Posttranscriptional Regulation of HLA-A Protein Expression By

Posttranscriptional Regulation of HLA-A Protein Expression by Alternative Polyadenylation Signals Involving the RNA-Binding Protein Syncrip This information is current as of September 27, 2021. Smita Kulkarni, Veron Ramsuran, Marijana Rucevic, Sukhvinder Singh, Alexandra Lied, Viraj Kulkarni, Colm O'hUigin, Sylvie Le Gall and Mary Carrington J Immunol 2017; 199:3892-3899; Prepublished online 20 October 2017; Downloaded from doi: 10.4049/jimmunol.1700697 http://www.jimmunol.org/content/199/11/3892 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/10/20/jimmunol.170069 Material 7.DCSupplemental References This article cites 66 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/199/11/3892.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Posttranscriptional Regulation of HLA-A Protein Expression by Alternative Polyadenylation Signals Involving the RNA-Binding Protein Syncrip

Smita Kulkarni,*,† Veron Ramsuran,*,‡,x,{ Marijana Rucevic,* Sukhvinder Singh,† Alexandra Lied,* Viraj Kulkarni,*,‖ Colm O’hUigin,‡ Sylvie Le Gall,* and Mary Carrington*,‡

Genomic variation in the untranslated region (UTR) has been shown to influence HLA class I expression level and associate with disease outcomes. Sequencing of the 39UTR of common HLA-A alleles indicated the presence of two polyadenylation signals (PAS). The proximal PAS is conserved, whereas the distal PAS is disrupted within certain alleles by sequence variants. Using 39RACE, we confirmed expression of two distinct forms of the HLA-A 39UTR based on use of either the proximal or the distal PAS, which differ Downloaded from in length by 100 bp. Specific HLA-A alleles varied in the usage of the proximal versus distal PAS, with some alleles using only the proximal PAS, and others using both the proximal and distal PAS to differing degrees. We show that the short and the long 39UTR produced similar mRNA expression levels. However, the long 39UTR conferred lower luciferase activity as compared with the short form, indicating translation inhibition of the long 39UTR. RNA affinity pull-down followed by mass spectrometry analysis as well as RNA coimmunoprecipitation indicated differential binding of Syncrip to the long versus short 39UTR. Depletion of Syncrip by small interfering RNA increased surface expression of an HLA-A allotype that uses primarily the long 39UTR, whereas an http://www.jimmunol.org/ allotype expressing only the short form was unaffected. Furthermore, specific blocking of the proximal 39UTR reduced surface expression without decreasing mRNA expression. These data demonstrate HLA-A allele-specific variation in PAS usage, which modulates their cell surface expression posttranscriptionally. The Journal of Immunology, 2017, 199: 3892–3899.

he HLA class I molecules are expressed on virtually all recently, a variant in an Oct1-binding site 800 bp upstream of the nucleated cells and present peptides to CTL, initiating an HLA-C coding region was also shown to regulate HLA-C ex- T adaptive immune response. They also serve as ligands for pression levels (2), and this variant along with the polymorphic

the killer cell Ig-like receptors expressed on NK cells, thereby miRNA-binding site account for 40% of the differential cell sur- by guest on September 27, 2021 regulating NK cell responses. HLA class I genes are the most face expression levels of HLA-C. Thus, variation in regulatory polymorphic loci in the human genome, and along with the HLA regions of the HLA class I loci that affect their expression levels class II genes, they associate with more human diseases than any may inﬂuence the immune response and disease susceptibility. other locus genome-wide (GWAS catalog; http://www.ebi.ac.uk/ The mRNA expression levels of HLA-A alleles were recently gwas). Disease associations with HLA class I variation have shown to vary in an allele-dependent manner as a function of the largely been attributed to variants encoding polymorphic amino degree of methylation in the promoter region of each allele (3). acid positions in the peptide-binding groove of the class I mole- Thus, epigenetic mechanisms account for a portion of the differ- cules. We have shown previously that a polymorphic microRNA ential mRNA expression patterns across HLA-A alleles. In this (miRNA)-binding site in the 39 untranslated region (UTR) of study, we describe, to our knowledge, a novel mechanism, the use HLA-C contributes to allele-speciﬁc variation in expression levels of alternative polyadenylation (APA) signals in the 39UTR of and associates with both HIV viral control and risk of Crohn HLA-A, which modulates expression levels of HLA-A protein disease independently of individual HLA allelic effects (1). More through regulation of translation. These data underscore the

*Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139; †Department of cation does not necessarily reflect the views or policies of the Department of Health Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227; ‡Cancer and and Human Services, nor does mention of trade names, commercial products, or Inflammation Program, Leidos Biomedical Research Inc., Frederick National Laboratory organizations imply endorsement by the U.S. Government. This research was sup- for Cancer Research, Frederick, MD 21702; xKwaZulu-Natal Research Innovation and ported in part by the Intramural Research Program of the National Institutes of Sequencing Platform, School of Laboratory Medicine and Medical Sciences, University Health, Frederick National Laboratory, and Center for Cancer Research. of KwaZulu-Natal, Durban 4001, South Africa; {Centre for the AIDS Programme of Address correspondence and reprint requests to Smita Kulkarni, Department of Ge- Research in South Africa, Nelson R. Mandela School of Medicine, University of ‖ netics, Texas Biomedical Research Institute, Building 12, Room 12.126, 7620 NW KwaZulu-Natal, Durban 4001, South Africa; and Department of Virology and Immu- Loop 410, San Antonio, TX 78227-5301. E-mail address: [email protected] nology, Texas Biomedical Research Institute, San Antonio, TX 78227 The online version of this article contains supplemental material. ORCIDs: 0000-0003-1590-9893 (V.R.); 0000-0002-2692-2180 (M.C.). Abbreviations used in this article: APA, alternative polyadenylation; miRNA, micro- Received for publication May 11, 2017. Accepted for publication September 25, RNA; MS, mass spectrometry; ORF, open reading frame; PAS, polyadenylation 2017. signal; PASD, distal PAS; PASP, proximal PAS; PASPD, proximal and distal PAS; This work was supported by institutional funds from the Texas Biomedical Research qPCR, quantitative PCR; RBP, RNA-binding protein; RIP, RNA immunoprecipita- Institute and the Ragon Institute of MGH, MIT and Harvard. V.R. is supported by tion; RNP, ribonucleoprotein; siRNA, small interfering RNA; UTR, untranslated South African Medical Research Council Grant MRC-RFA-UFSP-01-2013/UKZN- region. HIVEPI. S.S. is supported by the Cowles Postdoctoral Fellowship. This work has been supported in part with federal funds from the Frederick National Laboratory for Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Cancer Research under Contract HHSN261200800001E. The content of this publi- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700697 The Journal of Immunology 3893 complex regulatory mechanisms that dictate HLA class I expression Construction of the HLA-A open reading frame–c-Flag vector levels. HLA-A open reading frame (ORF) with a C-terminal DYKDDK (Flag) tag was amplified using PCR (clone OHu21196; NM_002116; GenScript). The Materials and Methods Renilla luciferase sequence was replaced by Flag-tagged HLA-A ORF in Samples the PASP, proximal and distal PAS (PASPD), and PASD vectors described earlier. Oligonucleotide sequences for the cloning primers used are listed Healthy European American donors recruited at the National Cancer In- in Supplemental Table I. stitute (Frederick, MD) were used for determination of HLA-A genotypes and the length of the HLA-A 39UTR. The respective Institutional Review Cell lines, cell transfection, luciferase reporter assays, and Boards approved the study, and all subjects gave written informed consent. Western blots HLA genotyping Transformed B cell lines from three individuals homozygous for HLA-A*11, A*03,orHLA-A*26, and the human T cell line Jurkat were grown in RPMI DNA samples were genotyped for HLA-A, -B,and-C genes by sequence- 1640 medium (Life Technologies) with 10% heat-inactivated FBS (Atlanta based typing of exons 2 and 3 and/or the PCR sequence-specific oligonu- Biologicals). PBLs were plated at a density of 1 3 106 cells per well in a cleotide probe typing protocol as recommended by the 13th International 96-well plate and transfected with 1 mM final concentration of either the Histocompatibility Workshop (4). The entire HLA-A 39UTR was amplified Apro1 morpholino oligonucleotide or the control morpholino oligonucleotide from genomic DNA by PCR-specific primers (Supplemental Table I). (Supplemental Table I) using Amaxa 4D-Nucleofector (Lonza). The cells The amplicons were sequenced in both directions using the same primers by were incubated for 36 h in a 37˚C CO2 incubator before determining cell capillary electrophoresis using an ABI 31730XL DNA analyzer (Applied surface expression of HLA-A. Jurkat cells were plated at a density of 0.5 3 Biosystems). 106 cells per well in a 12-well plate. One microgram per well of the 39UTR reporter constructs in the modified psichek2 vectors was transfected using the 39RACE and quantitative PCR Downloaded from optimized TransIT-X2 (Mirus Bio) protocol. The transfected Jurkat cells were Peripheral blood was obtained from healthy donors, and lymphocytes were incubatedfor18hat37˚CinaCO2 incubator. The cells were lysed and the separated using lymphocyte separation medium as per the manufacturer’s firefly and Renilla luciferase activity was measured using the Dual-Luciferase instructions (Lonza). Total RNA was extracted from PBLs (RNeasy uni- reporter assay system (Promega). Renilla luciferase activity was normalized versal kit; Qiagen). Each sample was treated with an eliminator to remove relative to the firefly luciferase activity for each transfection. Renilla luciferase genomic DNA. The RNAs were quantitated using HT RNA Lab Chip activity of each reporter construct is calculated as fold change relative to the (Caliper; Life Sciences), and all samples had an RNA quality score of .8. normalized activity of plasmid containing the 39UTR of A*01.Allexperi-

To determine the length of the 39UTR, 39RACE was performed using a kit ments were performed with six replicates in three independent experiments. http://www.jimmunol.org/ (Invitrogen) according to the manufacturer’s instructions. One microgram Jurkat cells transfected with HLA-A ORF-Flag plasmids were lysed 48 h of RNA was used to initiate the first-strand cDNA synthesis at the poly(A) after transfection. Western blot was carried out with DYKDDDK (Flag) tag tail of the mRNA. The HLA-A 39UTR was amplified using a gene-specific Ab (GenScript) and anti-firefly luciferase (GenTex) Ab. forward primer (HLA-A 39UTR 39RACE forward) and a universal primer (UAP) that targets the 39 end as the reverse primer (Supplemental Table I). Abs and flow cytometry Amplicons were electrophoresed on 2% agarose gels to determine their HLA-A expression on the surface of PBLs from an HLA-A*26 homozygous length, and then they were cloned and sequenced to confirm the specificity donor was analyzed by staining with a biotinylated mAb that specifically of amplification. binds the HLA-A*26 molecule (BIH0048; One Lambda) used in con- The mRNA expression level was measured by quantitative PCR (qPCR). junction with steptavidin-allophycocyanin (BioLegend), and expression Briefly, reverse transcription was performed with 900 ng of total RNA using was measured using an LSR II flow cytometer (BD Biosciences). Trans- by guest on September 27, 2021 the high-capacity RNA to cDNA kit (Applied Bioscience) in a volume of formed B cell lines expressing HLA-A*03 and HLA-A*11 were stained 10 ml. HLA-A coding region [amplified using previously published primers using respective Abs (BIH0269 [One Lambda] in conjunction with (3)], HLA-A 39UTR, and GAPDH transcripts were amplified (primer se- streptavidin-allophycocyanin [BioLegend] for A*03; 0544HA [One quences in Supplemental Table I) by SYBR Green qPCR using the Lambda] with PE-labeled secondary Ab [BioLegend] for A*11) and ex- threshold cycle method (5) in a Viia7 machine (Applied Bioscience). Each pression was measured using an AccuriC6 flow cytometer. The histograms qPCR reaction included 6 ml of power SYBR Green PCR master mix were plotted using FlowJo software version 10. (Applied Biosystems), 200 nM primers that specifically amplified the gene of interest (HLA-A) or the housekeeping gene (GAPDH), and 2 mlof RNA affinity pull-down, PAGE, and Western blot analyses cDNA (1:20 dilution) in a total volume of 12 ml. The genes were amplified using the following conditions: 50˚C for 2 min, 95˚C for 2 min, followed An RNA affinity pull-down protocol was adapted from a previously pub- by 40 cycles of 95˚C for 15 s and 60˚C for 30 s. The specificity of primers lished method (6). The 39UTR fragments of canonical A*03 (PASPD) and was verified by melt curve analysis using a dissociation step following the PASDD300–325 were in vitro transcribed, labeled with biotin (biotin la- qPCR protocol. Specificity of the primers was confirmed by sequencing beling mix; Sigma-Aldrich), and bound to the avidin agarose beads the HLA-A amplicons. The primers were found to amplify HLA-A spe- (Sigma-Aldrich). The RNA-coated beads were incubated with Jurkat cell cifically and did not cross-react with any other locus. The average ex- lysate and washed to remove unbound proteins. The proteins captured on pression levels of the genes were normalized to that of GAPDH RNA the beads were separated with Tris-glycine 4–12% gradient protein gels using the 22DDCt method (5). Oligonucleotide sequences of the primers (Invitrogen) electrophoresis and detected by Coomassie blue stain. Western are listed in Supplemental Table I. blot was carried out using anti-Syncrip Ab (Invitrogen). Construction of HLA-A 39UTR luciferase reporters Identification of proteins by nano liquid chromatography–tandem mass spectrometry The synthetic poly(A) signal downstream of Renilla luciferase gene in a psicheck2 vector (Promega) was deleted. The complete 39UTR frag- The gel bands were excised with gel-cutting racked tips and digested with ments from two HLA-A alleles (A*01 and A*03) were amplified from trypsin as described previously (7). Tryptic digests of proteins extracted genomic DNA and inserted downstream of the Renilla luciferase gene in from the gels (“in-gel” digests) were separated with a reversed-phase the modified psicheck2 vector. To create a functional distal promoter column using a linear gradient. Eluted peptides were subjected to only, mutations were introduced in the proximal polyadenylation signal reversed-phase microcapillary nano liquid chromatography–tandem (PAS) of HLA-A*03 39UTR using a site-directed mutagenesis kit (Stra- mass spectrometry (MS) analysis using an Eksigent HPLC system di- tagene). Sequential deletions in the distal PAS (PASD) sequence were rectly interfaced with an Orbitrap LTQ XL mass spectrometer (Thermo carried out and a strong PAS (AAUAAA) was added at the end of each Fisher). The eluted ions were analyzed by full precursor MS scans ac- fragment: 375 bp (PASD375), 350 bp (PASD350), 325 bp (PASD325), quired with the FT Orbitrap analyzer operated at a resolving power of and 300 bp (PASD300), respectively. A 25-bp region between the 30,000 (400–2000 m/z). The MS spectrum was followed by eight tan- proximal and the distal PAS spanning from position 300 to 325 bp dem MS spectra, where the eight most abundant multiply charged ions (i.e., immediately downstream of the proximal PAS [PASP]) was deleted were selected for tandem MS sequencing. Raw data were analyzed with to construct the PASDD300–325 mutant. All mutant amplicons were Proteome Discoverer 1.4 (Thermo Fisher; https://tools.thermofisher. cloned downstream of Renilla luciferase in the modified psicheck2 com/content/sfs/manuals/Man-XCALI-97506-Proteome-Discoverer-14- vector. Oligonucleotide sequences for the cloning primers used are listed User-ManXCALI97506-A-EN.pdf) software and searched against the in Supplemental Table I. SwissProt database restricted to human entries by using the MASCOT 3894 POSTTRANSCRIPTIONAL REGULATION OF HLA-A

HLA-A 39UTR sequences downstream of Renilla luciferase in a modified psicheck2 vector where the synthetic PAS downstream of Renilla luciferase was deleted (Supplemental Fig. 3A). Three clones were constructed using the following 39UTR sequences: HLA-A*01 39UTR, which has an intact proximal PAS and mutated distal PAS (PASP); A*03 39UTR, which carries intact proximal and distal PAS (PASPD); and HLA-A*03 39UTR with an experi- mentally mutated proximal PAS, which uses only the PASD (Fig. 2A). The three constructs did not differ significantly in mRNA FIGURE 1. Variation in the relative abundance of the two forms of the expression levels of luciferase (Fig. 2B). However, expression HLA-A 39UTR. RNA was obtained from PBLs of healthy donors. 39RACE level of Renilla luciferase protein was a function of the length of of 39UTRs of HLA-A alleles from six individuals with distinct HLA-A the 39UTR, with the long form (PASD) showing the least lucif- genotypes showed preferential use of either the proximal or distal PAS. erase activity, presence of both intact forms (PASPD) showing HLA-A*01 and A*11 use only the proximal PAS, resulting in the short form of the 39UTR, whereas others use various fractions of each. HLA-A intermediate activity, and the short form (PASP) producing the genotypes of the individuals are indicated below each amplicon. greatest luciferase activity (Fig. 2C). We further confirmed the effect of length of the 39UTR on HLA-A protein expression by (http://www.matrixscience.com/help/seq_db_setup_sprot.html) search replacing the firefly luciferase sequence with an HLA-A ORF se- engine. The precursor-ion tolerance was 10 ppm and the fragment-ion quence and C-terminal Flag tag (Supplemental Fig. 3B) in the PASP, tolerance was 0.8 Da. Enzymatic digestion was specified as trypsin, with PASPD, and PASD vectors (A-PASD, A-PASPD and A-PASD; Fig. Downloaded from up to two missed cleavages allowed. 2D). The HLA-A mRNA expression levels of the three plasmids RNA immunoprecipitation were similar (Fig. 2E). Concordant with the luciferase assay, however, A-PASD showed the least HLA-A protein expression as de- EBV-transformed B lymphoblastoid cell lines derived from individuals with termined by Western blot analysis (Fig. 2F). The effect of the length known HLA-A genotypes (HLA-A*11 or A*03 homozygotes) were transfected with a plasmid encoding Syncrip cDNA with a Myc-tag (RC217902; of the 39UTR on HLA-A protein expression in Jurkat (Fig. 2D–F)

OriGene) or a control plasmid (pcDNA3.1-eGFP; Addgene). For RNA was confirmed in HEK293 cells (Supplemental Fig. 3C). http://www.jimmunol.org/ immunoprecipitation of ribonucleoprotein (RNP) complexes from whole- To determine whether the length of the HLA-A 39UTR has an cell extracts, the transfected cells were lysed in 20 mM Tris-HCl at pH 7.5, effect on endogenous HLA-A surface expression levels, PBLs 100 mM KCl, 5 mM MgCl2, and 0.5% Nonidet P-40 for 10 min on ice and centrifuged at 10,000 rpm for 15 min at 4˚C. The supernatants were in- from an HLA-A*26 homozygous donor were transfected with ei- cubated with magnetic beads coated with anti-Myc Abs (Invitrogen) ther an anti-sense morpholino oligonucleotide that blocks the overnight at 4˚C. After the beads were washed with TBS-T buffer, the proximal PAS of HLA-A (APro1) or with a control morpholino complexes were incubated with 20 U of RNase-free DNase I (15 min at (control), and HLA-A mRNA as well as surface expression levels 37˚C) and further incubated with 0.1% SDS/0.5 mg/ml proteinase K (15 were measured 36 h later (Fig. 3A). Although there was no effect min at 55˚C) to remove DNA and proteins, respectively. The RNPs isolated from the RNA immunoprecipitation (RIP) were further assessed by of APro1 on expression levels of HLA-A*26 mRNA (Fig. 3B), an Western blot using an anti-Myc Ab (Invitrogen) for detection of the Myc- allele that uses both the proximal and distal PAS, cell surface by guest on September 27, 2021 tagged proteins and qPCR for detection of HLA-A 39UTR. expression of A*26 was significantly decreased upon blockage of the proximal PAS (Fig. 3C). Taken together with the data showing Results an effect of 39UTR length on luciferase activity and total protein Genomic DNA from white individuals homozygous for common expression of the constructs without a change in mRNA abun- HLA-A alleles were used to amplify and sequence the HLA-A 39UTR dance (Fig. 2), these data indicate that the length of the 39UTR using sequence-specific primers. HLA-A 39UTRs encode two PAS, a affects protein expression through a mechanism involving trans- canonical polyadenylation signal motif (59-AATAAA-39)aswellas lation of HLA-A, and not through differential stability of HLA-A its common variant (59-ATTAAA-39; Supplemental Fig. 1). The distal transcripts. PAS (59-AATAAA-39; 395–400 bp downstream of the stop codon) The lower level of luciferase expression associating with the was found to be polymorphic and disrupted in some alleles, including usage of the distal PAS suggested the presence of a translation HLA-A*01 and A*11 (Supplemental Fig. 1), but the proximal PAS inhibitory sequence located between the proximal and distal PAS (59-AATAAA-39; 294–299 bp) was conserved across alleles. 39RACE sequences, which are separated by 100 bp (nucleotide positions was carried out using RNA from PBLs of healthy donors, confirming 300–400 of the 39UTR). We therefore sequentially deleted 25-bp thepresenceoftwodistinctformsofthe39UTR that differ in length segments of the region between the proximal and distal PAS in the by 100 bp (Fig. 1), the sequence of which indicated usage of either PASD construct, leaving the distal PAS intact, but disrupting the the proximal PAS (294–299 bp; short 39UTR) or the distal PAS (395– proximal PAS (Fig. 4A). Deletion of the 25 bp (PASD375), 50 bp 400 bp; long 39UTR). Interestingly, HLA-A alleles vary in expression (PASD350), and 75 bp (PASD325) immediately upstream of the ratios of the short versus long forms, with some alleles expressing distal PAS resulted in low luciferase expression similar to that of only the short form due to a polymorphism in the distal PAS PASD, but deletion of the remaining 25 bp (PASD300) resulted in (HLA-A*01 and A*11), some expressing predominantly the long high levels of luciferase expression similar to that observed for form (HLA-A*03), and others expressing both forms to differing PASP, in which only the proximal PAS is intact (Fig. 4B). Deletion degrees. HLA-B and -C alleles, alternatively, encode only the distal of only the 25 bp immediately downstream of the proximal PAS PAS (Supplemental Fig. 2A) and express the long form of 39UTR and retaining the remaining downstream 75 bp (PASDD300–325) (Supplemental Fig. 2B, 2C). The patterns of alternative poly(A) resulted in high luciferase expression. Taken together, these data usage by distinct HLA-A alleles was consistent across CD4+ point to the presence of an inhibitory sequence located between T cells, CD8+ T cells, and monocytes from peripheral blood, as 300 and 325 bp. well as multiple cells lines (Jurkat, HEK293T, transformed B cell RNA-binding proteins (RBPs) (8, 9) and miRNAs (10, 11) are lines) (Supplemental Fig. 2B, 2C). knowntobindthe39UTR of many mRNA species and regulate their To determine whether the length of the 39UTR affects HLA-A translation. Bioinformatic analyses did not predict a strong miRNA- mRNA expression levels, constructs were cloned that contained binding site within the 300- to 325-bp inhibitory sequence, raising The Journal of Immunology 3895

long form (Fig. 5A, Supplemental Fig. 3E). Furthermore, the small interfering RNA (siRNA)–mediated knockdown of Syncrip (Fig. 5B) enhanced luciferase activity of the constructs expressing the long form(PASPDandPASD),buthadnosignificanteffectonluciferase activity of the construct that is missing the distal PAS (PASP) or is missing the Syncrip binding region (PASDD300–325; Fig. 5C). To confirm the interaction between Syncrip and cellular HLA-A 39UTR, a vector encoding Syncrip tagged with cMyc or a GFP control was transfected into transformed B cell lines (Fig. 6A) from individuals homozygous for HLA-A alleles that use either the long 39UTR (HLA-A*03) or the short 39UTR (HLA-A*11), and RIP was performed. The cMyc-tagged Syncrip was immunoprecipitated using anti-Myc Ab–coated magnetic beads and specific pulldown was confirmed using Western blot (Fig. 6B). Quantitative amplification (qPCR) of the coprecipitated, protein-bound RNA following RIP with Downloaded from

FIGURE 2. The long HLA-A 39UTR results in less luciferase activity and protein expression as compared with the short form. (A) Design of the http://www.jimmunol.org/ luciferase reporter constructs used in this study is shown. The active PAS are indicated by black triangles, whereas disrupted PAS are indicated by open gray triangles. (B) The HLA-A 39UTR luciferase constructs were transfected into Jurkat cell lines. Total RNA was extracted from the transfected cells after 18 h. Specific primers were used to measure the abundance of Renilla and firefly luciferase transcripts by qPCR. The normalized mRNA abundance is presented as a relative ratio of Renilla/ firefly mRNA. The data represent triplicates in each experimental group. (C) Renilla and firefly luciferase activity was estimated by Dual-Luciferase assays and presented as the normalized ratio of Renilla versus firefly by guest on September 27, 2021 luciferase activity. The data represent six replicates in each group. The mean 6 SE are depicted as horizontal and vertical bars for each group, respectively, and one of three comparable experiments that were performed is shown. Nonparametric Wilcoxon–Mann–Whitney U tests were used for statistical comparisons and two-tailed p values are indicated. (D) Design of the HLA-A ORF-Flag constructs used in this study is shown. The active PAS are indicated by black triangles, whereas disrupted PAS are indicated by open gray triangles. (E) Jurkat cells were transfected with HLA-A ORF-Flag plasmids. Total mRNA was extracted from the transfected cells, and cell lysates were extracted from the transfected cells after 24 h. Specific primers were used to measure the abundance of HLA-A and firefly luciferase transcripts by qPCR. The normalized mRNA abundance is presented as a relative ratio of HLA-A/firefly mRNA. The data represent triplicates in each experimental group. (F) Total cell lysates of Jurkat cells transfected with HLA-A ORF-Flag plasmids were used to carry out Western blot analysis with anti-Flag and anti–firefly luciferase Abs. One of two independent experiments is shown. FIGURE 3. Blocking the proximal PAS inhibited surface expression of HLA-A on PBLs. (A) PBLs from an individual expressing A*26 were the possibility that an RBP may be involved in translation inhibition transfected with either a morpholino oligonucleotide blocking the proxi- of the long form. To identify the RBPs specific for the inhibitory mal PAS of HLA-A (APro1) or with a control morpholino and cultured for sequence, we performed RNA pulldown experiments. The canonical 36 h. Apro1 inhibited the production of the short (proximal PAS) form of A*03 39UTR (PASPD) and the D300–325 mutant (PASDD300–325) the HLA-A 39UTR, but the control morpholino does not. (B) HLA-A*26 sequences were in vitro transcribed and biotinylated. Jurkat cell ly- mRNA expression is not affected by blockade of the proximal PAS. (C) sates were mixed with the biotinylated RNAs, precipitated with Cell surface expression of HLA-A*26 was reduced on cells transfected streptavidin beads, and separated by PAGE. To identify the RBPs with APro1 relative to the expression level on cells transfected with the control morpholino. One of three comparable experiments performed is that were precipitated specifically with the canonical A*03 39UTR ∼ depicted in the histogram. Fold change in expression levels shown in the (PASPD), we excised a protein band ( 70 kDa) that was selectively bar graph was calculated as ratio of mean fluorescence intensity (MFI) of precipitated with the PASPD (Supplemental Fig. 3D). We also ex- HLA-A*26 versus isotype control. The data represent three replicates in cised the corresponding gel regions in the PASDD300–325 and the each experimental group. The means 6 SE are depicted as horizontal and bead-only lane. The proteins in the excised gel bands were analyzed vertical bars for each group, respectively. A Student t test was used for by MS, identifying Syncrip as the protein that bound to the canonical statistical comparisons and a two-tailed p value is indicated. 3896 POSTTRANSCRIPTIONAL REGULATION OF HLA-A

Discussion Polyadenylation is essential for the stability, appropriate cellular localization, and efﬁcient translation of mRNA transcripts (12). Transcriptome-wide analyses have revealed that ∼70% of human genes use alternative polyadenylation to generate transcript isoforms with varying lengths of 39UTRs (13). In this study, we describe the identiﬁcation of two distinct forms of the HLA-A 39UTR that differ in length by 100 bp. Multiple cell subsets and cell lines of distinct tissue origins showed consistent alternative poly(A) usage by the given HLA-A alleles. Using a luciferase reporter assay, we showed that both forms produced similar mRNA expression levels, but the long 39UTR resulted in lower luciferase activity compared with the short form, suggesting the presence of a motif within the intervening sequence that results in Downloaded from http://www.jimmunol.org/

FIGURE 4. A translation inhibitor sequence is encoded within the 300- to 325-bp segment immediately downstream of the proximal PAS in the

HLA-A 39UTR. (A) Schematic representations of the luciferase reporter by guest on September 27, 2021 constructs used to identify the region causing inhibition of translation. Portions of PASD, the construct in which the proximal PAS of HLA-A*03 is mutated, were sequentially deleted by 25-bp increments with an intact PAS added at the end of each deletion. An intact PAS is shown as black triangles, and a disrupted PAS is shown as open gray triangles. (B) The various 39UTRs were cloned into the luciferase vector and transfected into Jurkat cells, and luciferase activity of each was compared with the full- length, canonical 39UTRs of HLA-A*01 (PASP) and A*03 (PASPD). De- letion of the 25 bp immediately downstream of the disrupted proximal PAS resulted in significantly higher luciferase activity than any other PAS construct in which this 25-bp segment was present, indicating the presence of an inhibitory sequence between 300 and 325 bp. The normalized luciferase activity is presented as the relative ratio of Renilla/firefly luciferase. The data represent six replicates in each experimental group. The FIGURE 5. Syncrip binds the long form of HLA-A 39UTR and inhibits mean 6 SE are depicted as horizontal and vertical bars for each group, luciferase activity of the constructs. (A) Western blot indicated the pres- respectively, and one of three comparable experiments performed is ence of Syncrip bound to the long form of HLA-A 39UTR. RNA affinity shown. Nonparametric Wilcoxon–Mann–Whitney U tests were used for pulldown was carried out with the in vitro–transcribed 39UTR fragments of statistical comparisons and two-tailed p values are indicated. canonical A*03 (PASPD) and PASDD300–325, labeled with biotin, and incubated with Jurkat cell lysate. RNA-bound protein was separated on PAGE, and Western blot was carried out using anti-Syncrip Ab. (B) Jurkat anti-cMyc Ab–coated magnetic beads showed that the 39UTR of the cells were transfected with either an siRNA targeting Syncrip (Syncrip allele expressing the long form (A*03), but not the short form (A*11), siRNA) or a control siRNA. Knockdown of Syncrip protein was confirmed coprecipitated with Syncrip (Fig. 6C), confirming the specific asso- after 72 h posttransfection using Western blot. (C) Seventy-two hours after ciation of the long form of 39UTR with Syncrip. Finally, we trans- siRNA transfection, Jurkat cells were transfected with various HLA-A fected B cell lines expressing HLA-A*03 or A*11 with siRNA 39UTR constructs and luciferase activity was measured after 18 h. Inhi- targeting Syncrip or a control siRNA. Syncrip knockdown was con- bition of Syncrip increased luciferase activity of the constructs encoding the distal PAS (PASPD; PASD). Luciferase activity of the constructs firmed using Western blot (Fig. 6D). Reduction in Syncrip increased containing only the proximal PAS (PASP) or lacking the inhibitory se- surface expression of HLA-A*03, which uses the long form of the quence (PASDD300–325) were not affected. The means 6 SE (n = 6) are 39UTR, whereas expression of A*11, which uses only the short form depicted as horizontal and vertical bars for each group, respectively, and of 39UTR, was unaffected (Fig. 6E). These data strongly point to one of three comparable experiments performed is shown. Nonparametric Syncrip as the RBP involved in decreased translation of HLA-A al- Wilcoxon–Mann–Whitney U tests were used for statistical comparisons, leles that use the distal PAS (i.e., long form). and two-tailed p values are indicated. The Journal of Immunology 3897 Downloaded from

FIGURE 6. Differential binding of Syncrip to the long form of HLA-A 39UTR regulates expression. RNA immunoprecipitation was carried out to confirm http://www.jimmunol.org/ differential binding of Syncrip to the long 39UTR of cellular HLA-A mRNA. (A) Transformed B cell lines from individuals with HLA-A*11 or HLA-A*03 homozygous genotypes were transfected with a c-Myc–tagged Syncrip encoding vector (Syncrip-Myc) or with pcDNA3-GFP (Control). Cells were lysed after 48 h and expression of c-Myc–tagged Syncrip as well as GFP control was confirmed using Western blot. (B) RNA immunoprecipitation was performed with the anti-c-Myc Ab–coated magnetic beads. Specific pull-down of myc-tagged Syncrip was confirmed by Western blot. (C) Fold enrichment of HLA-A 39UTR in the immunoprecipitated RNA was analyzed by qPCR. (D) EBV-transformed B cell lines from individuals with HLA-A*11 or HLA*03 homozygous genotypes were transfected with a Syncrip targeting siRNA and cultured for 72 h. Syncrip knockdown was confirmed by Western blot. (E) Cell surface expression levels of the HLA-A allotypes treated with the Syncrip siRNA (open black curve) or a control siRNA (gray curve) were measured using allele-specific Abs. Cell surface expression of HLA-A*03 (long form of 39UTR) was enhanced on the cells transfected with Syncrip-targeting siRNA, whereas expression of HLA-A*11 (short form of 39UTR) remained unaffected. The dotted curves indicate isotype control. Fold change in expression levels by guest on September 27, 2021 was calculated as the ratio of mean fluorescence intensity (MFI) of HLA-A versus isotype control. A histogram of one of three comparable experiments performed is shown for each allele. The means 6 SE (n = 3) are depicted as horizontal and vertical bars for each group, respectively. Student t tests were used for statistical comparisons, and a two-tailed p value is indicated. translation inhibition of the long 39UTR. Sequential deletion of proteins. Additionally, any quantitative differences in total RBPs segments within the 100-bp intervening sequence mapped the in- pulled down with the long or mutant 39UTR were not considered for hibitory sequence immediately downstream of the proximal PAS. this analysis. Thus, additional RBPs acting in association with or 39UTRs encode docking sites for miRNAs (10, 11) as well as RBPs independently of Syncrip may have a role in determining which PAS (8, 9), which are the major determinants of posttranscriptional gene is used, and in translation efficiency of HLA-A mRNA. regulation. Several reports indicate a role of 39UTR-associated RBPs The regulation of differential APA is yet unclear and several in the posttranscriptional regulation of HLA genes (14–17), including possible mechanisms have been proposed. Based on bioinformatic another member of the heterogeneous nuclear RNP family, HNRNP- analysis or limited functional evidence in cell lines, several factors R (17). We identified the RBP Syncrip as the translational inhibitor have been shown to influence the choice of the poly(A) site, in- of the long form of the HLA-A 39UTR. Depletion of Syncrip including the strength of the poly(A) signals (49), expression levels creased surface expression of HLA-A*03, an allotype that normally of poly(A) binding factors (50), sequence motifs surrounding the uses the distal PAS (i.e., long 39UTR),buthadnoeffectonex- poly(A) signals (51), and epigenetic alterations (52–54). Several pression level of HLA-A*11, an allotype associated with only the studies indicate coupling of APA regulation with transcription short form. Syncrip (alias HNRNPQ1, Nsap1) is a ubiquitously (55–57), which may be related to the relative increase in short expressed, cytoplasmic isoform of the RBP HNRNPQ (18–20) and 39UTRs across the transcriptome observed upon immune cell is known to regulate splicing (21–24), editing (25–27), transport activation (58). Recently, vesicular stomatitis virus infection was (28–32), translation (33–42), and stability (43–48) of mRNA. Syn- shown to induce shortening of 39UTRs globally and especially in crip binding sites are enriched in two core consensus sequences immune genes (59). The global shortening of 39UTRs is thought to (AYAAYY and UAUYRR; Y = C/U and R = A/G) (29) as well as facilitate rapid change in protein expression as well as increase in AU-rich elements (25). HLA-A 39UTR encodes one AU-rich elemet proliferation. Thus, this mechanism may enhance the strength of (AUUUA; 318–322 bp) located within the translation inhibitory immune responses under certain conditions, such as exposure to sequence that we identified in this study. Inherent limitations of pathogens and autoimmune Ags. It would be interesting to de- the RNA pulldown technique, such as incorrect folding of syn- termine whether the distinct patterns of PAS usage seen for HLA-A thetic biotinylated RNAs that does not allow optimal binding of alleles are stable across individuals and tissues, and whether they RBPs and insufficiency of the pulled down protein for detection vary with cell differentiation or activation. For example, HLA- by MS, may limit identification of all the endogenous RNA-bound A*03, which predominantly uses the distal PAS, may potentially 3898 POSTTRANSCRIPTIONAL REGULATION OF HLA-A switch to using the proximal PAS upon exposure to certain 19. Mizutani, A., M. Fukuda, K. Ibata, Y. Shiraishi, and K. Mikoshiba. 2000. SYNCRIP, HLA-A*01 HLA-A*11 a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts pathogens. and carry variants in the distal with ubiquitous synaptotagmin isoforms. J. Biol. Chem. 275: 9823–9831. PAS that prevent its use, so these alleles express only the short 20. Svitkin, Y. V., A. Yanagiya, A. E. Karetnikov, T. Alain, M. R. Fabian, 39UTR and have no flexibility in their surface expression levels A. Khoutorsky, S. Perreault, I. Topisirovic, and N. Sonenberg. 2013. Control of translation and miRNA-dependent repression by a novel poly(A) binding pro- through differential use of PAS. This may pose a disadvantage to tein, hnRNP-Q. PLoS Biol. 11: e1001564. individuals carrying these alleles if alternating expression levels of 21. Chen, H. H., J. G. Chang, R. M. Lu, T. Y. Peng, and W. Y. Tarn. 2008. The RNA a given allele is beneficial depending on the immune environment binding protein hnRNP Q modulates the utilization of exon 7 in the survival motor neuron 2 (SMN2) gene. Mol. Cell. Biol. 28: 6929–6938. at any given point. 22. Kabat, J. L., S. Barberan-Soler, and A. M. Zahler. 2009. HRP-2, the Caeno- Differential HLA expression levels have been associated with in- rhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleo- fectious (60–63), autoimmune, and inflammatory (61, 64–67) disease proteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements. J. Biol. Chem. 284: 28490–28497. outcome. Our data indicate that alternative polyadenylation is one 23. Mourelatos, Z., L. Abel, J. Yong, N. Kataoka, and G. Dreyfuss. 2001. SMN mechanism by which HLA-A cell surface expression levels are interacts with a novel family of hnRNP and spliceosomal proteins. EMBO J. 20: 5443–5452. regulated, potentially affecting HLA-A–mediated immune responses. 24. Neubauer, G., A. King, J. Rappsilber, C. Calvio, M. Watson, P. Ajuh, J. Sleeman, Blocking of the proximal or distal PAS has the potential to be used as A. Lamond, and M. Mann. 1998. 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