Identification of a Pre-Mrna Splicing Factor, Arginine/Serine-Rich 3 (Sfrs3

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Biochimica et Biophysica Acta 1762 (2006) 34 – 45 http://www.elsevier.com/locate/bba

Identification of a pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), and its co-expression with fibronectin in fetal and postnatal rabbit airway mucosal and skin woundsB,BB,1

Ha-Sheng Li-Korotky a,b,*, Patricia A. Hebda a,b,c, Lori A. Kelly a, Chia-Yee Lo a, Joseph E. Dohar a,b,c

a Division of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213, USA b Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA c McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA

Received 15 July 2004; received in revised form 28 June 2005; accepted 9 August 2005 Available online 26 August 2005

Abstract

Fibronectin (FN) is a multi-functional, adhesion protein and involved in multi-steps of the wound healing process. Strong evidence suggests that FN protein diversity is controlled by alternative RNA splicing; a coordinated transcription and RNA processing that is development-, age-, and tissue/cell type-regulated. We previously demonstrated that fetal rabbit airway mucosal healing is regenerative and scarless. Expression, regulation, and biological function of the FN gene and various spliced forms in this model are unknown. Airway and skin incisional wounds were made in fetal (gestation days 21–23), weanling (4–6 weeks) and adult (>6 months) rabbits. Non-wounded and wounded tissues were collected at 12 h (all age groups), 24 h and 48 h (weanling only) post-wounding. Expression profiles were obtained using mRNA differential display and cDNAs of interest were cloned, sequenced and validated by real-time PCR. Here, we report two rabbit cDNAs that showed similar expression patterns after wounding. One encodes a rabbit fibronectin gene, Fn1, and another shares a high sequence homology to a human pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), coding for a RNA binding protein, SRp20. Both Fn1 and Sfrs3 mRNAs were suppressed in fetal wounds but induced in postnatal wounds 12 h post-wounding. The increased levels of both Fn1 and Sfrs3 transcripts were sustained up to 48 h in weanling airway mucosal wounds. The augmentations of the two genes in postnatal airway mucosal wounds were more prominent than that in skin wounds, indicating that the involvement of Sfrs3 and Fn1 genes in postnatal airway mucosal wounds is tissue-specific. Literature provides evidence that SRp20 is indeed involved in the alternative splicing of FN and that the embryonic FN variants reappear during adult wound healing. A connection between the enhanced molecular activity of Sfrs3 and the regulation of the FN gene expression through alternative splicing during the early events of postnatal airway mucosal wound repair was proposed. D 2005 Elsevier B.V. All rights reserved.

Keywords: Airway mucosa; Fetal wound healing; Fibronectin; Differential gene expression; Splicing factor; SRp20

Abbreviations: 3V-UTR, 3V-untranslated regions; Ac #, GenBank accession number; CDS, cDNA coding sequence; Ct, cycle threshold; ECM, extracellular matrix; ED, extra domain; FN (human)/Fn (animal), fibronectin; Hs, Homo sapiens (human); Oc, Oryctolagus cuniculus (rabbit); rRNA, ribosomal RNA; RS, arginine/serine-rich; Sfrs3, splicing factor arginine/serine-rich 3; SGS, subglottic stenosis; SR, serine/arginine-rich; SRp20, serine/arginine-rich protein of 20-KD; V, variable region i GenBank accession number for Sfrs3 partial cDNA sequence: AF401290. ii GenBank accession number for Fn1 partial cDNA sequence: DQ022369. j Partial results were presented at the Meeting of the 19th Annual American Society of Pediatric Otolaryngology Meeting, Phoenix, AZ, USA, May 2–3, 2004. * Corresponding author. Children’s Hospital of Pittsburgh, Division of Pediatric Otolaryngology, 8140 Rangos Research Center, 3460 Fifth Avenue, Pittsburgh, PA 15213, USA. Tel.: +1 412 692 6664; fax: +1 412 692 5075. E-mail address: [email protected] (H.-S. Li-Korotky).

1. Introduction cellular FN filaments (cFN, a dimeric or multimeric form at the cell surface and in ECM) [27]. Upon wounding, FN, as a Subglottic stenosis (SGS) is a major clinical problem in major component of the primary ECM, is expressed at high premature infants following prolonged intubations [1–3] and levels in the wound site. The pFN is released from the alpha- in adults following percutaneous dilational tracheotomy [4]. granulates of platelets that are activated by damaged blood Promotion of airway mucosal wound healing with less or no vessels and the cFN is synthesized locally at the wound site scarring remains the best strategy and a research challenge. [28]. FN protein diversity is controlled by alternative pre- One of the most promising potential strategies is to recapitulate mRNA splicing; a coordinated transcription and RNA proces- the unique healing capacity of the developing fetus, that is the sing [29,30]. All isoforms of Fn are encoded by a single large fetal skin incisional wounds made in the early gestation heal gene, Fn1 (50 kb, 50 exons) [31], and have three regions more rapidly than adult wounds and they heal in a regenerative subject to alternative splicing including extra domain A (EDA, pattern without scar formation [5]. Distinct differences between EIIIA), extra domain B (EDB, EIIIB) and a variable region (V, fetal and postnatal tissue responses to wounding have been IIICS), depending on the cell type and stage of development demonstrated in human and animal models and in several types [29,32,33]. In human about 20 different mRNA-encoding of tissues [6,7]. protein subunits are produced [34]. The full-length nature of Study of normal skin embryogenesis and comparison some variants has not been determined. Alternative splicing between early scarless and late scarring fetal wounds have presumably allows a cell to produce the type of FN that is revealed distinct differences in the inflammatory response, most suitable for the needs of certain tissue or cellular cellular mediators, wound contraction, and extracellular matrix functions [35,36]. Nevertheless, development-, age-, and (ECM) modulators [8]. Tissue-specific wound regeneration or time-dependent, and tissue-specific expression and regulation repair was observed in fetal and postnatal wounds. Fetal of the FN gene in airway mucosal wound repair have not diaphragmatic wounds heal with scar formation [9]. Distinct previously been reported. tissue-specific profiles of cytokines and growth factors and Gene expression is one of the early cellular responses to associated healing process were observed comparing oral wound healing. During wound repair, tissue responses to mucosal and skin wounds [10,11]. Previous studies in variety wounding feature marked changes in gene expression [37].A of tissues from different species of animals suggest that key issue in understanding the differential molecular mechan- exposure to the components in amniotic fluid is not a crucial isms of scarless and scarring airway mucosal wound repair determinant in fetal scarless wound repair [12]. We previously between fetus and adult is to identify differentially expressed reported that fetal airway mucosal healing is regenerative and genes and associated signaling cascades that are preferentially scarless [13]. However, key molecules that govern fetal airway regulated in a development-, age-, tissue/cell type-, and time- mucosal scarless wound healing remain largely unknown. dependent manner during the early events of the wound Wound healing process is very complex and involves healing process. Therefore, our study goal is to define the multiple events, including homeostasis, angiogenesis, inflam- unique gene expression patterns by comparison between fetal mation, epithelialization, granulation, collagen deposition, scarless wound healing and postnatal fibrotic wound repair. fibroplasias, scar formation and tissue remodeling [14]. From the well-developed model of the fetal and adult rabbit Fibronectin (FN), an adhesive glycoprotein, is one of the airway and skin wound healing [13], using mRNA differential early expressed molecules found in wounds, involved in display profiling we isolated numerous genes that were multi-steps of wound healing process and scar formation. FN differentially expressed during wound repair [38,39]. Reasons is a modulator of various functions including cell attachment, for choosing mRNA differential display as a strategy for adhesion and spreading, migration, differentiation, prolifera- differential gene expression profiling are the lack of the tion, blood clotting, phagocytosis, ECM synthesis and genomic information in this species (rabbit) and the unknown formation, tissue remodeling and degeneration [15].FNis molecular paths that differentiate the regeneration in the fetal a constituent of the ECM early in embryonic development airway mucosal wounds or the scar formation in postnatal [16,17], is involved in the migration of all the major cell wounds. types into the wound site, including fibroblasts, keratino- From the differential display generated cDNA pool at cytes, endothelial cells, and macrophages [18], opsonizes 12 h after wounding, we isolated over 600 cDNA fragments tissue debris after injury [19], and acts as a provisional (manuscript in preparation). Among them, one cDNA tag (477 matrix for ECM assembly [20]. FN plays a major role in bp) shares high sequence homologies with multiple spliced vocal fold physiology, pathology and wound healing [21].In transcript variants of a human Fn1 gene. From the same cDNA airway, FN regulates the migration of the small airway pool we also isolated and cloned a pre-mRNA splicing factor, epithelial cells [22], fibroblasts [23], and airway smooth arginine/serine-rich 3 (Sfrs3), encoding a RNA binding protein, muscle cells [24] and is involved in the beta 1 integrin- a serine/arginine-rich protein of 20-KD (SRp20). The molec- mediated enhancement of human airway smooth muscle ular activities of the Fn1 and Sfrs3 genes in fetal airway cytokine secretion [25]. mucosal and skin wounds are strikingly different from their FN functional complexity is carried through its protein postnatal counterparts, suppressed in fetal wounds but induced diversity, which consists of multiple isoforms, including in postnatal wounds. FN protein diversity in wound healing plasma FN (pFN, a soluble dimeric form in blood) [26] and process is known to be modulated by alternative splicing. The 36 H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45 co-expression of Fn1 and Sfrs3 in fetal and postnatal airway examined in 1% agarose/Tris-borate-EDTA (1ÂTBE) gel. RNA samples from mucosal wounds in vivo has not been previously described. two or three individual rabbits of fetal and postnatal groups at 12 h after wounding were used for mRNA differential display profiling. Reverse Here, we report the identification, isolation and character- transcription reaction included 0.2 Ag DNA-free total RNA, a differential ization of a rabbit Fn1 cDNA, a molecule in provisional ECM display oligo(dT) primer and MMLV reverse transcriptase. The reaction was of wounds, and a rabbit arginine/serine-rich 3 (Sfrs3) cDNA incubated at 65 -C for 5 min, at 37 -C for 60 min, and at 75 -C for 5 min on a fragment, a pre-mRNA splicing factor, in fetal, weanling, and Perkin Elmer 480 thermocycler (Applied Biosystems Inc., Foster City, CA, adult rabbit airway mucosal and skin wounds during the early USA). The PCR reaction included 2 AL of cDNA, a differential display oligo(dT) primer and an arbitrary primer, [alpha-33P]-dATP (3000 Ci/mmol) phase of the wound healing process. We hypothesize that (ICN Biomedicals Inc., Casta Mesa, CA, USA), and AmpliTaq DNA SRp20 is co-regulated with FN and modulates FN gene polymerase (Perkin Elmer, Norwalk, CT, USA). Low-stringency PCR was expression through alternative splicing during the early events performed for 40 cycles with temperature at 94 -C for 15 s, at 40 -C for 2 min, of postnatal airway mucosal wound repair. and at 72 -C for 2 min on a 9600 thermocycler (Applied Biosystems Inc). PCR products were size-fragmented side-by-side on a 6% denaturing polyacrylamide gel (National Diagnostics, Atlanta, GA, USA) for 3.5 h. The gel was dried 2. Materials and methods under vacuum at 80 -C and exposed to a Kodak XAR-2 film (Scientific Imaging Systems, Rochester, NY, USA) for 24 h to 48 h. The autoradiogram 2.1. Animal model with bands showing differential expression was aligned with the gel assisted with Glogos II Autorad Markers (Stratagene, La Jolla, CA, USA). Differen- Adult pregnant and non-pregnant Pasteurella-free New Zealand white tially expressed bands were excised from the gel and the cDNA was extracted rabbits were obtained from a supplier (Hazelton Research Products, Denver, by ethanol precipitation and centrifugation, and re-amplified by PCR as PA, USA) approved by the United States Department of Agriculture (USDA). previously described [39]. At least 3 animals or fetuses were used in each group (non-wounded and wounded), including fetus at gestation days 21–23 (term=31 days), weanling 2.3. Cloning and sequencing (4 – 6 weeks) and adult (>6 months) rabbits. Animal maintenance and experiments were conducted following a protocol approved by the Animal PCR products containing cDNAs of interest were purified using Wizard Research and Care Committee of the Children’s Hospital of Pittsburgh, PCR Preps DNA Purification System (Promega, Madison, WI, USA). The Pittsburgh, PA (Animal Welfare Assurance Number: A3617–A3701). purified PCR products were cloned using pGEM-T Vector System II Rabbit airway and skin wounds were produced following well-established (Promega). Positive colony selection, plasmid DNA preparation and sequenc- techniques as previously applied in our laboratory [13]. Briefly, anesthesia was ing were performed as previously described [39]. Sequence analysis was induced with a mix of ketamine–HCl (35 mg/kg) and xylazine–HCl (5 mg/kg) performed using Sequencher software 4.1 (Gene Codes Co., Ann Arbor, MI, via intramuscular injection. For fetal surgery in pregnant rabbits, anesthesia was USA), BLAST program (http://www.ncbi.nlm.nih.gov/BLAST) and Entrez maintained with a mix of 1–3% halothane, 2% oxygen and 1% nitrous oxide cross-database search (http://www.ncbi.nlm.nih.gov/entrez) (National Center delivered by spontaneous mask ventilation at a rate of 1 L/min. The abdominal for Biotechnology Information, National Library of Medicine, National hair was shaved and the skin was prepared with povidone-iodine. The site for Institutes of Health, MD, USA). the incision was treated locally with subcutaneous infiltration of the skin with 2% lidocaine. A lower midline laparotomy incision was made extending from 2.4. Real-time PCR the umbilicus to the most caudal set of nipples. The size, number and position of the fetuses were determined by palpation. A surgery was performed on every 2.4.1. Gene-specific primers second fetus to reduce the risk of the spontaneous abortion and the every other Primers for the amplification of a rabbit Fn1 fragment (341 bp) were fetus was left un-wounded for controls. A purse-string suture was placed designed based on the sequence information of a rabbit Fn1 (Ac #AB004811) through all layers of the uterus and a hysterotomy incision was made within the (Nishimura, M, 1997, unpublished) at the region of a cDNA coding sequence borders of the suture. The fetal animal was partially delivered through the (CDS) that shares high sequence homologies across the species (Fig. 3), opening to expose the areas intended for wounding and then carefully replaced. including a forward primer (5V-ACC ATA CCT GCC GAA TGT AGA TGA-3V) All skin incisional wounds were made on the dorsal skin in an identical pattern and a reverse primer (5V-CAA CCT CCT CCC GCA CTT TGT GCC-3V). (¨1 cm long). For airway mucosal wounds, midline thyrotomy was made, Primers for the amplification of a rabbit Sfrs3 fragment (187 bp) were designed followed by cricoidotomy and circumferential mechanical injury of the based on the sequence information of the human SFRS3 (Ac #NM_003017) subglottic mucosa. Sterile normal saline was added to reconstitute the volume [40] at the CDS region that shares high sequence homologies across the species of the lost amniotic fluid and the purse-string suture was closed in layers with (Fig. 6), including a forward primer (5V-CGT GGC CCA CCT CCC TCT TG- running sutures. Similar procedures for generating skin and airway mucosal 3V) and a reverse primer (5V-AGA AGG ATC GGG ACG GCT TGT GAT-3V). wounds were applied to weanling and adult rabbits. Primers for the amplification of a 450-bp fragment of the rabbit 18S ribosomal After 12 h, a second surgery was carried out to deliver all the fetuses and RNA (rRNA) (Ac #X06778) [41] was designed and used as an endogenous the mother was euthanized under a deep anesthesia with an intracardiac control for data normalization, including a forward primer (5V-CCG CTA GAG injection of a mix containing sodium pentobarbital (50 mg/kg)/Euthasol (0.2 GTG AAA TTC TTG GAC-3V) and a reverse primer (5V-GCA TGC CAG AGT mL/kg). Non-wounded and wounded airway and skin tissues from individual CTC GTT CGT TAT-3V). age groups were immediately collected, rinsed in cold physiological saline, rapidly frozen in liquid nitrogen and stored at minus 80 -C. For weanling 2.4.2. Reverse transcription and real-time PCR rabbits, tissues were also collected at 24 h and 48 h after wounding. Gene expression levels were assessed using real-time PCR as that we previously described [42]. Briefly, a reverse transcription reaction included 250 2.2. mRNA differential display ng of DNA-free total RNA pooled from each group, random primers, and SuperScript II (GIBCO), and was incubated at 25 -C for 10 min, 48 -C for 30 Technical details about mRNA differential display were described in our min, and 95 -C for 5 min in a 9600 thermocycler (Applied Biosystems Inc.). previous publication [39] and company’s website (RNAimagei kits, SYBR Green PCR reagents (Applied Biosystems Inc.) were used for PCR GenHunter Co., Nashville, TN, USA; http://www.genhunter.com). Briefly, amplification. The PCR reaction (in triplicate) included 5 AL of 10X SYBR each tissue was individually homogenized and total RNA was extracted in PCR Buffer, 6 AL of 25 mM MgCl2,4AL of each dNTPs (blended with 2.5 TRIzol reagent (GIBCO BRL, Gaithersburg, MD, USA) following the mM dATP, dGTP and dCTP, and 5 mM dUTP), 2.5 AL of each gene-specific instructions of the manufacturer. The RNA was treated with RNase-free DNase primer (5 AM), 0.5 AL of AmpErase UNG (0.5 unit), 0.25 AL of AmpliTaq I to reduce the risk of genomic DNA contamination and the RNA integrity was Gold (1.25 units) and 5 AL of cDNA in a final volume of 50 AL. The conditions H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45 37 for the real-time PCR were as follows: 50 -C for 2 min, 95 -C for 12 min, and previously published rabbit Fn1 is only 303 bp [47]. Our Fn1 - - 40 cycles at 95 C for 15 s, and 60 C for 1 min in an ABI PRISM 7700 clone sequence has an ‘‘aYt’’ switch at the base 125 and one Sequence Detection system (Applied Biosystems Inc.). extra ‘‘g’’ at the base 128 (Fig. 2). These mismatches could be 2.4.3. Data analysis either generated from sequencing error or tissue-specific. We The 7700 Sequence Detection Software (Applied Biosystems Inc.) was are confident about the accuracy of our sequence data because used for instrument control, automated data collection, and data analysis. this clone sequence was generated from 8 independent colonies Relative quantification (fold difference) of the expression levels of each (16 sequences using either T7 or SP6 primer) of the clones transcript for each group was calculated using the 2-DDCt method [43]. The log cDNA copy number of a target gene that was automatically normalized derived from the two independent experiments (data not to the ROX internal passive reference (logDRn) was plotted as a function of shown). Since we only isolated partial cDNA sequence at the cycle number. The cycle number at which the signal crossed the mid-linear 3V-UTR, we do not know the full-length nature of this mRNA portion of the log DRn-cycle function was defined as the cycle threshold and its encoding Fn1 isoform. Based on the sequence (Ct)(Fig. 7). Because the input cDNA copy number and Ct are inversely homologies, this rabbit clone is most likely Fn1 transcript related, a sample that contains more copies of the template will have a data variant 1 or 2 (Fig. 3). line that crosses the Ct at an earlier cycle compared to one containing fewer copies of the template. The DCt represents the Ct of the target gene norma- Target 18S rRNA lized to the rabbit endogenous 18S rRNA (DCt =Ct ÀCt ). Rela- 3.2. Isolation of a rabbit Sfrs3 cDNA clone tive quantification (fold difference) of the mRNA expression levels of the Target 18S rRNA target gene was calculated using DDCt =(Ct ÀCt )non-wound À Another rabbit cDNA fragment (372 bp, Ac #AF401290) (CTarget ÀC18S rRNA) . t t wound (Fig. 4) showed a similar expression pattern as that of Fn1, 2.5. Search for promoter sequences, transcription factor binding sites down-regulated in fetal airway mucosal and skin wounds but (TFBSs), and transcription factors up-regulated in postnatal wounds. This clone shares high sequence homologies to a pre-mRNA splicing factor, arginine/ Similar expression patterns of the Fn1 and Sfrs3 genes in this model may serine-rich 3 (Sfrs3), encoding RNA binding protein, SRp20 suggest that during the wounding process, both genes are transcriptionally (serine/arginine-rich protein of 20-KD), at the 3V-UTRs across regulated by one (or more) common transcription factor(s). Search for the species including human (91%) (Ac #NM_003017) putative promoter-specific TFBSs and transcription factors for each gene _ might reveal conserved regulatory elements that are critical for their co- [40] (Fig. 5), dog (91%) (XM 532124) [51], Gecko (91%) expression during wound healing. This task was performed by computation- (Ac #AY880406) (Gu X, 2005, unpublished), rat (88%) (Ac based search engines of the Eukaryotic Promoter Database (EPD, Release 82, #XM_342107) (NCBI predicted), mouse (88%) (Ac http://www.epd.isb-sib.ch) [44], the Searching Transcription Factor Binding #NM_013663) (NCBI predicted), and cow (82%) Sites (TFSEARCH, version 1.3, Dr. Yutaka Akiyama) (http://www.cbrc.jp/ (Ac #XM_592905) (NCBI predicted) (Fig. 6). Sequence- research/db/TFSEARCH.html) [45], and the Multi-genome Analysis of Positions and Patterns of Elements of Regulation (MAPPER) (http:// based analysis suggests that the clone that was isolated mapper.chip.org) [46]. from our rabbit airway mucosal and skin wounds is indeed Sfrs3. 3. Results 3.3. Differential expression of Sfrs3 and Fn1 in airway mucosal 3.1. Isolation of a rabbit Fn1 cDNA clone and skin wounds

From the differential display generated cDNA pool, we 3.3.1. Reproducibility of the assays selected two cDNA tags that were preferentially suppressed Real-time PCR quantifications of the Sfrs3 and Fn1 gene in fetal airway mucosal and skin wounds but induced in transcripts were performed in non-wounded and wounded adult wounds. One cDNA (477 bp, Ac #DQ022369) (Fig. 1) airway mucosal samples (in triplicate) of the fetal (Fig. 7a), shares a high sequence homology to a fibronectin 1 (Fn1) gene weanling (Fig. 7b) and adult (Fig. 7c) rabbits 12 h after injury. at 3V-untranslated regions (3V-UTRs) across the species includ- The logarithm of the DRn-normalized cDNA copy number for ing rabbit (98%) (renal cortex, Ac #S80206) [47] (Fig. 2), transcripts encoding rabbit 18S rRNA (endogenous control), human (88%) (FN1 transcript variants 1 and 2, Acs Fn1 and Sfrs3 were plotted as a function of cycle number. The #NM_212482 and NM_212475) [48], rat (86%) (Ac overlapping amplification curves for the triplicate runs #NM_019143) [49], and mouse (84%) (Ac #NM_010233) demonstrate the reproducibility of the assay. For rabbit 18S [50] (Fig. 3). By comparison of the FN1 CDSs between rabbit rRNA, the data showed no difference between the respective and human, we noticed that the CDSs of the two human FN1 curves for the non-wounded and wounded samples. In transcript variants are over 7000 bp, whereas the CDS of the contrast, the differences between non-wounded and wounded

Fig. 1. Differential display generated rabbit Fn1 cDNA clone sequence (477 bp, Ac #DQ022369, this study). Flanking sequences of the differential display primers (AP8 and H-T11G) are underlined. 38 H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45

Fig. 2. Fn1 sequence homology (98%) between the rabbit cDNA clone (477 bp, Ac #DQ022369, this study) and rabbit renal cortex Fn1 (Ac #S80206) [47].An ‘‘ a Yt’’ switch at the base 125 and one extra ‘‘g’’ at the base 128 are shown in bold. tissues in these curves for Sfrs3 and Fn1 transcripts were weanling (skin>airway) and the least expressed in adult, observed. suggesting that the RNA alternative splicing is more active during fetal development. For the Fn1, there is no significant 3.3.2. Non-wounded tissue expression difference of the mRNA abundance between fetal and The abundances of the Sfrs3 and Fn1 mRNAs in non- weanling tissues, whereas Fn1 is the least expressed in adult wounded skin and airway mucosa were compared among the tissues, especially in adult skin. three age groups (Fig. 8). The cycle threshold (DCt) was normalized to the rabbit 18S rRNA of the same tissue of the 3.3.3. Wounded tissue expression same age. The input cDNA copy number and DCt are Quantitative data of the expression generated from the skin inversely related. Sfrs3 mRNA was the most abundantly and airway mucosal wounds 12 h after wounding assessed by expressed in the fetal tissues (airway>skin), followed by real-time PCR were summarized in Fig. 9. The expression level

Fig. 3. Fn1 sequence alignment. Rabbit cDNA clone (477 bp, Ac #DQ022369, this study) shares high sequence homologies to the Fn1 gene at 3V–UTRs across the species including rabbit (renal cortex, 98%) (S80206) [47], Homo sapiens (Hs) (human transcript variants 1 and 2, 88%) (NM_212482, NM_212475) [48], rat (86%) (NM_019143) [49], and mouse (84%) (NM_010233) [50]. Real-time PCR amplified fragment (341 bp) is shown against a rabbit Fn1 CDS (AB004811) (Nishimura,M, 1997, unpublished). H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45 39

Fig. 4. Differential display generated rabbit Sfrs3 cDNA clone sequence (372 bp, Ac #AF401290, this study). Flanking sequences of the differential display primers (AP6 and H-T11C) are underlined. for each gene was normalized to the rabbit 18S rRNA and 3.4. FN1 and SFRS3: predicted promoter sequences, referenced as a fold difference relative to the respective value transcription factor binding sites (TFBSs), and transcription measured for the non-wounded control sample of the same age. factors Similar expression patterns of the Fn1 and Sfrs3 genes in skin and airway mucosal wounds were observed. Both genes were Search for putative promoter-specific TFBSs for each gene suppressed in fetal wounds but induced in postnatal wounds, might reveal conserved regulatory elements that are critical for indicating that Fn1 is a potential target of the SRp20 alternative their co-expression during wound healing. Since there is no splicing and/or that the two genes are regulated by conserved full-length rabbit cDNA sequence available for either Fn1 or regulatory elements. The suppressed or silenced molecular Sfrs3, we examined the promoter sequences of the human FN1 activities of the two genes in fetal wounds may suggest a gene (Ac #EP16038: ccagaggggcgggagggccgtcccatataagccc- mechanism that prevents fetal wounds from fibrotic repair. It is ggctcccgcgctccgACGCCCGCGCC) and the human SFRS3 interesting to note that the augmentations of the postnatal gene (Ac #EP73723: attccagtatggcgtataaataaaggcgagga- expression of both Fn1 and Sfrs3 genes in airway mucosal gaaggcggtggtccgccATTTCGTGGAC). We found that the two wounds were more prominent than that in skin wounds, genes do not share a common TFBS. TFBSs for FN1 include indicating that the involvement of Sfrs3 and Fn1 genes in ADR1, Sp1, and STRE, whereas TFBSs for SFRS3 are CdxA, postnatal airway mucosal wounds is tissue-specific. In wean- HSF, TATA, CF2-II, XFD-1, and Pbx-1. Nevertheless, data ling airway mucosal wounds, increased levels (over 2-fold) of from MAPPER search showed 13 predicted transcription both Fn1 and Sfrs3 transcripts were sustained up to 48 h post- factors that may be shared by FN1 and SFRS3 at the promoter injury (Fig. 10). Results further suggest that the two genes are region of each gene, including ADF-1 (GeneID #47082), involved in the early events of the postnatal airway mucosal RUNX1 (861), PLAU (5328), XBP1 (7494), CAAT-BOX wound repair. (440973), CREB1 (1385), HEN-1 (191666), NFKB1 (4790),

Fig. 5. Sfrs3 cDNA sequence homology (91%) between rabbit (Oryctolagus cuniculus, Oc) clone (372 bp, Ac #AF401290, this study) and human (Homo sapiens, Hs) (Ac #NM_003017) [40,76]. 40 H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45

Fig. 6. Sfrs3 sequence alignment. Rabbit cDNA clone (372 bp, Ac #AF401290, this study) shares high sequence homologies to the Sfrs3 gene at 3V–UTRs across the species including human (91%) (NM_003017) [40,76], dog (91%) (XM_532124) [51], Gecko (91%) (AY880406) (Gu X, 2005, direct submission and unpublished), rat (88%) (XM_342107) (NCBI predicted), mouse (88%) (NM_013663) (NCBI predicted), and cow (82%) (XM_592905) (NCBI predicted). A real-time PCR fragment (187 bp) is shown against human FN1 CDS (NM_003017) [40,76].

PAX2 (5076), SPI1 (6688), SPIB (6689), Su(H) (34881), and fetal wounds but induced in postnatal wounds 12 h after injury. ZFP161 (7541). Therefore, we do not exclude the possibility The increased levels of the two genes were sustained in that the similar expression patterns of the two genes may be weanling airway mucosal wounds up to 48 h after wounding. regulated at the transcription levels by the same transcription Both cDNAs share high sequence homologies at 3V-UTRs factor(s). across the species including human, rats and mouse. The data of the cDNA pool generated from this in vivo model confirmed 4. Discussion that the mRNA differential display has a unique ability to identify gene-specific 3V-UTRs. The 3V-UTR is often gene-, 4.1. Summary of the results and validation of the differential tissue- and cell type-, gender-, and species-specific [52,53], display and animal model indicating that the differential display generated expression profile could reveal wound-model specific phenotypes. This Using the strategy of the mRNA differential display, feature also provides a great promise for gene therapy using simultaneous comparison of the cDNA tags between fetal microRNA targeting. MicroRNAs are small noncoding RNAs and postnatal rabbit airway mucosal and skin wounds during that recognize and bind to partially complementary sites in the the early events of the tissue responses to wounding, we 3V-UTRs of target genes and regulate protein production of the identified, isolated and cloned two co-expressed cDNA target transcript [54]. The suppression or silencing of the fragments, Fn1 and Sfrs3, that were selectively suppressed in molecular activities of the two genes in fetal wounds may

Fig. 7. Real-time PCR. 18S rRNA, Sfrs3 and Fn1 in triplicate samples from the non-wounded (gray lines) and wounded (black lines) airway mucosal samples of fetal (a), weanling (b) and adult (c) rabbit 12 h after injury. The logarithmic amplification functions were constructed by plotting the cDNA copy number normalized to the

ROX internal passive reference (log DRn) versus cycle number. Cycle threshold (Ct) is defined as a cycle number at which the signal crosses the mid-linear portion of the amplification. H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45 41

Fig. 8. Real-time PCR. The mRNA abundances of the Sfrs3 and Fn1 genes in non-wounded rabbit skin (white columns) and airway mucosal (black columns) tissues.

The DCt represents the cycle threshold Ct of the target gene normalized to the rabbit 18S rRNA. Input cDNA copy number and the Ct are inversely related. suggest a mechanism that prevents fetal wounds from fibrotic genes showed similar expression patterns in skin and airway repair. mucosal wounds, a striking difference between fetal and The rabbit Fn1 and Sfrs3 cDNAs share higher sequence postnatal wounds, and tissue-specific augmentations in homologies to human’s than any other species, suggesting that postnatal airway mucosal wound compared to skin wounds rabbit serves a valuable model relevant to human wound repair, (Fig. 9). particularly because of its short period of gestation and size In non-wounded airway mucosa, Sfrs3 mRNA was the most (easy for housing and fetus surgery), which are preferable to abundantly expressed in fetal tissues, followed by weanling, the study of fetal airway mucosal scarless wound healing. and the least expressed in adults. Our results suggest that the RNA alternative splicing is more active during fetal develop- 4.2. Age-dependent and tissue-specific expression of Sftrs3 and ment. Serine/arginine-rich (SR) proteins serve multiple roles in Fn the posttranscriptional control of gene expression, including as regulators of alternative splicing during cell differentiation and There are several reasons for us to focus on the Fn and development [57]. Sfrs3 gene expression in this report: (1) Fn is one of the early In our model at 12 h after wounding, expression levels of expressed molecules found in wounds; (2) Fn is one of the the Fn1 and Sfrs3 were suppressed in fetal airway mucosal and most important molecules in wound healing process as well skin wounds, whereas they were induced in the postnatal as scar formation; (3) potential role of Sfrs3 in mRNA wounds, indicating that Fn1 mRNAs were not active or even alternative splicing of Fn1 variants [55,56]; and (4) both inhibited in fetal wounds 12 h post-wounding. The Fn

Fig. 9. Real-time PCR. Sfrs3 and Fn1 in fetal (white columns), weanling (stripped columns) and adult (black columns) skin and airway mucosal wounds 12 h after injury. Data were normalized to the rabbit 18S rRNA of the same group and adjusted to a 0-fold level of the non-wounded group of the same kind. 42 H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45

synthesis and processing of pre-mRNA [69]. Serine/arginine- rich (SR) proteins are believed in alternative splicing of numerous transcripts because their expression levels can influence splice site selection. SR proteins have different specificities for subclasses of pre-mRNAs and the regulation of the levels of SR proteins in different cell types contributes to the regulation of cell-specific splice choices [70]. These proteins act as driving forces during spliceosome assembly and also play decisive roles in alternative splice-site selection, suggesting that they are crucial players in the regulation of splicing during cell differentiation and development [57].SR proteins can also influence pre-mRNA processing at the level Fig. 10. Real-time PCR. Sfrs3 (dot line) and Fn1 (solid line) in weanling airway of polyadenylation and generally participate in terminal exon mucosal wound 12 h, 24 h and 48 h after wounding. Data were normalized to recognition [71]. Therefore, this family of proteins may play a the rabbit 18S rRNA of the same specimen, adjusted to a 0-fold level of the role in recognition of 3V-terminal exons and perhaps in the non-wounded group of the same kind, and plotted relative to the level at the communication between polyadenylation and splicing. Differ- time zero. ent SR proteins have unique intracellular transport properties, suggesting that SR proteins may have roles not only in nuclear deposition was observed at 1 h–4 h in the fetal wounds pre-mRNA splicing but also in mRNA transport, cytoplasmic [58,59], suggesting that the regulation of the Fn genes in fetal events, and/or processes that involve communication between skin wounds of this study may occur much earlier. the nucleus and the cytoplasm [72]. SR proteins are involved The opposed expression patterns of Sfrs3 and Fn1 between in multi-steps of mRNA metabolism, including nuclear export, fetal and postnatal wounds at 12 h after injury indicate an RNA stability, mRNA quality control, and translation [73].SR importance of the molecular activities of these two genes in proteins play a crucial role in the selection of FN spliced differentiating fetal scarless wound regeneration from postna- domains during development, regeneration, and oncogenesis tal scar formation. It is interesting to note that the augmenta- [55,56,74]. tions of the postnatal gene expression of both Fn1 and Sfrs3 genes were more prominent in airway mucosal wounds than 4.3.3. SRp20 and Fn splicing that in skin wounds. Our results suggest that the two genes Our study demonstrated that Sfrs3, an mRNA encoding are involved in the early events of the postnatal airway SRp20, was co-expressed with Fn1 gene; induced as early as mucosal wound repair and that this process is tissue-specific. 12 h and sustained to 48 h post-injury in postnatal airway Similar expression patterns of the Fn1 and Sfrs3 genes in mucosal and skin wounds. In fetal wounds, however, the both skin and airway mucosal wounds may also indicate that molecular activity of this gene was either silenced or Fn1 is a potential target of the SRp20 alternative splicing and/ suppressed at 12 h post-wounding. SRp20 transcript was up- or that the two genes are regulated by conserved regulatory regulated within 3 days after peripheral nerve injury [75]. elements. SRp20, a 20-KD SR protein, is widely expressed in multiple tissues and cell lines [40,76], and is involved in the control of 4.3. Alternative splicing and SR proteins the early stages of embryonic development [77] and cell cycle [78]. SRp20 is also a nucleo-cytoplasmic shuttling protein, 4.3.1. Alternative splicing promoting the export of intronless RNAs in both mammalian Alternative splicing of pre-mRNA is a common mechanism cells and Xenopus oocytes [79]. of regulating gene expression and widely recognized to be a Expression of the SR protein SRp40, SRp20, or ASF/SF2 ubiquitous and crucial mechanism for generating protein stimulates EIIIB inclusion [56], whereas co-transfection diversity and regulating protein expression [60–63]. Bioinfor- studies employing cDNAs encoding individual SR proteins matics analysis of alternative splicing of both human and revealed that SRp20 decreased mRNA accumulation in HeLa mouse data indicates the presence of alternative spliced forms cells, whereas SRp40 increased exon EIIIA inclusion in in up to 80% of human genes [64]. The FN protein diversity chondrocytes, but not in HeLa cells [55], suggesting that the and associated multi-functions are known to be modulated by splicing activity of SR proteins is cell-type dependent. In alternative splicing [29,30,33,65–68]. Our recent study humans, inclusion or exclusion of the FN-EIIIA exon is showed that the three common domains (EIIIA, EIIIB and V) mainly regulated by a polypurinic enhancer element (exonic of Fn1 that are subjective to alternative splicing were splicing enhancer) and a nearby silencer element (exonic differentially, selectively included in this model wounds splicing silencer). This is confirmed by RNA–protein (manuscript in preparation). interaction analysis of levels of SR protein binding to each exon [80]. How a cell achieves the precise tissue-specific 4.3.2. RS domain and SR proteins control of an Fn splicing pattern is poorly understood. It is Proteins containing arginine/serine-rich (RS) domains play important to define the high affinity targets of SRp20 and to a fundamental role in coordinating different steps in the evaluate its ability to discriminate between defined RNA H.-S. Li-Korotky et al. / Biochimica et Biophysica Acta 1762 (2006) 34–45 43 targets of the different isoforms of Fn in the context of the 5. Conclusion cellular responses to fetal and postnatal airway mucosal wound healing. The mRNA differential display provided a unique tool to identify candidate genes that may play key roles in fetal 4.3.4. Fn pre-mRNA splicing and scar formation airway mucosal scarless wound healing or postnatal wound Enhanced alternative splicing of FN variants was observed repair. Rabbit serves a valuable model relevant to human in cutaneous wounds [81,82]. The expression of EIIIA is wound repair. Sfrs3, a pre-mRNA splicing factor, and Fn1, a increased in the skin of patients with cutaneous graft-versus- provisional ECM, were co-expressed in skin and airway host disease, suggesting that FN-EIIIA is a marker of skin mucosal wounds in vivo. The expression patterns of the two fibrosis [83]. Expression of EIIIA and EIIIB spliced variants in genes were strikingly different between fetal (suppressed) bone indicates that these two splice variants are strong markers and postnatal (induced) wounds and tissue-specific. We for active fibrogenetic and osteoid-forming processes in human suggest that the molecular activities of Sfrs3 and Fn1 during bones [84]. FN spliced variants containing the EIIIA and/or the early events of the airway mucosal wound healing EIIIB exons are prominently expressed in the vasculature of a process are crucial in differentiating between fetal wound variety of human tumors but not in normal adult counterpart regeneration and postnatal scar formation. It is important to [85]. The deletion of the alternatively spliced EIIIA domain in define the high affinity targets of SRp20 and to evaluate its mice reduces atherosclerosis [86]. Results from previous ability to discriminate between defined RNA targets of the studies and our in vivo model of wound healing suggest that different isoforms of Fn in the context of the cellular alternative splicing of Fn domains is involved in the multi-steps responses to fetal and postnatal airway mucosal wound of the postnatal wound healing process and associated with healing. postnatal wound scar formation. Acknowledgments 4.4. Regulatory elements of the co-expression of Fn1 and Sfrs3 cDNAs This work was supported by the Lester A. Hamburg Endowed Fellowship in Pediatric Otolaryngology and the Previous studies and our data provide evidence that Sfrs3 is Eberly Family Endowed Chair through Children’s Hospital of one of the SR proteins that may selectively exclude or include Pittsburgh (Dr. Charles D. Bluestone). Authors thank Dr. David Fn1 variants [55,56] during wound healing process. However, C. Whitcomb (Director of the Center for Genomic Sciences) such a similar expression pattern of Fn1 and Sfrs3 in our in for his support to this project and Nancy Lo (University of vivo model may suggest that during the wounding process, Chicago) for her assistance in gene expression assays during both genes are transcriptionally regulated by one (or more) her Summer Student Research Program sponsored by the common transcription factor(s). Search for putative promoter- Children’s Hospital of Pittsburgh. specific transcription factor binding sites (TFBSs) and transcription factors of each gene might reveal conserved References regulatory elements that are critical for their co-expression during wound healing. We found that the two genes do not [1] J.E. Dohar, E.C. Klein, J.L. Betsch, P.A. Hebda, Acquired subglottic share a common TFBS. 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