 1-116 SerRS1-C2 464-514 SerRS1-C3 425-514 SerRS1-N1 1-152 SerRS1-N2 1-472 SerRS1-N4 1-45 aa SerRS1-N5 1-46 + 33 aa SerRS1-N6 1-46 + 5 aa SerRS1-N9 1-147 ThrRS1-C1 307-723 ThrRS1-C5 294-723 ThrRS1-N1 1-322 ThrRS1-N2 1-146 ThrRS1-N3 1-328 + 1aa ThrRS1-N4 1-109 + 9aa TyrRS1-C1 390-528 TyrRS1-C2 340-528 ValRS1-N11 1-214 ValRS1-N12 1-229 ValRS1-C4 940-1264 ValRS1-N2 1-230 ValRS1-N3 1-300