Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants.

Am J Physiol Lung Cell Mol Physiol 308: L58–L75, 2015. First published October 17, 2014; doi:10.1152/ajplung.00058.2014.

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants Nikolaos Tsotakos,1 Patricia Silveyra,1 Zhenwu Lin,2 Neal Thomas,1,3 Mudit Vaid,1 and Joanna Floros1,4 1

Center for Host Defense, Inflammation and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; 2Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; 3Department of Public Health Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; and 4Department of Obstetrics and Gynecology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania Submitted 3 March 2014; accepted in final form 16 October 2014

Tsotakos N, Silveyra P, Lin Z, Thomas N, Vaid M, Floros J. Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants. Am J Physiol Lung Cell Mol Physiol 308: L58 –L75, 2015. First published October 17, 2014; doi:10.1152/ajplung.00058.2014.—Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5=-untranslated regions (5=-UTR) due to differential splicing. The 5=-UTR variant ACD= is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/ SP-A2 ratio, with potential for clinical implication. uAUG; translation regulation; ribosome binding site; scanning mechanism

(SP-A), a molecule with surfactant- and immune-related functions, is encoded by two similar, yet distinct, genes (SFTPA1 and SFTPA2). The two gene products, SP-A1 and SP-A2, have several distinct characteristics, both functional and structural (59, 87, 88, 91). The nucleotide sequence similarity of the two genes is 98% in the coding regions, but the difference in the untranslated regions is up to 15% (25). There are several nucleotide polymorphisms that result in a number of variants for each gene (14, 16, 17, 76). These SP-A variants have been denoted as 6An for the SFTPA1 (SP-A1) gene and as 1An for the SFTPA2 (SP-A2) gene (15), with the most frequent being 6A2 for SP-A1, and HUMAN SURFACTANT PROTEIN A

Address for reprint requests and other correspondence: J. Floros, Pennsylvania State Univ., College of Medicine, 500 Univ. Dr., Rm. C4752, H085, PO Box 850, Hershey, PA 17033-0850 (e-mail: [email protected]). L58

1A0 for SP-A2 (27, 68). The SP-A1 and SP-A2 gene-specific products are distinguished by four amino acid differences within the collagen-like region of the molecules. To add to the complexity of SP-A genetic variability, several different 5=untranslated region (UTR) splice variants have been reported (15, 36). These result from alternative splicing of a number of different untranslated exons in the 5=-UTR (A, A=, A⬙, B, B=, C, C=, D, D=) (36, 55, 76) and have been shown to differentially affect SP-A regulation (89, 90). For instance, exon B of SP-A2 5=-UTR has been recently shown to have cis-acting regulatory roles, enhancing SP-A2 transcription and protein translation (62, 77). The ACD= splice pattern has been described as a minor variant of SP-A1 (36, 37, 55). It includes the 60nucleotide (nt)-long exon C, which contains two AUG sites that, in addition to the primary translation start site, could potentially be responsible for translation initiation. In eukaryotes, translation initiation starts when the 40S ribosomal subunit binds the mRNA with the help of several initiation factors. The initiator factor eIF2 binds to GTP to promote binding of the initiator tRNA (Met-tRNAi) to the 40S subunit. Both eIF1 and eIF1a stimulate binding of eIF2-GTPMet-tRNAi to 40S ribosomal subunits, thus forming the 43S preinitiation complex (31). This complex scans the 5=-UTR of the mRNA for the first AUG codon. Once there, eIF2-bound GTP is hydrolyzed, leading to recruitment of the 60S ribosomal subunit and formation of elongation-competent ribosomes. This procedure, termed “ribosome scanning,” is most efficient when the AUG codon is found in an optimal environment; Kozak (45, 47) found that a purine (A or G) in position ⫺3 and a G in position ⫹4 (the adenine of the AUG being nucleotide ⫹1) constitute the best context for translation initiation. AUG or other alternative codons (e.g., CTG, GTG) that are flanked by a least favorable context may be skipped, a mechanism known as “leaky scanning” (2). Other than the trans-acting initiation factors mentioned above, cis features act to modulate the translation initiation mechanism. The presence of secondary structures downstream from any putative start codon can suppress bypassing an AUG that lies in a suboptimal nucleotide context (43). On the other hand, hairpin structures within the 5=-UTR itself may compromise translation initiation (48) and require the recruitment of eIF4A (80). Other features in the 5=-UTR that play a regulatory role include the presence of small upstream open reading frames (uORF) and/or upstream AUG codons (uAUG). These elements often exert a suppressing role in translation efficiency (56). This can be explained by the scanning mechanism, as uAUG sites (both in frame and out of frame with the primary

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REGULATION OF SP-A1 BY UPSTREAM AUGs

start site) can be occupied by ribosomes; these ribosomes cannot fully be recruited to reinitiate the process at the primary AUG. If there is no termination codon upstream of the primary start codon, in-frame upstream start codons can generate NH2terminally extended protein products, as is the case of VEGF (29) and the murine complement factor B (20). In the case of the out of frame uAUGs where the ORF overlaps with the frame of the main coding sequence, deleterious effects on translation can be observed (28). In the present work, we employed a number of in vitro techniques to understand the effect of exon C uAUGs on SP-A1 expression. Our findings demonstrate that uAUGs in exon C negatively regulate the translation of SP-A1, contributing to the complexity of SP-A regulation. MATERIALS AND METHODS

Cell and Lung Tissue Cultures The human lung adenocarcinoma cell line NCI-H441 was cultured in RPMI-1640 Glutamax medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gemini Bioproducts, West Sacramento, CA) and 1⫻ antimycotic-antibiotic solution (Sigma, St. Louis, MO). The human adenocarcinoma cell line A549 was cultured in DMEM (Gibco) supplemented with 1% L-glutamine (Sigma), 10% FCS, and 1⫻ antimycotic-antibiotic solution. CHO-K1 cells were maintained in Glasgow minimum essential medium supplemented with 10% dialyzed FCS (Gibco), NaHCO3 (2.75 g/l, Fischer Scientific, Fair Lawn, NJ), proline (40 mg/l, Sigma), 1⫻ nonessential amino acids solution (Life Technologies), glutamate, and asparagine (6 mg/l, Sigma) sodium pyruvate (1 mM, Gibco), 1⫻ Nucleoside Mix, and 1⫻ antimycotic-antibiotic solution. Cells were grown at 37°C in the presence of 5% CO2 and were trypsinized (0.25% trypsin/EDTA, Gibco) when they reached 90% confluence. The human lung tissue used in this study was obtained from two sources. The first set included surgical samples [n ⫽ 4, termed #1787, 1797, 1798, 1799 (35)] that were acquired from patients who underwent lung resections, primarily for tumors. The lung tissues used were grossly examined and denoted healthy, “normal” tissues by a pathologist. The second set of samples consisted of human lungs deemed unsuitable for transplantation (n ⫽ 11). These were obtained from the Gift of Life Donor Program (Philadelphia, PA). Lung tissue was cultured in Waymouth’s medium, as described previously (34). Lung tissue was minced, unless otherwise stated, and kept in Waymouth’s medium for 96 h. In certain experiments, tissue was exposed to lipopolysaccharide (LPS) or dexamethasone, as indicated in the respective figure legends. All protocols were approved by the Pennsylvania State University Institutional Review Board.

RNA Isolation, Reverse Transcription, and PCR RNA was isolated from lung tissue cultures with a combined TRIzol-PureLink protocol, according to manufacturers’ instructions. In brief, lung tissue pieces were snap-frozen with liquid nitrogen, pulverized, and homogenized in TRIzol (Ambion, Carlsbad, CA) with a Polytron homogenizer. The homogenate was passed 5–10 times through a 26-gauge needle to shear the genomic DNA and left at room temperature for 5 min. Chloroform was added and the tubes were vigorously shaken. After a 15 min centrifugation at 12,000 g, the aqueous phase was transferred to fresh, RNase-free tubes, and the RNA was precipitated overnight at ⫺20°C with isopropanol. The pellet was washed with 75% ethanol and resuspended in RNase-free water. The RNA was then purified with the PureLink RNA Mini kit (Ambion) following the manufacturer’s protocol. RNA was quantified spectrophotometrically, and its quality was microfluidically analyzed (Bioanalyzer 2100, Agilent Technologies) at the Penn State College of Medicine Genome Sciences Core Facility. All samples used had an RNA integrity number ⬎6 [range: 6.0 –9.8, with 10 being fully intact RNA (74)]. Reverse transcription was performed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) with 1 ␮g of RNA as template. For the nested PCR of surgical samples, SP-A was amplified with primers 32A and 781 (see Table 1) with the use of FastStart High Capacity Taq DNA polymerase (Roche, Indianapolis, IN) under the following conditions: 95°C for 2 min, 95°C for 30 s, 58°C for 30 s, 72°C for 30 s (the last three steps were repeated for a total of 30 cycles), 72°C for 7 min. For the amplification of ACD=-containing transcripts, 1⁄10 of the product from the first reaction was amplified with the same heating profile as above for 28 cycles with primers 1413 and 293. Primer 1413 specifically hybridizes with exon C and primer 293 is specific for SP-A1. Table 1 shows the sequence, specificity, and location of the primers used. PCR products were analyzed by 1% agarose gel electrophoresis. For the quantitative PCR of lung donor RNA samples, SP-A1 5=-UTR regions that contained ACD= (GenBank accession number: HQ021440) were amplified with the use of primers 2140 and 2141. The accuracy of the reaction was assured with the use of the hydrolysis (Taqman) probe (Table 1). Primer concentration was 300 nM, and probe concentration was 250 nM in the final reaction. The efficiency of the reaction was tested by a standard curve with serial dilutions of ACD= plasmids (described below) and was within the range of 104 –110% (acceptable range is 90 –110%). To ensure that the probe would not hybridize with transcripts that did not contain exon C, serial dilutions of AD= plasmids were tested, and no amplification was observed in the experimental conditions used. SP-A1specific and 18S rRNA-specific (used as reference gene) primer-probe mixes and the reaction master mix were purchased from Applied Biosystems. An ABI 7900HT cycler was used for the quantitative

Table 1. Oligonucleotides used in the present study Primer Name

Sequence (5= - 3=)

32A 781 1413 293 2140 2141

CAG GGT AGA CCA TGG GGC

PCR hydrolysis probe Toeprint primer

5=-VIC-TGT GGC CTG GGA GAG ACC CCA CAA CCT CCA-3= 5=-FAM-CAG GAG GAC ATG GCA TTT CTC-3=

CTG ACC CCC TCA AGG ACA

GAG AGT CAC TTT CAG GCC

GCTCTG TGT GTG G TGG TGT AGT TCA CAG AAC CTC CA CCA GGA GGA CAT GGC A AGA CCC AAG C ACA TGG CTC T

Specificity

Orientation

Location

SP-A SP-A SP-A1 SP-A1 SP-A1 SP-A1

sense antisense sense antisense sense antisense

untranslated exon A coding exon IV untranslated exon C coding exon II spanning untranslated exons A and C spanning coding exon I and untranslated exon D’

SP-A1 SP-A1

sense antisense

spanning untranslated exons A and C coding exon II

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(q)PCR reactions (Penn State University College of Medicine Core Facilities). Data were analyzed with the ⌬⌬Ct method (DataAssist software, v. 3.01, Applied Biosystems). For the SP-A1 content, 18S rRNA was used as a reference gene. We calculated the frequency of ACD=-containing transcripts in SP-A1 mRNAs by normalizing them to SP-A1. Reverse transcription and qPCR reactions were performed in triplicate. For the RT-qPCR of NCI-H441 cells, the cells were treated with dexamethasone (100 nM), tunicamycin (2 ␮g/ml), diesel particulate matter (20 ␮g/ml diesel PM powder 2975; NIST, Gaithersburg, MD), LPS (100 pg), or the respective vehicles for 12 and 24 h before the RNA isolation (performed as described above).

reaction was carried out in a total volume of 50 ␮l containing the entire ribosome binding reaction, 50 mM Tris·HCl (pH 7.5), 75 mM KCl, 3 mM MgCl2, 4 mM DTT, 500 ␮M each dNTPs, 20 U RNAseOUT, and 200 U of M-MLV RT (Invitrogen). Reactions were incubated at 25°C for 30 min. Primer extension products were extracted with the use of DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA), diluted in Hi-Di formamide containing GeneScan 600 Liz size standards (Invitrogen), and analyzed by capillary gel electrophoresis independently at the Penn State College of Medicine Core Facilities and by Genewiz (South Plainfield, NJ). The resulting electropherograms (n ⫽ 9) were analyzed with the PeakScanner software (v.1.0, Applied Biosystems).

Plasmids

Transient mRNA Transfection

Previously generated rpcDNA3/5=-UTR/Luc constructs (89) were modified by digestion with HindIII and NruI to replace the SV40 promoter with the T7 promoter. Each of the three ATG sites present in the ACD= variant of the SP-A1 5=-UTR was mutated by sitedirected mutagenesis using the QuikChange II XL site mutagenesis kit (Agilent Technologies), and the modified vectors were confirmed by DNA sequencing. Moreover, the SP-A1 (6A2) coding sequence was cloned downstream of the 5=-UTR replacing the luciferase coding region in these vectors to create rpcDNA3/5=-UTR/SP-A constructs (Fig. 1A). The sequence alignment of these constructs is shown in Fig. 1B.

Transfection of in vitro transcribed mRNAs (n ⫽ 3) was performed using the TransIT-mRNA transfection kit (MirusBio, Madison, WI) per manufacturer’s instructions. NCI-H441, A549, and CHO-K1 cells were plated 24 h before transfection at ⬃60% confluence. In brief, 1 ␮g of in vitro transcribed experimental mRNAs and 100 ng of Renilla luciferase control mRNA were preincubated with the mRNA Boost and TransIT-mRNA reagents in OptiMEM I (Gibco) for 3 min at room temperature and delivered to the cells in complete growth medium. Cells were harvested after 90 min, washed with PBS, and rocked for 15 min at room temperature in 500 ␮l of 1⫻ Passive Lysis Buffer (Promega). Luciferase activity was measured in an aliquot using the Dual-Luciferase reporter assay system kit (Promega).

In Vitro Transcription To generate capped mRNA, plasmids were linearized with XhoI and purified. DNA was transcribed in vitro with mMESSAGE mMACHINE Ultra kit (Ambion, Austin, TX). In brief, 1 ␮g of linear plasmid was incubated with 7.5 mM dNTPs in the presence of a cap analog and T7 polymerase for 4 h at 37°C. Following treatment with 2 units of TURBO DNase for 15 min at 37°C, RNA was precipitated overnight at ⫺20°C by addition of LiCl precipitation solution. RNA quality was assessed by 1% denaturing agarose gel electrophoresis and quantified by UV light absorbance with Nanodrop. A second reporter mRNA, Renilla luciferase, was prepared by in vitro transcription and used as an internal control in the transient transfections. In Vitro Translation In vitro translation reactions (n ⫽ 6) were performed with the rabbit reticulocyte lysate (RRL) system (Promega, Madison, WI). Reaction mixtures were assembled on ice in a total volume of 80 ␮l containing 50% (vol/vol) RRL, 20 ␮M amino acid mixture, and 40 units RNAseOUT (Invitrogen, Carlsbad, CA). In the case of radioactive reactions (n ⫽ 6), the amino acid mixture was methionine free, and 65 ␮Ci [35]S-methionine (Perkin Elmer, Boston, MA) were included per reaction. The mixture was incubated at 30°C for 90 min. Translation products were analyzed by 15% SDS-PAGE, and the gels were fixed in 50% methanol-10% acetic acid and equilibrated in 7% methanol-7% acetic acid before being dried for 4 h at 75°C and autoradiographed. In certain experiments (n ⫽ 3), tunicamycin (10 ␮g/ml) and canine pancreatic microsomal membranes (Promega) were added per manufacturer’s instructions in the reactions. Primer Extension “Toeprinting” Assay of Initiation Complexes The toeprint primer 5=-FAM-CAGGAGGACATGGCATTTCTC-3= (20 pmol) that binds to positions 318 –338 of SP-A1 mRNA (GenBank accession no. HQ021440, Fig. 1B) was preannealed with 3 ␮g of in vitro transcribed RNA by heating at 68°C for 2 min and cooled at 37°C for 8 min. The translation mix, containing 50% RRL, 20 ␮M amino acid mixture, 500 ng/␮l cycloheximide, and 20 U of RNAseOUT (Invitrogen), was added to a final volume of 30 ␮l. The reactions were incubated at 30°C for 20 min. The reverse-transcriptase

Secondary Structure Prediction, Kozak Sequence Scoring, and Signal Peptide Prediction We used the AUG_hairpin program, via which one can evaluate the presence of secondary structure downstream of AUG sites that might influence the recognition of a translation initiation site (38). We assessed whether regions downstream of AUGu1, AUGu2, and AUGp form such secondary structures. Kozak sequence scoring was calculated with several different tools: NetStart (64), weakAUG (82), and TIS Miner (52). For the prediction of signal peptide cleavage site, we used SignalP 4.1 (65). This is a neural network-based method designed to recognize signal peptides in eukaryotic proteins. RESULTS

Description of the Natural ACD= and the ACD= Mutants The ACD= splice variant has only been described as a 5=-UTR variant of the SP-A1 gene product and has a total length of 127 nucleotides (GenBank accession number: HQ021440). The natural AUG sites of the ACD= and the mutants generated are depicted in Fig. 1A. Exon C contains two distinct AUG sites. One is closest to the transcription start site (AUGu1) and in frame with the primary AUG site of the coding sequence (AUGp), and the other (AUGu2) is out of frame with AUGp and introduces an ORF that overlaps with the primary ORF. The AUGu2-guided ORF meets a termination codon inside the coding sequence, in positions 343–345 of the SP-A1 transcript, which corresponds to amino acid positions 72–73 of the protein product of the primary ORF. The wild-type sequence is referred to as u1p_SP-A1 (WT), because AUGu1 and AUGp are in frame. In the WT, the sizes of the expected protein products from each putative AUG are 248 amino acids (aa) for AUGp, 263 aa for AUGu1, and 84 aa for the out-of-frame AUGu2. The respective names can be seen on the left of each sequence in Fig. 1A. We used site-directed mutagenesis to create a number of mutant constructs. Construct u2p_SP-A1 has a 2 nt insertion

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Fig. 1. A: nomenclature of ACD= containing constructs used in this study. ATGs above each box are in frame with the coding sequence, and ATGs below each box are out of frame. The respective sizes of all in-frame and out-of-frame putative products are shown next to them under the column labeled “protein size.” The untranslated exon C of the hSP-A1 ACD= sequence contains 2 ATG codons; the 1st one (AUGu1) is in frame with the primary start codon (AUGp), while the 2nd (AUGu2) is not. The wild-type (WT) sequence is referred to as u1p_SP-A1 (WT), and the translation products would be 248 amino acids (aa) (AUGp) and 263 aa (AUGu1) long. The AUGu2-starting frame ends by a termination codon, giving a putative product of 84 aa. In mutant u2p_SP-A1, site-directed mutagenesis was employed to shift ATGu2 in frame with ATGp by inserting 2 nt (GG) 8 bp downstream from the ATGu2. This places ATGu1 out of the coding sequence frame. The in-frame translation product for the u2p_SP-A1 would be 261 aa, whereas the putative out of frame product would be 27 aa. In mutant p, both uATGs present in exon C were mutated to ACGs. In mutant u1_SP-A1, ATGp was mutated to GTG, leaving only ATGu1 in frame with the coding sequence potentially giving a translation product of 263 aa. In mutant no5=, the 5=-untranslated region (UTR) was completely removed. B: sequence alignment of the constructs used. SP-A1 coding sequence (6A2) is given as reference. The sites of mutagenesis are marked by boxes. The toeprint primer annealing site is indicated.


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(GG was inserted 8 nt downstream of AUGu2) to place AUGu2 in frame with AUGp, and AUGu1 shifted out of the reading frame, initiating an ORF with a putative product of 27 aa. The expected protein sizes from this mutant are 248 aa for AUGp and 261 aa for AUGu2. p_SP-A1 has both uAUGs mutated so that translation can only be initiated by AUGp (expected protein size: 248 aa). u1_SP-A1 was created with a mutation in AUGp, to leave AUGu1 as the sole translation initiation codon, with an expected translation product size of 263 aa. In mutant no5=_SP-A1, ACD= is completely removed and AUGp is immediately downstream of the transcription start site, thus having an expected protein size of 248 aa. The alignment of the sequences of the constructs used for this study is shown in Fig. 1B. ACD= Variant Is Not Present in All Individuals Two different sets of RNA samples were screened for the presence of ACD=. The first set consisted of four surgically obtained samples, which were analyzed by both nested and qPCR. Although nested PCR was positive for three out of four samples, ACD= was detected by RT-qPCR in all of them, albeit in different quantities. The number of ACD=-containing transcripts varied from 0.23 to 2.59 ⫻ 106, while its contribution to the pool of SP-A1 ranged from ⬍0.01% to ⬎7%(Fig. 2). The second set of samples consisted of RNA from lungs from deidentified human donors. qPCR specific for the presence of ACD= showed that ACD=-containing variants were present in eight out of 11 samples tested (i.e., quantification cycle ⬍40, data not shown). Thus, collectively, the data from two separate sets of samples indicate that ACD= transcripts vary in relative amount among individuals and are not present in all individuals. Levels of ACD=-containing Transcripts Can Be Regulated by Environmental Stimuli Dexamethasone and LPS have been shown to affect SP-A mRNA levels in lung tissue culture (34, 49, 72, 85). To determine whether ACD= levels can be affected by external factors, such as dexamethasone and LPS, we cultured donor lung tissue under different conditions. In one instance, we isolated RNA from unminced and minced tissue before culture and from tissues cultured for 1 h in the presence or absence of LPS (Fig. 3, A and B). Although mechanical injury such as mincing seems to reduce total SP-A1 levels, this reduction is not significant (Fig. 3A). Most importantly, the levels of ACD=-containing SP-A1 transcripts in the minced tissue were significantly lower than the ones of the uncut tissue (Fig. 3B). This may be attributed to either decreased transcription rate, altered splicing events, or reduced stability of the ACD=containing mRNAs. A combination of all these events seems more than likely, given the nature of the stimulus. In the samples shown in Fig. 3, C–H, we modified the time and concentration of stimuli. A number of tissues and stimuli did not show significant changes in SP-A1 or ACD= levels. Tissues from a very young patient (Fig. 3, C and D) and a 57 yr old patient with chronic obstructive pulmonary disease medical history (Fig. 3, E and F) were cultured in the presence of 100 nM dexamethasone, 100 pg LPS, or without stimuli for 0.5, 1, or 2 h. In both cases, neither SP-A1 (Fig. 3, C and E) nor ACD= mRNA levels (Fig. 3, D and F) were affected. However, an increase in ACD= levels was observed in a lung tissue cultured for 2 h in the presence of dexamethasone or LPS (Fig.

Fig. 2. Detection of ACD= by nested and quantitative (q)PCR in surgically obtained human lung samples. Primers 32A and 781 were used to amplify total surfactant protein (SP)-A transcripts in the 1st reaction (top panel). For the nested reaction, an aliquot of the PCR product from the 1st reaction was amplified with primers 1413 and 293, in order to detect exon C-containing SP-A1 transcripts (bottom panel). The observed size for the ACD= amplicon was the same as expected for the ACD= splice variant of the 5=-UTR. Plasmids containing ACD=- and AD=-SP-A1 were used as positive and negative controls, respectively. A no-template control for each reaction is shown in the last lane of each gel. The number of copies of ACD=-containing variants and total SP-A1 per 100 ng of RNA, as calculated by a standard curve in qPCR experiments, are noted at bottom. The ACD= contribution was calculated as the ratio of the number of ACD= to the number of SP-A1 transcripts.

3H). This change was not accompanied by statistical differences in SP-A1 content (Fig. 3G). The respective patient had undergone lung transplantation ⬃2 yr prior to the donation of the lung due to Langerhans’ cell histiocytosis and had been under chronic immunosuppressive treatment. In this case, the ACD=-containing transcripts are highly abundant after a 2 h treatment with either LPS or dexamethasone. Dexamethasone induces a ⬃30-fold increase, while LPS increases ACD= levels nearly 100-fold (Fig. 3H). At the same time, the levels of AD= were reduced by ⬃50% in both the LPS- and dexamethasonetreated tissue (data not shown). Of interest, in lung tissues that lacked ACD=, treatment with dexamethasone or LPS did not induce ACD= expression (data not shown). Since the cultured tissue samples did not give conclusive results (presumably because of patient-to-patient differences) regarding the regulation of ACD= variants, we employed an in vitro system to assess whether ACD= is subject to regulation by studying the impact of environmental stimuli on ACD= mRNA levels. H441 cells were treated for 12 and 24 h with different agents. The agents selected were dexamethasone, which is used in the clinic to treat for surfactant deficiency in newborns, bacterial LPS as a lung inflammatory stimulus, diesel particulate matter (PM) as an environmental stressor, and tunicamycin as an endoplasmic reticulum (ER) stress inducer (8). The time


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Fig. 3. Relative quantification of SP-A1 (left panels) and frequency of ACD=-containing SP-A1 transcripts (right panels) in human donor tissues. Results from the same tissue are grouped horizontally. A and B: RNA was isolated from uncut tissue before culture (white bars), cut tissue before culture (light gray bars), and after 1 h in culture under control conditions (dark gray bars) or in the presence of 100 pg LPS (black bars). C–H: RNA was isolated from 3 different tissues, after the respective treatment (C, control; LPS, 100 pg LPS; Dex, 100 nM dexamethasone) for the indicated times. SP-A1 levels are expressed relative to 18S rRNA, and ACD= levels are normalized against total SP-A1 levels. For each sample, at least 3 experiments were performed in triplicate. Results were analyzed by ANOVA (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001).

points selected were chosen based on the half-life of SP-A mRNA, which is ⬃12 h (7). The different factors affected ACD=- and AD=-containing variants as well as total SP-A1 in various ways (Fig. 4). LPS increased the levels of SP-A1 following 12 h of treatment, and the contribution of AD= variants in these elevated levels is higher than its baseline contribution, whereas the opposite effect is observed in ACD=

variants. The level of contribution of ACD=-containing variants in the LPS-induced increased pool of SP-A1 is lower than that in the vehicle-treated cells. However, following 24 h of exposure to LPS, the ACD= contribution to SP-A1 levels remained significantly lower than baseline levels, despite the fact that total SP-A1 levels reverted to the normal levels (Fig. 4A). The effect of dexamethasone on ACD=-containing transcripts


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Fig. 4. RT-qPCR of NCI-H441 cells for the levels of ACD=- and AD=-containing as well as total SP-A1 mRNA. A: cells were treated with LPS or equal amount of water (vehicle, ctrl) for 12 and 24 h. B: cells were treated with dexamethasone (dex), tunicamycin (TNM), or ethanol (vehicle, EtOH) for 12 and 24 h. RNA was isolated by cells following the respective treatments and reverse transcribed, and the cDNA was used as template for qPCR. SP-A1 levels are expressed relative to 18S rRNA and ACD= levels are normalized against total SP-A1 levels. For each treatment, 3 experiments were performed in triplicate. Results were analyzed by ANOVA (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001).

seemed to be time-dependent, as their contribution is increased after 12 h of exposure but significantly downregulated at the 24 h time point (Fig. 4B). The AD= levels are only affected after 24 h of exposure to dexamethasone. Total SP-A1 levels are increased at 12 h but are not significantly different from control at 24 h. Tunicamycin seemed to have a regulating effect as well, increasing SP-A1 at both time points under study, but the ACD=- and the AD=-containing variants were not differentially regulated (Fig. 4B). Diesel PM did not seem to have any effect on either AD= or ACD= variants (data not shown).

uAUGs Affect Translation Activity in Cell Culture To determine whether SP-A1 uAUGs have any effects on translation in a cell system, we used the same in vitro transcribed luciferase RNAs as above to perform transient mRNA transfection in three cell lines: 1) NCI-H441, a lung adenocar-

uAUGs Modulate Translation in an In Vitro System The translation activity of the mutant and WT ACD= UTRs was investigated with the use of the RRL system and RNAs carrying the 5=-UTRs upstream of the firefly luciferase gene (Fig. 5). In this system, the mutant RNA p_luc, which carries mutations in both AUGu1 and AUGu2, shows increased luciferase activity compared with the WT RNA. The u2p_lucguided translation shows reporter activity levels comparable to the ones observed by the WT (u1p_luc) and significantly lower than p_luc. Thus, both u1p_luc and u2p_luc exhibit similar levels of luciferase activity but significantly lower activity than p_luc, indicating that the inhibitory effect of AUGu1 and AUGu2 on protein translation using AUGp is similar. When translation is initiated only from the AUGu1 (u1_luc), the luciferase activity does not show statistically significant differences compared with the WT, indicating that mutation of AUGp to GUG does not abolish the translation capacity of the primary site. The RNA lacking any 5=-UTR showed increased luciferase activity levels similar to the ones resulting from the p_luc RNA, and significantly different from the WT RNA, but also significantly higher than either u1_luc or u2p_luc.

Fig. 5. Comparison of firefly luciferase activity of WT and mutant hSP-A1 5=-UTRs. Capped mRNAs containing WT and mutant ACD= UTRs upstream of the luciferase coding sequence were generated by in vitro transcription with the mMESSAGE mMACHINE Ultra Kit (Ambion) and translated in vitro by RRL (Promega) per manufacturer’s instructions. Firefly luciferase activity was measured with use of the Luciferase Assay System (Promega). Results are shown as means ⫾ SE of 3 independent experiments each performed in triplicate. Pairwise differences against the WT with P ⬍ 0.05 were considered significant (by ANOVA, with Dunnett post hoc comparisons, *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001).



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Fig. 6. SP-A1 WT and mutant 5=-UTR variants differentially regulate the translation of the reporter gene. Translational efficiency was measured in the lung cell lines NCI-H441 (A), A549 (B), and Chinese hamster ovary CHO-K1 (C) as the ratio of firefly and Renilla luciferase activities, after 90 min of transfection with mRNA transcripts containing hSP-A1 5=-UTR variants. Each cell line was transiently cotransfected with the experimental (firefly luciferase) and the control (Renilla luciferase) mRNA using the TransIT-mRNA transfection kit (MirusBio), and the respective activities were measured with the Dual Luciferase Reporter Assay System (Promega). Results are shown as means ⫾ SE of normalized fLuc/Rluc ratios of 3 independent experiments, each performed in triplicate. Pairwise differences against the WT with P ⬍ 0.05 were considered significant (by ANOVA, with Dunnett post hoc comparisons, *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001).

cinoma cell line that expresses SP-A and is therefore expected to have the necessary factors for the translation of the specific transcripts; 2) A549, another lung adenocarcinoma cell line of alveolar origin that does not express SP-A; and 3) CHO-K1 cells, used as an control cell line. Cells were cotransfected with 100 ng of Renilla luciferase (Rluc) and 500 ng experimental RNA and then the cells were lysed 90 min after transfection as described in MATERIALS AND METHODS. Luciferase activity calculated as fLuc/Rluc ratio is shown in Fig. 6. The p_luc shows increased luciferase activity in all three cell lines compared with the WT, indicating that the AUGu1 plays a regulatory role in the translation of the WT construct. This is similar to that observed in the cell-free RRL system (Fig. 5). Interestingly, for the u2p_luc RNAs, significant differences were observed in luciferase activity in the cell lines (H441, CHO-K1, and A549 cells) compared with the respective WT (P ⬍ 0.05). The AUGu1 (u1_luc) efficiently initiated translation by itself in all three cell lines, with levels of luciferase activity nearly equal to the ones obtained by the WT (u1p_luc) RNA.

at AUGu1 and one starting at AUGp. The results of samples u1p_SP-A1 and u2p_SP-A1 indicate that the ribosomes scan the RNAs for the first available AUG, where they begin translation. As far as the intensity of the bands is concerned, the most prominent bands were obtained by p_SP-A1 (lane 3) and no5=_SP-A1 (lane 5); these data are consistent with the ones in Fig. 5. Together, these indicate that uAUGs have a negative impact on translation from the primary AUG. Ribosome Binding Site Is Not Altered in Mutated SP-A1 Transcripts Because the data in Fig. 7 showed that the different 5=-UTR mutants can give rise in vitro to different protein size products, we wished to determine whether translation initiation occurs in

Upstream Elements Give Rise to NH2-terminally Extended Primary Translation Products We next investigated in vitro whether the different 5=-UTRs used in this study can generate primary SP-A products of various lengths. For this, we generated and used constructs with the described 5=-UTRs (WT and mutants) upstream of the SP-A1 coding sequence (6A2). The AUGu1 is located 15 codons upstream of the AUGp in the u1p_SP-A1 (WT) construct, and the AUGu2 is 13 codons upstream of the AUGp in the u2p_SP-A1 construct (AUGu2 is in frame with AUGp). The constructs were in vitro transcribed as described in MATERIALS AND METHODS and translated in vitro by the RRL system in the presence of 65 ␮Ci of [35]S-methionine. The reaction products were electrophoretically separated by a 15% SDS-PAGE, and the gel was dried and autoradiographed. Our results indicate that translation of the SP-A1 WT mRNA (u1p_SP-A1) is guided by AUGp and an upstream element, possibly AUGu1, as seen by the double band in the first lane of Fig. 7. The same appears to be true for u2p_SP-A1 RNA, which includes AUGu2 and AUGp (lane 2). p_SP-A1 (lane 3) and u1_SP-A1 (lane 4) RNAs generate primary translation products with a slight difference in the molecular size, in compliance with the theoretical difference in length of 15 aa between a protein starting

Fig. 7. Polyacrylamide gel electrophoresis of in vitro synthesized SP-A. Capped RNAs containing the WT and mutant 5=-UTRs upstream of SP-A1 coding sequence were generated by in vitro transcription with the mMESSAGE mMACHINE Ultra Kit (Ambion) and translated in vitro in the presence of 65 ␮Ci [35]S-methionine by rabbit reticulocyte lysate (RRL, Promega). An aliquot of each reaction was precipitated with acetone and loaded in a 15% polyacrylamide gel. The gel was fixed, dried for 3 h at 70°C, and autoradiographed. The upper bands of ⬃48 kDa are nonspecific and have been reported elsewhere (32). The gel shown is indicative of ⬎6 experiments performed.


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the various uAUGs. To address this, we mapped the site of ribosome binding on the 5=-UTR using a toeprint assay with a fluorescent primer as described previously (23). The assay is based on the fact that ribosomes bound to the RNA inhibit the extension of a downstream antisense primer by reverse transcriptase as depicted in Fig. 8A. Due to the size of the translation initiation apparatus, the position of the P site of the ribosome (the peptidyl-tRNA binding site that binds the tRNA carrying the growing peptide chain) is typically 15–17 nt upstream of the toeprint peak (69). This means that the fragment size given out by the capillary gel electrophoresis is 15–17 nt shorter than the actual size. Smaller magnitude discrepancies between the observed and expected fragment size can be seen with the use of this technique and can be explained by 1) the existence of the FAM label of the primer (23), 2) and the fact that the ribosomes may be able to move along the mRNA for one codon (one translocation step) in the presence of cycloheximide (73), and 3) the nucleotide size calibration (96). In our case, for example, the FAM-labeled primer anneals to positions 318 –338 from the first nucleotide of the 5=-UTR (190 –210 nt downstream of the AUGp, Fig. 1B), and this is expected to give a size of 338 nt. We obtained a peak that corresponded to the full-length transcript in all reactions performed, indicating RNAs that were not occupied by ribosomes. In the presence of 5=-UTR, this peak has an observed size of 334 –335 nt (Fig. 8B). The difference between observed (334 – 335 nt) and expected size (338 nt) is explained by the reasons mentioned above. Similarly, the full-length RNA fragment transcribed from construct no5=_SP-A1 has an observed size of 212 nt due to the absence of the ACD= 5=-UTR, which is 127 nt long (predicted size: 338-127 ⫽ 211 nt, Fig. 8B). In nearly all the experimental samples a major peak was observed at 185 nt and is consistent with the position of AUGp. Because the ribosome occupies the first 15–17 nt downstream of the start site, the observed peak of a 185 nt fragment would mean that the distance between the primer annealing site and the start codon would be 200 –202 nt. However, the expected fragment size in this case is 210 nt because the primer anneals at 190 –210 nt downstream of AUGp. The reasons mentioned above can explain this difference as well. Surprisingly, we also observed a peak of lower intensity in all samples with a 5=-UTR (marked with an asterisk in Fig. 8B) that corresponds to a fragment size of ⬃276 nt. There are two possible explanations for the existence of this peak. The toeprinting assay may demonstrate false positive peaks generated by mRNA binding complexes other than ribosomes that can impede the progress of the reverse transcription reaction. If however, the observed peak corresponds to ribosome binding, the cis element would be the sequence CTGTGTGTG near the 3=-border of exon A (positions 35– 41 in Fig. 1B) (62, 90). Although it has been noted previously that in vitro translation by RRL may lead to artifacts due to ectopic translation initiation (23), this element could attract and bind ribosomes either at its CTG or GTG sites. This site has been found to play a cis regulatory role in previous work of our laboratory on SP-A2 (62, 90) and has the unique characteristic of including alternative start codons in all three reading frames. In the current study, the reading frame starting from the alternative initiation codon CTG (positions 35–37 in Fig. 1B) runs into a stop codon within exon C (TGA, nucleotides 77–79 in Fig. 1B), thus

forming a uORF that might also have a modulatory role for SP-A1 translation. Concurrently, the alternative start codon GTG (positions 37–39) initiates an ORF that ends at nucleotides 343–345 (TGA), well into the coding region of the SP-A1 transcript. Lastly, the GTG in positions 39 – 41 starts another uORF that terminates with the TGA in positions 84 – 86 and may have a regulatory role as well. The unexpected peak of the toeprint data from the CTGTGTGTG sequence found in the 3=-end of exon A (positions 35– 41, Fig. 1B) may reflect a regulatory role of this sequence and can become the subject of further studies in the future. However, in all cases in Fig. 8B, the major translation initiation site used is that of the primary AUG (AUGp). The fact that this site is the primary initiation site even in the RNA from the construct u1_SP-A1, where the primary site is mutated, may be explained by the nature of this mutation; the initial mutation was ATG to GTG, which has the potential of being a translation start site. We further mutated this site to make sure that the observed peak does not mask potential ribosomal binding in the uAUGs. The RNA designated as u1ATC is transcribed from a construct that carries an ATG-to-ATC mutation, while the one designated as u1weak carries the same mutation (ATG to ATC), plus the flanking nucleotides have been modified to represent the weakest Kozak context (final sequence: TTTTA⫹1TCT⫹4)(45). Toeprint analysis of these constructs shows that the ribosomes still bind around the primary site, but the more mutations are made in the RNA, the fewer ribosomes bind to the site, based on the fluorescence intensity of the respective peaks (Fig. 8B). Bioinformatic Prediction of the Translation Initiation Site The data in Fig. 8B indicate that the primary initiation site was preferred in all cases, regardless of the presence or absence of uAUGs. To better understand this usage advantage of the primary translation start site, we performed a bioinformatic analysis to assess the importance of context and secondary structure on this advantage. Sequence context. The flanking sequences of AUGs have been shown to affect the degree of translation initiation from the specific site (45, 47). The optimal sequence has been A determined to be GCC CC A⫹1UGG, in which the purine G at position ⫺3 and the guanine at position ⫹4 are the most important residues. These are conserved elements (41). Specific interactions of nucleotides in these positions with proteins of the translation initiation mechanism (mostly eIF1 and eIF2) have been demonstrated with UV crosslinking experiments (66). To compare the different sequences surrounding AUGu1, AUGu2, and AUGp, we used several tools available online (52, 64, 82). Over the last few years there have been several attempts to increase the specificity and efficiency of algorithms that allow the scoring of potential translation initiation (24, 83). Most of these algorithms are based on neural networks. In the present study, the important positions at ⫺3 and ⫹4 do not differ among AUGu1, AUGu2, and AUGp (Fig. 9A); all putative start codons have a purine at position ⫺3 (A for AUGu1, G for AUGu2 and AUGp), but none of them has a G at position ⫹4. Based on this fact, all potential initiation codons would be characterized as “weak” sites according to the guidelines is-


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Fig. 8. A: diagrammatic representation of the fluorescent toeprinting assay. i: an antisense fluorescent primer preanneals to the coding sequence (CS). ii: RRL is added (as source of ribosomes) in the presence of cycloheximide. The ribosomes occupy the putative translation initiation sites (u1, u2, p) in the 5=-UTR, but elongation is stalled. iii: primer gets extended by reverse transcriptase, and the extension is inhibited by the presence of ribosomes on the AUGp site. iv: if ribosomes are not present in AUGp, the primer will get extended until it meets the upstream binding site of the ribosomes. Fragment size analysis of the RT products maps the translation initiation sites. B: electropherograms of fluorescently labeled toeprint assays. All the constructs show a prominent peak with size that corresponds to ribosome binding to the AUGp site (185 bp). The full-length peak (335 bp) corresponds to transcripts that were not occupied by ribosomes during the RRL reaction. The minor peak that is marked with an asterisk corresponds to a 9 nt element (CTGTGTGTG) and contains alternative translation initiation codons. The u1p, u2p, p, no5=, and u1 RNAs are described in Fig. 1A. u1ATC carries the mutation A⫹1TG to A⫹1TC in the primary (p) AUG site, and u1weak in addition to A⫹1TC mutations has the mutated flanking sequence TTTTA⫹1TCT⫹4, as the weakest consensus sequence possible.


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A Comparison of AUG context in ACD' with optimal Kozak sequence to +4) Kozak

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Fig. 9. A. Sequences surrounding the putative start sites within the 5=-UTR of SP-A1. The consensus Kozak sequence is given in the 1st row. Putative start codons are underlined and consensus positions at ⫺3 and ⫹4 are boldfaced. The column with the heading “Strength” is in accordance with the Consensus Coding Sequence guidelines (67). NetStart score ⬎0.5 or weakAUG score ⬃1 indicate that translation is likely to initiate at the respective site. TIS Miner has similar precision to NetStart with a threshold of 0.7. B: prediction of downstream secondary structures compensating for suboptimal AUG context by AUG_hairpin. For the calculation, 10 nt upstream of the AUG and 100 nt of the coding sequence were used. The setting for hairpin start position was ⫹12 to ⫹18 (the A of the respective AUG being ⫹1), denoted by green letters. Red letters delineate the putative start codon (positions ⫹1–⫹3), and blue letters indicate double strands. Ess denotes the energy of the secondary structure, and Edsh denotes the energy of double strands in hairpin.

sued by the Consensus Coding Sequence project (67). We used the following three approaches to determine AUG context strength, the results of which are shown in Fig. 9A: 1) the NetStart algorithm is based on artificial neural networks that predict vertebrate translation start sites with a success rate of 85%, taking into account the frame and local context of the AUG as well as global sequence data. The algorithm’s output score values range between 0 and 1. Scores ⬎0.5 indicate a probable translation start site (64). In the case of SP-A1, NetStart-predicted scores indicate that only AUGp (score ⬎ 0.5) can be a functioning start codon (Fig. 9A). 2) The weak AUG score is the output of a different algorithm that takes for granted the fact that the Kozak context is weak (82). This algorithm gives a similar score for AUGu2 and AUGp (Fig. 9A), but none of the AUGs has a score high enough to qualify as a predicted initiation site according to this algorithm. This may be explained by the presence of a purine at position ⫺4 in all the AUGs under study which may interfere with the algorithm process, as the context may not be recognized as weak. 3) The third algorithm used, TIS Miner, is based on data

mining technology and gives a score between 0 and 1. The AUGp scores 0.705, which is the highest among the three putative start codons, whereas the uAUGs had scores ⬍0.7. With a threshold setting at the value of 0.7, the specificity of TIS Miner is 92.2%, and its precision is 85.9% (52). These TIS Miner rates are comparable to those of NetStart, and both identify AUGp (but not AUGu1 or AUGu2) as a functioning start codon. Secondary structure. It has been reported that hairpins starting 12–15 nt downstream of AUG sites with suboptimal context facilitate translation by pausing the 40S ribosomal subunit with its AUG recognition site exactly over the initiation codon, thus diminishing the possibility of leaky scanning of that specific site (43). We checked for differences in secondary structures downstream of each putative start site of SP-A1 that might affect translation initiation. AUG_hairpin is an algorithm that has been developed as a tool to predict such hairpins that may affect translation initiation (38). In ACD= 5=-UTR of SP-A1, all three putative start sites have moderately stable downstream secondary structures that would be able to



pause the translation initiation machinery at a site that would result in increased translation efficiency (Fig. 9B). The double strand of the hairpin downstream of the AUGp is less stable than the ones of the hairpins located downstream of the upstream AUGs (Edsh ⫽ ⫺3.0 kcal/mol for AUGp vs. ⫺20 or ⫺15 kcal/mol for AUGu1 and AUGu2, respectively, Fig. 9B). It is possible that the lower stability of the double strand in the downstream hairpin in AUGp provides less resistance for the RNA helicases of the translation initiation machinery, eIF4A and eIF4B. u1 AUG Does Not Affect the Predicted Cleavage Site of the Signal Peptidase To assess whether the mature protein is the same regardless of its precursor protein size or initiation site of the precursor molecule, we used bioinformatic analysis to determine the cleavage site of the signal peptide. Previous experimental data in our laboratory have shown that the first amino acid of the signal peptidase-cleaved SP-A1 protein is either Val 19, Cys 20, or Glu 21 (number of aa refers to the number of the respective aa in the SP-A1 precursor protein, given that the methionine translated by AUGp is the first residue) (87). Considering that longer primary translation products can be generated in vitro when there are uAUGs in frame with the primary start codon (Fig. 7, lanes 1 and 2), we used the SignalP 4.1 algorithm (65) to determine whether the AUGu1- and AUGu2-guided translation products are cleaved in the same position as the AUGp product. Figure 10A shows the output of the SignalP algorithm. The left panel shows the translation product of AUGp, while the right panel shows the one of AUGu1. The y-axis depicts algorithm prediction scores, and the x-axis depicts residue position. Each panel contains three plots. The raw cleavage site score (C score) is indicated by red lines. The algorithm is trained to discern cleavage sites and assign high C score to the first residue after the signal peptide cleavage site, i.e., the first aa of the mature protein. In the case of the AUGp-initiated product, the C score identified three probable aa cleavage sites (Ala18, Val19, and Glu21). The signal peptide score (S score, green lines) is used to distinguish positions among residues that belong to the signal peptide and residues that are part of the mature protein. A steep slope of this plot is indicative of the signal peptidase target site. In our case, the steep slope of the S score is between aa 18 and 21. The Y score (blue lines) is the geometric mean of the C and S scores, which provides a more accurate indication of the signal peptidase cleavage site. The Y score predicts that the target site of the signal peptidase is either Ala18 or Glu21. Thus, the algorithm used here predicts that the cleavage site of the primary translation product led by the AUGp can be between Ala 18 and Glu 21, consistent with published experimental data (left panel) (87). The AUGu1translated peptide is predicted to be cleaved at Ala 33 or Glu 36 (right panel). These residues are the exact same ones that are cleaved in the AUGp-guided product when one takes into account the 15 residues of NH2-terminal extension of this translation product, indicating that the cleaved protein is the same in length regardless whether the translation is initiated by AUGu1 or AUGp. Finally, in the case of the construct that contains AUGu2 in frame with AUGp (not found in nature), there is no evident signal peptide (data not shown).

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To experimentally verify the above bioinformatic predictions, we performed in vitro translation reactions using RRL in the presence of microsomal membranes, to obtain the mature protein product, after the cleavage of the signal peptide (12). As SP-A is heavily glycosylated, and the process of glycosylation is co- and posttranslational, we also included the Nlinked glycosylation inhibitor tunicamycin in some reactions. The reaction products were electrophoretically analyzed and immunoblotted with an SP-A antibody of goat origin (Fig. 10B). There is no evident difference in the size of the translation products obtained in the presence or absence of microsomal membranes and tunicamycin. There is also no difference in the size of products generated by the different RNA templates used in the reactions. The results show that the mature SP-A1 protein has the same size regardless of which site is used as translation start site by the ribosomal machinery. DISCUSSION

SP-A, shown to play an important role in lung innate immunity, is regulated at multiple levels. There are two distinct genes coding for two different products, SP-A1 and SP-A2. These differ in their structure and function (17, 71, 88). SP-A1 exhibits lower activity than SP-A2 in several cases. For instance, SP-A2 showed higher biological activity in inducing TNF␣ production by THP-1 cells (92, 93), stimulating Pseudomonas aeruginosa association with rat alveolar macrophages (58, 59) and inhibiting phosphatidylcholine secretion from alveolar type II cells (87). The relative amounts of SP-A1 and SP-A2 vary and seem to be affected by age and health status (81, 94). The functional difference between the two products can probably be explained by structural and oligomerization differences (71, 87, 91). Apart from the differences in functional activity, the two genes exhibit differences in regulation (34, 49, 89, 90). Exon C is exclusively found in the ACD= conformation of SP-A1 5=-UTR (36, 55). In this study, we determined 1) its frequency in human samples, 2) that ACD=containing variants can be regulated by environmental factors, and 3) that the upstream AUG elements of exon C have a negative effect on SP-A1 translation. Moreover, using various algorithms and an in vitro approach we showed that the mature protein is the same size regardless of whether the translation is initiated by an upstream AUG (AUGu1 or AUGu2) or the primary AUG (AUGp). Exon C was not detected in all the human samples tested. In the samples without ACD=, neither LPS nor dexamethasone could induce its expression (data not shown). When exon C was detected, some samples did not show any difference in SP-A1 mRNA content or ACD= levels after dexamethasone or LPS treatment, except in one case of a patient with severe lung disease and transplantation history. In this specific case, ACD=containing variants were upregulated in LPS- and dexamethasone-treated lung tissues in culture. The unique medical history of this case (chronic inflammation followed by chronic immunosuppression) may be a factor that contributes to the specific ACD= regulation pattern. Furthermore, to determine whether the ACD= variants are subject to regulation, we used NCIH441 cells, an adenocarcinoma cell line that expresses SP-A. This cell line as shown in the present report expresses ACD= variants. We found that multiple factors may regulate ACD= variants and that there may be differential regulation between


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Fig. 10. A: signal peptide analysis prediction using SignalP 4.1 software (http://www.cbs.dtu.dk/services/SignalP/). The amino acid sequence of the N-termini of p_SP-A and u1_SP-A are shown (left and right panel, respectively). x-Axes represent the aa position, and y-axes show the scores returned from the software. Three scores (C, S, and Y) for potential cleavage sites are provided as shown in the scheme. The C score or “cleavage site” score is calculated for each sequence position (red lines). The S score (green line) predicts the signal peptide, and high values indicate that the respective amino acids are part of the signal peptide. A precipitous drop of S score value indicates the end of the signal peptide. The Y score is the geometric mean acquired from the C and S scores for each residue position, and predicts the actual signal peptidase cleavage site. The cleavage site is assigned from the Y score where the slope of the S score is still steep and a significant C score exists. The residues that are predicted to be the first in the mature protein are marked with a dot. The target residues of signal peptidase are the same in both products, indicating that there is no difference in the length of the mature protein. B: Western blot of in vitro translation products. Purified RNAs from different constructs, as shown, were translated in vitro by the RRL system in the presence or absence of canine pancreatic microsomal membranes (MM) and TNM. A fraction of the reaction products was analyzed in 4 –12% gradient SDS-PAGE, immunoblotted in nitrocellulose membrane and probed with a goat anti-SP-A polyclonal antibody. The data show that in all cases a translation product of the same size was obtained.

AD= and ACD= in response to several stimuli. These include dexamethasone, LPS, and tunicamycin, which is a potent ER stress inducer (70) that induces eIF2a phosphorylation, a generalized mechanism for inhibition of protein synthesis. Together, these indicate that there may be a cross talk of external

factors with genetic determinants in regulating splice events that lead to differential expression of SP-A 5=-UTRs. Previous work on our lab is consistent with this observation: SP-A genotype affects mRNA levels and splice variants of SP-A (35), and dexamethasone has been shown to play a role in



SP-A expression as well (34). It is well known that splice variant ratios are regulated by external stimuli through cell signaling pathways in pathological conditions, such as cancer (61). Moreover, such interplay between genetic complexity and signaling events has been demonstrated in other systems as well (57, 63). Recently, in a pilot study the SP-A expression levels and specific SP-A genotypes were shown to associate with differential outcome in patients with lung transplants (10). In ACD=-containing SP-A1 transcripts, there are two upstream AUG codons; the first one (AUGu1) is in the same ORF as the primary AUG, whereas the second one (AUGu2) is out of frame. Previous work performed in our laboratory has shown that there is no internal ribosome entry site activity in A=CD=containing transcripts (90) and that they exhibit similar stability with other SP-A1 5=-UTR variants, but their translational efficiency index is lower than that of the other variants (89). In this study, the results obtained by the cell-free RRL system indicate that when uAUGs are mutated (p_luc) or when there is no 5=-UTR (no5=_luc), luciferase activity is enhanced, indicating that uAUGs may have a negative impact on translation. In fact, upstream translation start codons have been shown to regulate translation of mRNAs in multiple systems, and the regulatory role of uAUGs is becoming increasingly evident. Recently, a systematic study in Saccharomyces cerevisiae showed that uAUGs exert potent and widespread regulation in both transcription and translation levels (97). Mapping of transcript leaders (5=-UTRs that may contain upstream open reading frames) in translation (TL-seq) revealed that uAUGs are rare in yeast, but when present, they are conserved and functional (4). Usually, the presence of uAUGs is associated with inhibition of expression of the relevant mRNA, which is consistent with the results from the cell-free system of the present study. Regulatory control through uAUGs is exerted in systems that require fine-tuning of protein expression, such as SAPAP3 in synaptic signal transduction (9), BACE1 in APP processing (99), PPAR␥ in differentiation and metabolism (54), and oncogenes related to various types of cancer (42, 98). There are many molecules with immunity-related functions that are regulated via uAUGs as well (6, 41, 42). For instance, the p35 subunit of IL-12 in mice is encoded by alternative transcripts that have up to four uAUGs with differential impact on protein translation (5). However, the expression of uAUG-containing transcripts is itself under tight control. We show in the present study that under certain conditions LPS and dexamethasone are able to regulate the frequency of ACD= transcripts of SP-A1 in human lung tissue culture and in NCI-H441 cell culture. In fetal lung explants, high concentrations of dexamethasone decreased SP-A expression and the SP-A1/SP-A2 ratio (34, 49). This is in accordance with the biphasic effects of dexamethasone as shown in fetal lung; this was shown to depend on the duration of treatment and concentration (30, 33, 51). Similarly to our results, dexamethasone has been shown to upregulate the mRNA of p27 (Kip1) (11, 22). This mRNA has uAUG sites, and the dexamethasone responsive element is found in the 5=-UTR of the p27 mRNA, overlapping with an upstream ORF (22). The upregulation by dexamethasone seems to be achieved at the translation level, by activation of a pathway that involves PI3K, Akt, and mTOR (11). In vitro translation of SP-A1 transcripts by RRL shows that there are differences in the size of the primary translation

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products, depending on which AUGs are part of the reading frame of the coding sequence. The protein translation machinery uses the scanning mechanism to identify the first AUG site that is in frame with the coding sequence. Toeprint assays showed major peaks that correspond to ribosome binding to AUGp in every transcript studied. This binding inhibited further extension of the primer, resulting in the absence of toeprint peaks generated by the upstream AUGs. However, the u1_SP-A1 transcript, where the AUGp was mutated, showed the same toeprint pattern with the rest of the transcripts. This may be explained by the fact that our initial mutation of the primary AUG to GUG did not affect levels of luciferase activity, indicating that this mutation does not completely abolish the potency of the translation start codon. In fact GUG has been shown in other systems to function as an alternative start codon (19). Further mutation of the start site and the flanking nucleotides attenuated ribosome binding, but it did not completely abolish it. Even in the RNA that carries the weakest possible Kozak sequence and shows reduced binding of ribosomes in the primary translation initiation site, there were no peaks that correspond to the uAUGs, suggesting that the selectivity of the ribosomes for the specific region depends on factors other than the RNA sequence. At the same time, we purposely opted to use the M-MLV reverse transcriptase (instead of AMV RT) during the toeprint reactions in order to be able to exclude any interference of secondary structure effects in this assay (46). Along with the peak of the major site, we observed a toeprint peak that corresponds to an upstream sequence (CTGTGTGTG, positions 35– 41 in Fig. 1B). This is of particular interest, as it has been shown in previous studies from our laboratory to play a regulatory role in SP-A2 translation (62, 90). In the present study, we show that this element attracts ribosomes or other proteins that can exert regulatory role in SP-A1 translation by introducing uORFs. The ribosome scanning mechanism sufficiently explains the repressing effect that uAUGs and uORFs have on translation. Ribosome scanning depends on the attachment of the 40S ribosomal subunit on the mRNA and its inspection for the first AUG codon. The presence of an uAUG means that the ribosomes are engaged in translating the reading frame guided by that uAUG and thus unable to attach to the primary AUG. Although there are exceptions to this rule, the context of the putative start codon appears to be of high importance as far as the association of ribosomes to the mRNA is concerned (26, 45). Recent work mechanistically proved the importance of nucleotides in positions ⫺3 and ⫹4 by associating these with the recruitment of certain initiation factors (53, 66). The bioinformatic analysis of the SP-A1 uAUG context performed herein with several different algorithms is consistent with the prevalence of the leaky scanning mechanism in the translation of SP-A1. Despite being surrounded by what would be considered “equivalent” context, AUGp scored higher than the uAUGs in all the algorithms used to predict the initiation site. Secondary structures either in the 5=-UTR or downstream of the initiation site also play an important role in determining whether an AUG site is capable of functioning as translation initiation site (48). The algorithm used in this study to assess the implication of such secondary structures (AUG_hairpin) returned scores favoring AUGp as the translation initiation site. This may mean that the uAUGs are skipped in favor of translation initiation by AUGp. If we take into account the fact


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that codons other than AUG can act as alternative start sites, another possibility emerges; ribosomes scan the SP-A1 mRNA according to the classically described mechanism, but before encountering any AUG site, they are bound to the upstream element (CTGTGTGTG) identified in Fig. 7B of the present study. The uORFs that are introduced by the start sites of this element interfere with the unrestrained ribosomal scanning across the 5=-UTR. Although the termination codons for these uORFs are upstream of all AUGs, the ribosomes are less likely to reinitiate translation from the uAUGs because of their proximity to the release site of the ribosome subunits from the uORFs. The distance between the termination codon in positions 84 – 86 and the AUGp on the other hand may allow the reinitiation of translation by the ribosomes (95). The role of these uORFs in the regulation of SP-A1 expression is of interest for further research. Initiation factor recruitment may not be the only type of regulation exerted by potential initiation sites and their context; microRNAs are generally considered to bind to 3=-UTRs, but it has been suggested that microRNAs (miRNAs) can also bind to uAUGs and inhibit the ribosomes from reaching the ORF (1). As stated above, mutation of the uAUGs leads to increased luciferase activity. However, the absence of the whole 5=-UTR has different effects on the cell lines used. No significant difference in reporter activity is observed between the no5= and the WT RNA in A549 and CHO-K1 cells, whereas a significantly lower activity of no5=_luc compared with u1p_luc (WT) was observed in H441 cells. Interestingly, SP-A is only expressed by H441 cells. Our results indicate that there may be regulating factors, specific to this particular cell line, that interact with the 5=-UTR of SP-A transcripts. The nature of these factors (proteins, miRNAs, etc.) can only be speculated at the present time and would be the focus of further research. However, there is evidence in the literature to show that the expression systems (e.g., different cell lines, site of transcription, etc.) can affect translation mediated by the same uAUGcontaining 5=-UTR sequence, as is for instance the case of BACE1 mRNA (39). Previous work suggested that AUGu1 can give rise to a NH2-terminally extended form of SP-A1 (36, 55), and the data presented support this hypothesis (Fig. 7). The additional 15 aa would increase the hydrophilicity of the protein, and this might affect ER translocation (55). However, as shown in the present study, the size of the mature protein does not depend on the AUG used as initiation site, as the size of the in vitro translated protein in the presence of microsomal membranes and tunicamycin was the same for all SP-A1 constructs studied. These data are also supported by an in silico analysis with SignalP 4.1, an online algorithm that predicts the site of signal peptidase cleavage. This prediction does not show any difference in the size of the mature protein, regardless of whether the translation is guided by AUGp or AUGu1. Moreover, the weak context of the AUGs cannot verify or discard the notion of an NH2-terminally extended precursor protein. Such proteins may be the products of in-frame uAUGs as has been reported for RNase H1 (79), estrogen receptor-␣ (40), FGF-2 (3, 84), and C/EBP␣ (60). This form of regulation sometimes associates with sequestration in a specific cellular compartment, but sometimes is the result of response to cellular stress (50, 75, 86).

Recently, a global approach to discover uORF and NH2terminally extended proteins using ribosomal footprinting in a monocytic cell line (THP-1) showed that about half of the annotated coding sequences have uORF that might have an effect on their expression (18). The same technique was employed to assess the effects of oxidative stress on translation in yeast (21). The results of these studies associate the presence of uAUGs with pathophysiology. This is further acknowledged by the identification of pathogenic mutations that introduce uAUGs in several genes (44, 95). There are ⬎509 human genes identified with single nucleotide polymorphisms that either create or delete an uORF, and some of these are related to a wide spectrum of diseases, ranging from cancer to hereditary thrombocythemia (78). Recently this observation led to accurate regulation of ectopic protein expression (p21) with the use of small synthetic uORF, to control the ribosome flux to the primary ORF (13). Although this type of intervention is still at its early stages, the potential for therapeutic targeting is undeniable. In summary, our results indicate that 1) ACD=-containing SP-A1 transcripts are present in the majority of individuals; 2) The uAUGs of exon C play a repressing role in SP-A1 expression, presumably lowering the SP-A1/total SP-A ratio, and this may affect the surfactant-related and immune functions of SP-A; 3) The mature protein size was not affected by uAUGs; 4) ACD= expression can be affected by mechanical injury, and its contribution to the SP-A1 pool under certain conditions can be regulated by external stimuli, such as dexamethasone and LPS; 5) AD= and ACD= expression may be differentially regulated in response to various stimuli. Taken together, these data indicate that exon C, although found only in a minor variant of SP-A1, may play an important role in the regulation of SP-A1 in the human lung. Further work is needed to discern the conditions under which ACD= is formed, the way it is regulated, and its overall impact on SP-A content. ACKNOWLEDGMENTS The authors thank the Pennsylvania State University College of Medicine core facility for RNA quality analysis, qPCR, and fluorescent capillary gel electrophoresis services. We deeply thank the Gift of Life Donor Program (Philadelphia, PA), and we acknowledge the families of the organ donors for allowing the use of organs in research to understand the mechanisms of human disease. Present address for M. Vaid: Tumor Biology Group, Southern Research Institute, Birmingham, AL. GRANTS This work was funded by National Heart, Lung, and Blood Institute Grant HL-34788. DISCLOSURES No conflicts of interest, financial or otherwise are declared by the author(s). AUTHOR CONTRIBUTIONS N. Tsotakos, P.S., Z.L., and J.F. conception and design of research; N. Tsotakos, P.S., Z.L., N. Thomas, and M.V. performed experiments; N. Tsotakos analyzed data; N. Tsotakos interpreted results of experiments; N. Tsotakos prepared figures; N. Tsotakos drafted manuscript; N. Tsotakos, P.S., Z.L., and J.F. edited and revised manuscript; N. Tsotakos, P.S., Z.L., N. Thomas, M.V., and J.F. approved final version of manuscript. REFERENCES 1. Ajay SS, Athey BD, Lee I. Unified translation repression mechanism for microRNAs and upstream AUGs. BMC Genom 11: 155, 2010.


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Regulation of plant translation by upstream open reading frames.

Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity.

Inducible suppression of global translation by overuse of rare codons.

Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

Measurements of translation initiation from all 64 codons in E. coli.

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons.

Co-regulation of translation in protein complexes.

Primary translation products of pulmonary surfactant protein D.

Epigenetic Regulation of the Protein Translation Machinery.

Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae.

High-level translation initiation.

Translation: Start upstream to relieve stress.

A var gene upstream element controls protein synthesis at the level of translation initiation in Plasmodium falciparum.

Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation.

Quantitative analysis of mammalian translation initiation sites by FACS-seq.

Translation initiation of the HIV-1 mRNA.

The scanning mechanism of eukaryotic translation initiation.

Overview: mechanism of translation initiation in eukaryotes.

Translation initiation of IS50R read-through transcripts.

Letter: Specificity in initiation of mRNA translation.

Elimination of truncated recombinant protein expressed in Escherichia coli by removing cryptic translation initiation site.

Phosphorylation of Escherichia coli translation initiation factors by the bacteriophage T7 protein kinase.

Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein.

Transcription and translation of APOL1 variants.