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5′-Heterogeneity of mouse Dda3 transcripts is attributed to differential initiation of transcription and alternative splicing
ABB Archives of Biochemistry and Biophysics 425 (2004) 221–232 www.elsevier.com/locate/yabbi
50 -Heterogeneity of mouse Dda3 transcripts is attributed to differential initiation of transcription and alternative splicing Pang-Kuo Lo and Fung-Fang Wang* Institute of Biochemistry, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan Received 12 February 2004, and in revised form 22 March 2004
Abstract We have previously shown that mouse Dda3 gene is a p53 and p73 transcriptional target whose expression suppresses tumor cell growth. Here, we report the identification of multiple variants of Dda3 transcripts with diverse 50 sequences through 50 rapid amplification of cDNA ends (50 -RACE) and RT-PCR. Analysis by primer extension and RNase protection revealed that the 50 heterogeneity was generated by transcription initiation at multiple sites in exon 1 and intron 1 and by alternative splicing. These transcripts, both coding and non-coding, exhibited distinct expression patterns in various adult tissues and were developmentally regulated. Furthermore, they were induced in a p53-dependent manner by various stress signals. These data demonstrated that differential initiation of transcription and alternative splicing both participate in the regulation of Dda3 gene expression. Ó 2004 Published by Elsevier Inc. Keywords: Dda3; p53; Multiple transcription initiation sites; Alternative splicing
Eukaryotic cells are constantly exposed to a myriad of environmental stresses that cause DNA lesions. The maintenance of genomic stability depends on the ability of cells to sense and recognize damaged DNA and to elicit cellular responses to the damage [1]. The tumor suppressor p53 is a key modulator of stress responses; it guards the genome through either apoptosis induction or arresting cell cycle progression at specific checkpoints. It has been shown that the stability and activity of p53 are enhanced by a variety of DNA-damaging agents as well as by many other forms of cellular stresses [2–4]. A p53 homologue, p73, has recently been identified [5] and shown to participate in DNA-damage response induced by a limited subset of agents, including IRs and cisplatin [6–8], but not by UV-radiation or actinomycin D [5]. A complex pattern of posttranslational modifications, including phosphorylation and acetylation, has been shown to modulate p53/p73 functions in response to genotoxic stress [6–10]. Although transcription-independent functions have been described, modulation of specific target gene expression mediates most of the p53 and p73 effects [9,11,12]. p53- and p73* Corresponding author. Fax: +886-2-28264843. E-mail address: ff[email protected] (F.-F. Wang).
0003-9861/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.abb.2004.03.026
induced G1/S cell cycle arrest is mainly mediated by the p21 cyclin-dependent kinase inhibitor [13]. The proapoptotic Bcl-2 family member Bax [14], the Killer/DR5 death receptor [15], and the mitochondrial proteins p53AIP1 [16] and Noxa [17], all shown to induce apoptosis when overexpressed, are activated by p53 and p73 and may mediate apoptosis in response to DNA damage [9,11,12]. Through mRNA differential display on IW32 murine erythroleukemia cells transfected with a temperaturesensitive p53 mutant gene tsp53Val-135 [18], we have previously identified a novel p53- and p73-inducible mouse gene Dda3 [19,20]. When overexpressed in mammalian cells, Dda3 is able to suppress cell growth [19]. Mouse Dda3 encodes a serine- and proline-rich cytoplasmic protein of 329 amino acids, containing a coiled-coil region plus six putative SH3-domain binding motifs PXXP; it therefore may provide a link between p53 and components of other signal transduction pathways [19]. The mouse and human DDA3 genes have been cloned, both contain eight exons with similar genomic organization [21]. Despite recent advances in the genomic research, many of the problems in control of transcription and splicing remain elusive. It is clear that a single gene can
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generate multiple transcripts through the combination of alternative usages of promoters and of splicing sites. While some of the transcripts may retain the complete open reading frame, others have altered reading frames, still others are non-coding which lack an open reading frame. Recently, it has become clear that certain noncoding RNAs are functioning in regulating gene expression, including transcriptional regulation, RNA processing, RNA stability, and translation [22–25]. Therefore, identification of the diverse transcripts generated from a gene and elucidating their functions are central in understanding the multiple functions of the gene. In this study, we have identified multiple forms of coding and non-coding mouse Dda3 transcripts and shown that the heterogeneity is due to the usage of multiple transcriptional start sites plus alternative splicing of the pre-mRNA. These Dda3 transcripts exhibit tissue- and developmental stage-specific expression; in addition, they were differentially induced by genotoxic agents in a p53-dependent manner. The possible roles of these non-coding transcripts on Dda3 expression remain to be elucidated.
50 Rapid amplification of cDNA ends
Materials and methods
To clone the full-length mouse Dda3 cDNA, the Marathon cDNA amplification kit (Clontech) was adopted using the poly(A)þ RNA prepared from IW32tsp53 cells cultured at 32 °C for 4 h. For the first strand cDNA synthesis, 1 lg of the poly(A)þ RNA was incubated with 100 U of M-MuLV reverse transcriptase and 10 pmol oligo(dT) primer in 10 ll of 50 mM Tris, pH 8.3, containing 6 mM MgCl2 , 75 mM KCl, and 1 mM dNTP for 1 h at 42 °C. After that a mixture of enzymes containing DNA polymerase I (24 U), DNA ligase (4.8 U), and RNase H (1 U) was added, and the reaction was carried out in 80 ll final volume of 100 mM KCl, 10 mM ammonium sulfate, 5 mM MgCl2 , 0.15 M b-NAD, 0.2 mM dNTP in 20 mM Tris–HCl, pH 7.5, at 16 °C for 45 min. The cDNA was purified and ligated with the cDNA adaptor according to the protocol recommended by the manufacturer. The 50 rapid amplification of cDNA ends (50 -RACE) reaction utilized a Dda3-specific antisense primer RACE-30 R 50 -CTCCTGGCCTTTC CAAACAGACC-30 , complementary to the 30 -UTR of Dda3 (Fig. 1B), in conjunction with the adaptor primer. The RACE products were purified by agarose gel electrophoresis, cloned into pGEM-T vector (Promega), and sequenced from both directions.
Cell culture
Cloning of the mouse Dda3 cDNA isoforms
The IW32 murine erythroleukemia cells stably expressing the temperature-sensitive p53 mutant allele tsp53Val-135 (IW32-tsp53) [18] were grown in RPMI medium containing 10% fetal bovine serum (FBS)1 and 50 lg/ml gentamicin in a humidified chamber of 5% CO2 at 38 °C. Temperature-shifting to 32 °C was performed by transferring subconfluent cultures to a pre-equilibrated incubator. Wild-type and p53-knockout murine embryonic fibroblast (MEF) cells were grown in DMEM containing 10% FBS, 100 U/ml penicillin and 100 lg/ml streptomycin, and maintained at 37 °C in a humidified chamber of 5% CO2 . All cells were subcultured every 3 days.
To confirm the 50 -RACE products and to clone other isoforms of Dda3, RT-PCR was performed using the RNA prepared from IW32-tsp53 cells cultured at 32 °C for 4 h, with the sense primer (Fig. 1A) Dda3-I-50 F (for Dda3-I cDNA cloning), or Dda3-II-50 F (for Dda3-II cDNA cloning), in conjunction with the antisense primer Dda3-30 R (Fig. 1B). The PCR was carried out using the Advantage KlenTaq polymerase mix (Clontech) according to the protocol provided. The PCR products were purified and ligated into pGEM-T vector. The sequence of each cloned cDNA was verified by autosequencing. Primer extension assay
RNA isolation Total RNA was isolated by lysing the cells in REzol C&T Reagent (PROtech Technology) according to the manufacturerÕs recommendation. Poly(A)þ RNA was isolated from total RNA preparation using an Oligotex mRNA kit (Qiagen) according to the procedures described by the manufacturer.
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Abbreviations used: FBS, fetal bovine serum; MEF, murine embryonic fibroblast; 50 -RACE, 50 rapid amplification of cDNA ends; AD, adriamycin-treated; EST, expressed sequence tag.
The 50 -ends of the Dda3-I and -II mRNA isoforms were mapped by a primer extension assay kit (Primer Extension System-AMV Reverse Transcriptase, Promega). Two antisense oligonucleotides (PE1 and PE2, Fig. 3) were synthesized and 50 -end-labeled with [c32 P]ATP (3000 Ci/mmol) and T4 polynucleotide kinase (Promega). After purification on a Sephadex G25 spin column, the end-labeled primer (20 fmol) was added to 10 lg of poly(A)þ RNA isolated from IW32-tsp53 cells incubated at 32 °C for 0 and 4 h. RNA samples were precipitated with ethanol and dissolved in 11 ll of 1 AMV reverse transcriptase primer extension buffer
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Fig. 1. Murine IW32 erythroleukemia cells express two types of Dda3 transcripts with distinct 50 -end sequences. (A) 50 -nucleotide sequences of two mouse Dda3 cDNA clones. The sequence common to both cDNA clones is underlined. The 50 -end sequence unique to Dda3-II is indicated by lowercase letters and is shown to be derived from intron 1. (B) The 30 -end sequence of the Dda3 cDNA. Primers used for 50 -RACE and RT-PCR are underlined with arrows pointing to the 30 -end of the primers. (C) RT-PCR analysis of the Dda3-I and Dda3-II cDNAs. RT-PCR was performed using the cDNAs prepared from IW32-tsp53 cells incubated at 32 °C for 4 h as templates. The primer pairs were: for Dda3-I, Dda3-I-50 F/Dda3-30 R and for Dda3-II, Dda3-II-50 F/Dda3-30 R. The PCR products were separated on 1% TAE agarose gel, stained with ethidium bromide, and visualized by UV illumination. Lane M is the 1-kb ladder DNA markers.
(Promega). The samples were denatured at 80 °C for 2 min and hybridization was carried out at 60 °C for 1 h. After adding 9 ll of 1 AMV reverse transcriptase primer extension mix containing 1.2 U AMV reverse transcriptase, 6 mM sodium pyrophosphate, 20 U RNasin (Promega), and 1 lg actinomycin D, the extension reaction was carried out at 42 °C for 45 min. The
reaction was terminated by adding EDTA to a final concentration of 2.5 mM. The RNA templates were treated with pancreatic ribonuclease A for 30 min at 37 °C. Samples were extracted once with phenol/chloroform, precipitated with ethanol, and analyzed on a 6% polyacrylamide sequencing gel. The gel was dried and exposed to an X-ray film at )70 °C for 2 days.
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RNase protection assay To prepare the antisense RNA probes for RNase protection assay, cDNA fragments of Ia (174 bp), IIa (411 bp), IIb (323 bp), and IIc (253 bp) (shown in Fig. 2) were amplified by PCR using the isoform-specific primers, including two sense primers 50 -CGGGAACTG GGAGATTGTAGAAGA-30 (for Dda3-I cDNA isoforms) and 50 -GATGGAGGTGAAGACTGACCGA CTG-30 (for Dda3-II cDNA isoforms), as well as an antisense primer 50 -CCTCCGAAGTCCAAGGTCTCG TC-30 (complementary to the exon 3, identical to the PE1 primer), and ligated into the pGEM-T TA-cloning vector (Promega). Before in vitro transcription reaction, the plasmid constructs were previously linearized with SalI (for using T7 promoter) or NcoI (for using SP6 promoter). The antisense RNA probes were synthesized from these templates by in vitro transcription using T7 or SP6 RNA polymerases and [a-32 P]CTP according to the manufacturerÕs instructions (Riboprobe in vitro transcription systems, Promega). For verification of equal loading, the linearized pTRI-Actin-Mouse DNA (Ambion) was used as a template for in vitro transcription using T7 RNA polymerase to synthesize a 304 nt RNA probe containing 250 nt mouse b-actin in the antisense orientation. The RNase protection assay was performed using a RPA II kit (Ambion) according to the product protocol. Briefly, 20,000–80,000 cpm of riboprobes was mixed with 10 lg of total RNA from IW32-tsp53 cells maintained at 32 °C for 0 or 4 h, de-
natured at 95 °C for 5 min, and then hybridization was carried out at 45 °C overnight. The RNA probes were also hybridized to 10 lg of yeast tRNA as a negative control. The samples were then treated with a mixture of RNase A and T1 at 37 °C for 30 min. Protected RNA fragments were separated on a 6% polyacrylamide gel containing 8.3 M urea. The fragments were detected by overnight exposure to X-ray film. Semiquantitative RT-PCR The expression of the mouse Dda3 mRNA isoforms was analyzed by PCR using mouse multiple tissue cDNA panels containing 8 adult tissues and day-7 to day-17 embryos (Clontech) and cDNAs synthesized from total cellular RNA of wild-type and p53-knockout MEF treated with UV-radiation or adriamycin. Total cellular RNA (5 lg) was reversely transcribed to cDNA using 2.0 lM oligo(dT)15 -primer (Promega), 0.5 mM of each dNTP, 40 U RNase inhibitor (Promega), and 200 U SuperScript II reverse transcriptase (Invitrogen) in a final volume of 20 ll. The isoform-specific primers (shown in Fig. 7A) used for semiquantitative PCR were: PF-Ia (50 -AGTGGAAACCGAGTGTGCGCTGGAG30 ), sense primer for Ia and Ic; PF-Ib (50 -CCTGCGTA GACTGTGAAGCGAGGTCTAAGC-30 ), sense primer for Ib and Id; PR-I (50 -GGCTTCATCAAGAATCTC TTCCAGCTTCTCTG-30 ), antisense primer for all Dda3-I isoforms; PF-II (50 -AAGGATGGAGGTGAA GACTGACCGACTG-30 ), sense primer for all Dda3-II
Fig. 2. Schematic representation for the 50 heterogeneity of mouse Dda3 transcripts. Exons (E1–E4) and introns (IN1–IN3) are shown as hatched and open rectangles, respectively. Regions of the antisense RNA probes (Ia and IIa to IIc) used for RNase protection assay are indicated with their sizes in nucleotides (nt) shown in parentheses. Positions of the antisense primers (PE1 and PE2) used for primer extension assay are depicted by arrows. Two of the alternative splice donor sites located in intron 1 and exon 1 (detailed sequences are shown in Fig. 3) are indicated by asterisks.
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isoforms; and PR-II (50 -GCCCTCCGAAGTCCAAG GTCTCGTCC-30 ), antisense primer for all Dda3-II isoforms. We also used the sense (50 -GGACCGACTA GGTAAGAAGAGCACG-30 ) and antisense (50 -CAAG TTCTGTACATCAGCTGTCCTCCTGGC-30 ) primers, both derived from the sequence of exon 8, to quantitate the expression levels of all Dda3 isoforms. For determining the p53 levels, the sense (50 -CGAA GACTGGATGACTGCCATGGAGGAGTC-30 ) and antisense (50 -ATGCAGAGGCAGTCAGTCTGAGT CAGG-30 ) primers were used. Quantitation of p21WAF1 expression in MEF cells was carried out using the sense (50 -CATGTCCAATCCTGGTGATGTCCGAC-30 ) and antisense (50 -AAGACACACAGAGTGAGGGCTAA GG-30 ) primers. As an internal control, expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed using the sense (50 -TGAAGGTCGGTG TGAACGGATTTGGC-30 ) and antisense (50 -CAT GTAGGCCATGAGGTCCACCAC-30 ) primers. PCR was performed using 0.2 mM of each dNTP, 5 U Titanium Taq DNA polymerase (Clontech), with 0.4 lM of each primer. PCR was carried out for 30 s at 94 °C, 30 s at 68 °C, and 1 min at 72 °C. The PCR products were separated by electrophoresis on 1.6% agarose gels and visualized after staining with ethidium bromide.
Results Cloning and characterization of the alternatively spliced forms of mouse Dda3 We have previously cloned the mouse Dda3 cDNA (GenBank Accession No. AF156598), hereafter designated Dda3-I, from IW32 erythroleukemia cells stably expressing the temperature-sensitive mutant p53 (IW32tsp53) by 50 -RACE using RACE-30 R as a primer (Fig. 1B) [19]. Along the course of that study, a cDNA clone, designated Dda3-II, containing a 50 untranslated region (UTR) distinct from Dda3-I was also identified (Fig. 1A). Comparison of the Dda3-II 50 -UTR to the mouse Dda3 genome (GenBank Accession No. AF322890) [21] revealed that the Dda3-II 50 -UTR was derived from intron 1 (starting at position )279 relative to the translation start site) (Fig. 1A). Based on these findings, it appeared that in addition to the 50 nucleotide(s) of exon 1, transcription of Dda3 gene can also initiate at the 50 -end of intron 1. To confirm the results from 50 -RACE, RT-PCR was performed using two sense primers, Dda3-I-50 F and Dda3-II-50 F (Fig. 1A), corresponding to regions of exon 1 and intron 1 of Dda3, respectively, and an antisense primer, Dda3-30 R, which is complementary to the 30 -end region of exon 8 (Fig. 1B). As shown in Fig. 1C, the Dda3-I-specific primer amplified a DNA fragment of approximately 1.7-kb in size, and Dda3-II-specific pri-
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mer produced fragments with sizes of around 1.9 kb. By cloning and sequencing of these cDNA fragments, we were able to identify four Dda3-I transcripts (designated Dda3-Ia, -Ib, -Ic, and -Id) and three Dda3-II transcripts (designated Dda3-IIa, -IIb, and -IIc). Comparison of these cDNA sequences with the Dda3 genomic sequence (GenBank Accession No. AF322890) [21] indicated that transcripts within each subgroup were the products of differential splicing, as indicated in Fig. 2. Dda3-Ia (GenBank Accession No. AF156598) was produced by usage of the previously reported 50 splice donor site positioned at )340 to )339 (Fig. 3) [21], whereas usage of the donor site at )371 to )370 (Fig. 3), internally located in exon 1 (Fig. 2, asterisk), resulted in the generation of Dda3-Ib; inclusion of intron 2 converted Dda3-Ia and -Ib to Dda3-Ic and -Id, respectively (Fig. 2). The transcripts of Dda3-II subgroup started at intron 1, inclusion of intron 2 resulted in the production of Dda3-IIa, whereas the normal splicing out of intron 2 led to the generation of Dda3-IIb (Fig. 2). Dda3-IIc was produced by using the alternative 50 splice-donor site at the 30 region of intron 1 (asterisk, Fig. 2; nucleotides )51 to )50, Fig. 3), that spliced out a segment containing the last 20 nucleotides of intron 1 plus the entire regions of exon 2 and intron 2 (Fig. 2). Sequence analysis of the newly identified Dda3 isoforms indicated that only Dda3-Ia, -Ib, and -IIb retained the complete open reading frame (ORF) capable of encoding the DDA3 protein of 329 amino acids. The intron 2 containing transcripts Dda3-Ic, -Id, and -IIa contained an in-frame stop codon ‘‘TGA’’ at positions 52–54 (Fig. 3, bold italic), resulting in premature termination of translation within intron 2. Dda3-IIc isoform lacked the entire exon 2, and is also unable to encode the Dda3 protein. Although an internal ORF of 100 amino acids was predicted for all the non-coding transcripts, we were unable to detect this alternative ORF in the human DDA3 (GenBank Accession No. AF223000) [21], suggesting that the alternative ORF is probably non-functional. Verification of the expression of Dda3-I isoforms and mapping of their transcription start sites To verify that Dda3-I cDNA isoforms we isolated were expressed in cells and to map their transcription initiation sites, we concomitantly performed the primer extension assay and RNase protection analysis. A synthetic oligonucleotide (PE1, Figs. 2 and 3) complementary to nucleotides 125–147 was used as an antisense primer for primer extension assay; for RNase protection analysis, a 174-nt antisense RNA probe (Ia probe, Fig. 2), encompassing the 30 -terminus 84 nt of exon 1, the entire 50 nt of exon 2, and the 50 -end 40 nt of exon 3, was synthesized by in vitro transcription with PE1 as primer. Because PE1 was used for both the primer
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Fig. 3. Part of the DNA sequence of Dda3 gene encompassing the upstream genomic region to exon 3. Exons and introns are indicated by upper- and lowercase letters, respectively, with exons underlined. The putative CCAAT- and GC-box elements are boxed as indicated and the major transcriptional start sites identified by primer extension and RNase protection analyses are indicated by arrowheads. Two of the alternative splice donor sites are boxed. The occurrence of the stop codon (tga) in intron 2-containing variants Ic, Id, and IIa is shown in italic bold letters. Nucleotides are numbered relative to the translation start codon which is designated as +1.
extension assay and antisense RNA probe synthesis, identical extension products and the protected fragments were therefore expected for any given transcription initiation site. The RNA templates for both assays were derived from IW32-tsp53 cells cultured at 32 °C for 0 and 4 h. As shown in Fig. 4, multiple extension products (Fig. 4A) and protection fragments (Fig. 4B) were obtained and their intensities were significantly elevated with RNA templates prepared from IW32tsp53 cells cultured at 32 °C for 4 h. These results were consistent with our previous report that Dda3 was a transcriptional target of p53 [19]. By comparing the results from primer extension (Fig. 4A) to those from RNase protection (Fig. 4B), we have identified seven RNase protection fragments that were identical in size to the primer extension bands designated as Ia-2 (164 nt), Ia-3 (149–152 nt), Ia-4 (136 nt), and Ia-5 (125 nt). Analysis of these results revealed that the transcription of Dda3 gene could be initiated at multiple sites located at nucleotides )414, )402 to )399, )386, and )375 relative to the translation start codon ATG (Fig. 3). In addition, a weak extension band Ia-1 (180 nt) was also detected that represented the farthest tran-
scription start site at )430 (Figs. 3 and 4A). Interestingly, analysis of the Dda3 sequence did not show the presence of a TATA-box in the upstream genomic region, instead, a CCAAT-box and two tandem GC-box elements, which is the potential binding site for SP1, were found near the identified transcription initiation sites (Fig. 3). Examination of the primer extension products of Dda3-I also revealed the presence of alternatively spliced transcripts that utilized transcription start sites at nucleotides )402 to )399 (Fig. 4A). These include Ib3 that were 118–121 nt in size, equivalent to the length of Ia-3 minus 31 nt from the 30 -end of exon 1; Id-3 that were 206–209 nt in size, corresponding to the size of Ib-3 plus that of intron 2 (88 nt); and Ic-3 that were 237– 240 nt in size, equivalent to the size of Ia-3 plus intron 2 (88 nt). The presence of some of these splice variants was further confirmed by the results from RNase protection analysis. Protected fragments of 109–112 nt in size were obtained that correlated to the intron 2 containing transcript Ic; and the 90 nt band may result from protection by the E2/E3 region of the transcripts Ib and IIb (Fig. 4B). Taken together, these results demonstrated
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Fig. 4. Identification of Dda3-I mRNA variants and mapping of their transcription initiation sites by primer extension and RNase protection analyses. (A) Primer extension analysis. A 23-nt end-labeled antisense primer (PE1) complementary to the sequence from +125 to +147 (Fig. 3) was annealed to 10 lg of the poly(A)þ RNA prepared from IW32-tsp53 cells incubated at 32 °C for 0 (lane 2) or 4 h (lane 3) and extended upstream with reverse transcriptase. A reaction containing 10 lg of yeast RNA was included as a negative control (lane 1). The extension products were separated on a 6% (w/v) polyacrylamide gel containing 8.3 M urea and visualized by autoradiography. The extension bands on lane 3 that derived from Dda3Ia, -Ib, -Ic, and -Id mRNAs are depicted by arrows with their sizes shown in parenthesis. The DNA ladders shown on the left lanes were sequencing results from Dda3-Ia cDNA, whose 50 -end begins at )424, using PE1 as primer. Enlargement of the sequence ()424 to )372) is shown on the left of the sequencing ladders and the nucleotides corresponding to the mapped transcriptional start sites are indicated by arrow. (B) RNase protection analysis. The antisense Ia probe (174 nt, Fig. 2) was synthesized by in vitro transcription as described in the ‘‘Materials and methods.’’ Total RNA (10 lg) from IW32-tsp53 cells with (lane 2) or without (lane 1) incubation at 32 °C for 4 h was hybridized to the Ia probe, treated with RNase, and separated by electrophoresis on a 6% (w/v) polyacrylamide sequencing gel. The sizes of the protected fragments as shown in parentheses were calculated from a sequencing ladder run in parallel. The RNase protection analysis for b-actin mRNA was also included as an internal control for equal loading.
that the four alternatively spliced Dda3-I mRNA isoforms we have identified whose transcription initiated at multiple sites scattered throughout exon 1 are indeed expressed in IW32 cells. Validation of the expression of Dda3-II isoforms and mapping of their transcription start sites To validate the expression of the cloned Dda3-II transcripts in cells, RNase protection assay was performed using three isoform-specific antisense RNA
probes IIa (411 nt), IIb (323 nt), and IIc (253 nt) (Fig. 2) with RNAs isolated from IW32-tsp53 cells cultured at 32 °C for 0 or 4 h. The 411-nt antisense IIa probe protected mRNA fragments of 411, 283, and 213 nt in length, consistent with the expected protection sizes of Dda3-IIa, -IIb, and -IIc (Fig. 5). Using the 323-nt antisense IIb probe, protected fragments of 323, 283, and 213 nucleotides were obtained, which were derived, respectively, from Dda3-IIb, -IIa, and -IIc; in addition, the 90-nt protected fragment was derived from Dda3-Ia and -Ib (Fig. 5). As expected, the 253-nt antisense IIc probe
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Fig. 5. Identification of Dda3-II mRNA variants by RNase protection assay. The antisense IIa (411 nt), IIb (323 nt), and IIc (253 nt) probes (shown in Fig. 2) were synthesized by in vitro transcription as described in the ‘‘Materials and methods.’’ Total RNA (10 lg) from IW32-tsp53 cells cultured at 32 °C for 0 or 4 h was hybridized to the 32 P-labeled probes, reacted with RNase, and separated on 6% (w/v) polyacrylamide sequencing gels. The transcripts responsible for the protected bands are indicated by arrows on the right, with protected sizes calculated from a sequencing ladder run in parallel shown in parentheses. Protected fragments for b-actin were shown as an internal control for equal loading.
protected a 253-nt fragment from Dda3-IIc and a 213-nt fragment from Dda3-IIa and -IIb (Fig. 5). Similar to the Dda3-I mRNA isoforms, the levels of all these Dda3-II transcripts were significantly induced by wild-type tsp53Val-135 expression. To map the transcription start sites, primer extension analysis was performed using the antisense primer PE2 (Figs. 2 and 3) complementary to nucleotides )111 to )138 of the Dda3 intron 1; the poly(A)þ RNAs derived from IW32-tsp53 cells cultured at 32 °C for 0 or 4 h were used as templates. Four prominent extension fragments of 187, 186, 178, and 177 nt in length were generated from template derived from IW32-tsp53 cells cultured at 32 °C for 4 h (Fig. 6, lane 3), but not from the same cells
Fig. 6. Mapping of the transcriptional start sites for Dda3-II mRNA isoforms by primer extension analysis. A 28-nt end-labeled antisense primer (PE2) complementary to the sequence from )138 to )111 (shown in Fig. 3) was annealed to 10 lg of poly(A)þ RNA from IW32tsp53 cells cultured at 38 °C (lane 2) or shifted to 32 °C for 4 h (lane 3) and extended upstream with reverse transcriptase. A reaction containing 10 lg yeast RNA was included as a negative control (lane 1). The extension products were analyzed on a 6% (w/v) polyacrylamide sequencing gel and visualized by autoradiography. The DNA ladders shown on the right are sequencing results from Dda3 genomic DNA containing intron 1 using PE2 as primer. The transcription start sites are indicated by arrows in the enlargement of the corresponding sequence shown to the right of the sequencing ladders. The sizes of 32 Plabeled /X174 HinfI DNA markers are shown on the left.
cultured at 38 °C (Fig. 6, lane 2). These results demonstrated that the transcription initiation sites for Dda3-II transcripts start at )297, )296, )288, and )287 (Fig. 3) and their expression is tightly correlated with the presence of functional p53 (Fig. 6). Expression of the mouse Dda3 transcripts in various tissues and developmental stage embryos We next examined the tissue distribution and developmental stage-specific expression of the Dda3 transcripts by PCR using cDNAs prepared from different mouse tissues and embryos of various developmental stages. Specific primers as described below and in Fig. 7A were used to determine the level of each mRNA isoform. For Dda3-1a and -Ic, the sense primer PF-Ia, corresponding to the 30 -end 25 nucleotides of exon 1
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Fig. 7. Tissue- and developmental stage-specific expression of the mouse Dda3 mRNA isoforms. (A) Schematic representation of the partial genomic structure of mouse Dda3 gene and the primers used for PCR. Exons (E1–E4) and introns (IN1–IN3) are shown as hatched and open rectangles, respectively. Two of the alternative splice donor sites (detailed sequences in Fig. 3) are indicated by asterisks and the PCR primers are depicted by arrows. Nucleotide sequences of the primers are shown in the ‘‘Materials and methods.’’ The sequence of the 30-nt PF-Ib primer is shown, which is complementary to the 22-nt sequence 50 upstream of the alternative splice site of exon 1 and the 8-nt 50 -end sequence of exon 2. (B) Determination of the levels of mouse Dda3 mRNA isoforms in tissues and embryos during various developmental stages by PCR. PCRs were performed with cDNAs (0.25 ng) prepared from various mouse tissues and embryos at different developmental stages (Clontech) using primer pairs shown in (A). The isoform-specific primers amplified fragments of Ia (299 bp), Ib (294 bp), Ic (387 bp), Id (384 bp), IIa (416 bp), IIb (328 bp), and IIc (258 bp), respectively, whereas primers complementary to exon 8 produced a fragment of 490 bp from all forms of Dda3 transcripts. Lanes are: M, 100-bp ladder markers; H, heart; B, brain; Sp, spleen; Lu, lung; Li, liver; Sk, skeletal muscle; K, kidney; T, testis; E7–E17, day-7 to day-17 embryos; and N, no template control. Results from PCR analysis on p53 expression at different developmental stages of embryos were also shown. Analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as an internal control. Expression of GAPDH was more abundant in heart and skeletal muscle.
()365 to )341), and an antisense primer PR-I complementary to the central region of exon 4 were used; for Dda3-Ib and -Id, the sense primer PF-Ib, corresponding to the 22 nucleotides upstream of the internal splice donor site ()371 and )370) of exon 1 plus the first 8 nucleotides of exon 2, and an antisense primer PR-I were used; and for Dda3-IIa, -IIb, and -IIc, the sense primer PF-II, corresponding to nucleotides )267 to )240 of intron 1, and the antisense primer PR-II complementary to the nucleotides +124 to +149 of exon 3 were used. To determine the expression level of total Dda3, primers that amplify a region in exon 8 were used. As shown in Fig. 7B using exon 8-specific primers, the expression of Dda3 was abundant in the brain, spleen,
and lung, and, to a lesser extent, in the heart, kidney, and testis. On the other hand, liver and skeletal muscle expressed very little Dda3. Among the Dda3-I subgroup transcripts, Dda3-Ic and -Ia were more abundant and were expressed in most tissues, in contrast, Dda3-II expression was absent in the brain and liver (Fig. 7B). These results suggest that the expression of Dda3 transcripts is regulated in a tissue-specific manner. It has been shown by Komarova et al. [26] that the transactivation activity and mRNA expression of p53 are under developmental regulation. We examined the expression of Dda3 transcripts in embryos of various stages, and showed that total Dda3 expression was detectable in day-7 embryos, increased significantly from
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day 11 to 15, and decreased at day 17 (Fig. 7B). The expression of Dda3-I and II isoforms essentially followed this pattern except that Dda3-Ib and -Id as well as all Dda3-II isoforms were not detectable in day-7 embryos. Using the p53-specific primers, we demonstrated that the expression of p53 mRNA during embryonic development closely correlated with that of the Dda3 transcripts (Fig. 7B), suggesting that Dda3 expression is tightly regulated by p53 during development.
Expression of the Dda3 mRNA isoforms is induced by DNA damage in a p53-dependent manner We next examined if the expression of Dda3 transcripts was regulated by p53 in cells undergoing DNA damage using the mouse embryonic fibroblast cells (MEF) with or without wild-type p53 expression. Levels of p21WAF1 mRNA were determined by RT-PCR as a positive control for p53-induced target. As shown in Fig. 8, transcripts of p21WAF1 and total Dda3 levels were significantly elevated in wild-type MEF cells (p53þ=þ ) undergoing UV- and adriamycin-induced DNA damage; but not in p53-knockout MEF (p53= ) cells under the same treatments, suggesting stress-induced activation of these transcripts is p53 dependent. It is noteworthy that UV irradiation, when compared to adriamycin treatment, had a lesser effect on Dda3-I mRNA induction (Fig. 8), and in the absence of p53, it suppressed the expression of all forms of Dda3 transcripts, including Dda3-I and Dda3-II (Fig. 8). On the other hand, induction of Dda3-IIa and -IIb transcripts appeared more prominent by UV irradiation than by adriamycin treatment, whereas level of Dda3-IIc transcript was down-regulated by UV irradiation (Fig. 8). These results demonstrated that Dda3 mRNA isoforms are regulated distinctively by stress signals in a p53-dependent manner.
Discussion
Fig. 8. p53-dependent induction of Dda3 transcripts by DNA-damaging agents. RT-PCR analysis was performed using isoform-specific primer pairs to determine the levels of the Dda3 transcripts in UVirradiated (UV) or adriamycin-treated (AD) wild-type (p53þ=þ ) and p53-knockout (p53= ) MEF cells. Levels of total Dda3 mRNA were determined using exon 8-specific primers. Cells were irradiated with 60 J/m2 of UV for 10 h or treated with 400 ng/ml adriamycin for 16 h, total RNA was prepared and used for cDNA synthesis. As a positive control of p53 transcriptional target, levels of p21WAF1 were determined using specific primers as described in the Materials and methods. The GAPDH mRNA level was shown to assure equal amounts of RNAs used for RT-PCR analysis. Numbers on the right are PCR amplification cycles for the indicated isoforms.
We have demonstrated the presence of multiple mouse Dda3 transcripts with 50 -heterogeneity, and shown that they are the consequence of alternative splicing in conjunction with transcription initiation at multiple sites scattered throughout exon 1 and the 50 region of intron 1. In addition to the previously reported isoform Dda3-Ia [19], two newly identified transcripts Dda3-Ib and -IIb also retain the complete ORF of Dda3. On the contrary, this ORF is disrupted in the rest of transcripts due to premature termination resulting from intron 2 inclusion or exon 2 exclusion. All of these identified Dda3 transcripts are expressed in a tissuespecific manner and are developmentally regulated, moreover, their expressions are differentially affected by different stress signals. We have mapped the transcription start sites and shown that Dda3 transcripts can be classified into two subgroups that use transcription initiation sites located in exon 1 and intron 1, respectively, suggesting the existence of two promoters for Dda3 gene. This hypothesis is supported by the finding of distinctive expression patterns of these subgroups in some tissues examined. For example, whereas the expression of subgroup I transcripts is observed in the brain, the subgroup II transcripts are absent from the brain, indicating that the
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transcription initiation of these two subgroups may be differentially regulated in some tissues. We have searched the human expressed sequence tag (EST) database of the NCBI and encountered two EST cDNA clones that starts at nucleotides 27 and 44 of intron 1 (GenBank Accession Nos. BX644686 and BM014528, respectively), suggesting that the use of intron 1 as transcription initiation is conserved in mouse and human. Multiple transcription initiation sites are features typical of constitutively expressed housekeeping genes or proto-oncogenes [27–29]. These genes have characteristics of a high GC-rich upstream promoter that lacks the apparent CAAT or TATA motif, and the presence of SP1 binding sequence preceding the transcription initiation sites [27–29]. Examination of the 200-nt upstream sequence of Dda3 gene indicates that this region lacks a consensus TATA box and is rich in GC content (61%); moreover, two putative SP1-like GC boxes contiguous to the transcription initiation sites are detected (Fig. 3). Whether these SP1-like elements are involved in controlling the transcription of Dda3 gene remains to be elucidated. It has been shown that the length and nucleotide composition of the 50 -UTR of a gene may affect its interaction with protein factors involved in the regulation of mRNA transports, turnover and protein translation [30,31]. The coding isoforms Dda3-Ia, -Ib, and -IIb all contain an intact DDA3 ORF, however, they vary in the sequence of the 50 -UTR; Dda3-Ib has a shorter 50 -UTR as compared to Dda3-Ia, in contrast, Dda3-IIb contains intron 1, but not exon 1, as its 50 UTR. The possibilities that these transcripts are translated with different efficiencies or possess distinct stability or are localized to discrete intracellular compartments cannot be excluded. There is growing evidence that non-coding mRNAs constitute a major population of expressed transcripts in mammalian cells [32]. The non-coding RNAs have been implicated in a variety of cellular processes including control of chromosome architecture, RNA processing and modification, mRNA turnover and translation, and even transcription and alternative splicing [22–25]. The functional significance of coexistence of non-coding Dda3 mRNAs among the coding isoforms is not clear, it is possible that by changing the relative ratios of coding to non-coding transcripts, the expression levels of Dda3 may be modulated. In this sense, differential initiation of transcription and alternative splicing provide alternative pathways for regulating the expression of a gene. We have shown that the extent of induction of the Dda3 isoforms differs in cells subjected to distinct DNAdamaging agents, suggesting that both promoter and splicing mechanisms of the Dda3 gene are under differential regulation by various cellular stresses. It is worth noting that the levels of the non-coding Dda3 isoforms are subjected to regulation by DNA damage. For ex-
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ample, the expression of the non-coding IIa and IIc transcripts responded in a p53-dependent manner to UV irradiation and adriamycin treatment (Fig. 8), which cause different types of DNA damage and trigger distinct damaging signals. Dda3-IIa was induced to a greater extent by UV irradiation than by adriamycin, unlike the subgroup I transcripts that showed opposite response; on the other hand, Dda3-IIc was suppressed by UV treatment (Fig. 8). The findings that these noncoding Dda3 mRNAs respond distinctively to various cellular stresses raise the possibility that as RNA regulatory molecules, they may play roles in stress-signaling.
Acknowledgments This work is supported by Grant NHRI-EX929124BI from National Health Research Institute, Taiwan, Republic of China.
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50 -Heterogeneity of mouse Dda3 transcripts is attributed to differential initiation of transcription and alternative splicing Pang-Kuo Lo and Fung-Fang Wang* Institute of Biochemistry, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan Received 12 February 2004, and in revised form 22 March 2004
Abstract We have previously shown that mouse Dda3 gene is a p53 and p73 transcriptional target whose expression suppresses tumor cell growth. Here, we report the identification of multiple variants of Dda3 transcripts with diverse 50 sequences through 50 rapid amplification of cDNA ends (50 -RACE) and RT-PCR. Analysis by primer extension and RNase protection revealed that the 50 heterogeneity was generated by transcription initiation at multiple sites in exon 1 and intron 1 and by alternative splicing. These transcripts, both coding and non-coding, exhibited distinct expression patterns in various adult tissues and were developmentally regulated. Furthermore, they were induced in a p53-dependent manner by various stress signals. These data demonstrated that differential initiation of transcription and alternative splicing both participate in the regulation of Dda3 gene expression. Ó 2004 Published by Elsevier Inc. Keywords: Dda3; p53; Multiple transcription initiation sites; Alternative splicing
Eukaryotic cells are constantly exposed to a myriad of environmental stresses that cause DNA lesions. The maintenance of genomic stability depends on the ability of cells to sense and recognize damaged DNA and to elicit cellular responses to the damage [1]. The tumor suppressor p53 is a key modulator of stress responses; it guards the genome through either apoptosis induction or arresting cell cycle progression at specific checkpoints. It has been shown that the stability and activity of p53 are enhanced by a variety of DNA-damaging agents as well as by many other forms of cellular stresses [2–4]. A p53 homologue, p73, has recently been identified [5] and shown to participate in DNA-damage response induced by a limited subset of agents, including IRs and cisplatin [6–8], but not by UV-radiation or actinomycin D [5]. A complex pattern of posttranslational modifications, including phosphorylation and acetylation, has been shown to modulate p53/p73 functions in response to genotoxic stress [6–10]. Although transcription-independent functions have been described, modulation of specific target gene expression mediates most of the p53 and p73 effects [9,11,12]. p53- and p73* Corresponding author. Fax: +886-2-28264843. E-mail address: ff[email protected] (F.-F. Wang).
0003-9861/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.abb.2004.03.026
induced G1/S cell cycle arrest is mainly mediated by the p21 cyclin-dependent kinase inhibitor [13]. The proapoptotic Bcl-2 family member Bax [14], the Killer/DR5 death receptor [15], and the mitochondrial proteins p53AIP1 [16] and Noxa [17], all shown to induce apoptosis when overexpressed, are activated by p53 and p73 and may mediate apoptosis in response to DNA damage [9,11,12]. Through mRNA differential display on IW32 murine erythroleukemia cells transfected with a temperaturesensitive p53 mutant gene tsp53Val-135 [18], we have previously identified a novel p53- and p73-inducible mouse gene Dda3 [19,20]. When overexpressed in mammalian cells, Dda3 is able to suppress cell growth [19]. Mouse Dda3 encodes a serine- and proline-rich cytoplasmic protein of 329 amino acids, containing a coiled-coil region plus six putative SH3-domain binding motifs PXXP; it therefore may provide a link between p53 and components of other signal transduction pathways [19]. The mouse and human DDA3 genes have been cloned, both contain eight exons with similar genomic organization [21]. Despite recent advances in the genomic research, many of the problems in control of transcription and splicing remain elusive. It is clear that a single gene can
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generate multiple transcripts through the combination of alternative usages of promoters and of splicing sites. While some of the transcripts may retain the complete open reading frame, others have altered reading frames, still others are non-coding which lack an open reading frame. Recently, it has become clear that certain noncoding RNAs are functioning in regulating gene expression, including transcriptional regulation, RNA processing, RNA stability, and translation [22–25]. Therefore, identification of the diverse transcripts generated from a gene and elucidating their functions are central in understanding the multiple functions of the gene. In this study, we have identified multiple forms of coding and non-coding mouse Dda3 transcripts and shown that the heterogeneity is due to the usage of multiple transcriptional start sites plus alternative splicing of the pre-mRNA. These Dda3 transcripts exhibit tissue- and developmental stage-specific expression; in addition, they were differentially induced by genotoxic agents in a p53-dependent manner. The possible roles of these non-coding transcripts on Dda3 expression remain to be elucidated.
50 Rapid amplification of cDNA ends
Materials and methods
To clone the full-length mouse Dda3 cDNA, the Marathon cDNA amplification kit (Clontech) was adopted using the poly(A)þ RNA prepared from IW32tsp53 cells cultured at 32 °C for 4 h. For the first strand cDNA synthesis, 1 lg of the poly(A)þ RNA was incubated with 100 U of M-MuLV reverse transcriptase and 10 pmol oligo(dT) primer in 10 ll of 50 mM Tris, pH 8.3, containing 6 mM MgCl2 , 75 mM KCl, and 1 mM dNTP for 1 h at 42 °C. After that a mixture of enzymes containing DNA polymerase I (24 U), DNA ligase (4.8 U), and RNase H (1 U) was added, and the reaction was carried out in 80 ll final volume of 100 mM KCl, 10 mM ammonium sulfate, 5 mM MgCl2 , 0.15 M b-NAD, 0.2 mM dNTP in 20 mM Tris–HCl, pH 7.5, at 16 °C for 45 min. The cDNA was purified and ligated with the cDNA adaptor according to the protocol recommended by the manufacturer. The 50 rapid amplification of cDNA ends (50 -RACE) reaction utilized a Dda3-specific antisense primer RACE-30 R 50 -CTCCTGGCCTTTC CAAACAGACC-30 , complementary to the 30 -UTR of Dda3 (Fig. 1B), in conjunction with the adaptor primer. The RACE products were purified by agarose gel electrophoresis, cloned into pGEM-T vector (Promega), and sequenced from both directions.
Cell culture
Cloning of the mouse Dda3 cDNA isoforms
The IW32 murine erythroleukemia cells stably expressing the temperature-sensitive p53 mutant allele tsp53Val-135 (IW32-tsp53) [18] were grown in RPMI medium containing 10% fetal bovine serum (FBS)1 and 50 lg/ml gentamicin in a humidified chamber of 5% CO2 at 38 °C. Temperature-shifting to 32 °C was performed by transferring subconfluent cultures to a pre-equilibrated incubator. Wild-type and p53-knockout murine embryonic fibroblast (MEF) cells were grown in DMEM containing 10% FBS, 100 U/ml penicillin and 100 lg/ml streptomycin, and maintained at 37 °C in a humidified chamber of 5% CO2 . All cells were subcultured every 3 days.
To confirm the 50 -RACE products and to clone other isoforms of Dda3, RT-PCR was performed using the RNA prepared from IW32-tsp53 cells cultured at 32 °C for 4 h, with the sense primer (Fig. 1A) Dda3-I-50 F (for Dda3-I cDNA cloning), or Dda3-II-50 F (for Dda3-II cDNA cloning), in conjunction with the antisense primer Dda3-30 R (Fig. 1B). The PCR was carried out using the Advantage KlenTaq polymerase mix (Clontech) according to the protocol provided. The PCR products were purified and ligated into pGEM-T vector. The sequence of each cloned cDNA was verified by autosequencing. Primer extension assay
RNA isolation Total RNA was isolated by lysing the cells in REzol C&T Reagent (PROtech Technology) according to the manufacturerÕs recommendation. Poly(A)þ RNA was isolated from total RNA preparation using an Oligotex mRNA kit (Qiagen) according to the procedures described by the manufacturer.
1
Abbreviations used: FBS, fetal bovine serum; MEF, murine embryonic fibroblast; 50 -RACE, 50 rapid amplification of cDNA ends; AD, adriamycin-treated; EST, expressed sequence tag.
The 50 -ends of the Dda3-I and -II mRNA isoforms were mapped by a primer extension assay kit (Primer Extension System-AMV Reverse Transcriptase, Promega). Two antisense oligonucleotides (PE1 and PE2, Fig. 3) were synthesized and 50 -end-labeled with [c32 P]ATP (3000 Ci/mmol) and T4 polynucleotide kinase (Promega). After purification on a Sephadex G25 spin column, the end-labeled primer (20 fmol) was added to 10 lg of poly(A)þ RNA isolated from IW32-tsp53 cells incubated at 32 °C for 0 and 4 h. RNA samples were precipitated with ethanol and dissolved in 11 ll of 1 AMV reverse transcriptase primer extension buffer
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Fig. 1. Murine IW32 erythroleukemia cells express two types of Dda3 transcripts with distinct 50 -end sequences. (A) 50 -nucleotide sequences of two mouse Dda3 cDNA clones. The sequence common to both cDNA clones is underlined. The 50 -end sequence unique to Dda3-II is indicated by lowercase letters and is shown to be derived from intron 1. (B) The 30 -end sequence of the Dda3 cDNA. Primers used for 50 -RACE and RT-PCR are underlined with arrows pointing to the 30 -end of the primers. (C) RT-PCR analysis of the Dda3-I and Dda3-II cDNAs. RT-PCR was performed using the cDNAs prepared from IW32-tsp53 cells incubated at 32 °C for 4 h as templates. The primer pairs were: for Dda3-I, Dda3-I-50 F/Dda3-30 R and for Dda3-II, Dda3-II-50 F/Dda3-30 R. The PCR products were separated on 1% TAE agarose gel, stained with ethidium bromide, and visualized by UV illumination. Lane M is the 1-kb ladder DNA markers.
(Promega). The samples were denatured at 80 °C for 2 min and hybridization was carried out at 60 °C for 1 h. After adding 9 ll of 1 AMV reverse transcriptase primer extension mix containing 1.2 U AMV reverse transcriptase, 6 mM sodium pyrophosphate, 20 U RNasin (Promega), and 1 lg actinomycin D, the extension reaction was carried out at 42 °C for 45 min. The
reaction was terminated by adding EDTA to a final concentration of 2.5 mM. The RNA templates were treated with pancreatic ribonuclease A for 30 min at 37 °C. Samples were extracted once with phenol/chloroform, precipitated with ethanol, and analyzed on a 6% polyacrylamide sequencing gel. The gel was dried and exposed to an X-ray film at )70 °C for 2 days.
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RNase protection assay To prepare the antisense RNA probes for RNase protection assay, cDNA fragments of Ia (174 bp), IIa (411 bp), IIb (323 bp), and IIc (253 bp) (shown in Fig. 2) were amplified by PCR using the isoform-specific primers, including two sense primers 50 -CGGGAACTG GGAGATTGTAGAAGA-30 (for Dda3-I cDNA isoforms) and 50 -GATGGAGGTGAAGACTGACCGA CTG-30 (for Dda3-II cDNA isoforms), as well as an antisense primer 50 -CCTCCGAAGTCCAAGGTCTCG TC-30 (complementary to the exon 3, identical to the PE1 primer), and ligated into the pGEM-T TA-cloning vector (Promega). Before in vitro transcription reaction, the plasmid constructs were previously linearized with SalI (for using T7 promoter) or NcoI (for using SP6 promoter). The antisense RNA probes were synthesized from these templates by in vitro transcription using T7 or SP6 RNA polymerases and [a-32 P]CTP according to the manufacturerÕs instructions (Riboprobe in vitro transcription systems, Promega). For verification of equal loading, the linearized pTRI-Actin-Mouse DNA (Ambion) was used as a template for in vitro transcription using T7 RNA polymerase to synthesize a 304 nt RNA probe containing 250 nt mouse b-actin in the antisense orientation. The RNase protection assay was performed using a RPA II kit (Ambion) according to the product protocol. Briefly, 20,000–80,000 cpm of riboprobes was mixed with 10 lg of total RNA from IW32-tsp53 cells maintained at 32 °C for 0 or 4 h, de-
natured at 95 °C for 5 min, and then hybridization was carried out at 45 °C overnight. The RNA probes were also hybridized to 10 lg of yeast tRNA as a negative control. The samples were then treated with a mixture of RNase A and T1 at 37 °C for 30 min. Protected RNA fragments were separated on a 6% polyacrylamide gel containing 8.3 M urea. The fragments were detected by overnight exposure to X-ray film. Semiquantitative RT-PCR The expression of the mouse Dda3 mRNA isoforms was analyzed by PCR using mouse multiple tissue cDNA panels containing 8 adult tissues and day-7 to day-17 embryos (Clontech) and cDNAs synthesized from total cellular RNA of wild-type and p53-knockout MEF treated with UV-radiation or adriamycin. Total cellular RNA (5 lg) was reversely transcribed to cDNA using 2.0 lM oligo(dT)15 -primer (Promega), 0.5 mM of each dNTP, 40 U RNase inhibitor (Promega), and 200 U SuperScript II reverse transcriptase (Invitrogen) in a final volume of 20 ll. The isoform-specific primers (shown in Fig. 7A) used for semiquantitative PCR were: PF-Ia (50 -AGTGGAAACCGAGTGTGCGCTGGAG30 ), sense primer for Ia and Ic; PF-Ib (50 -CCTGCGTA GACTGTGAAGCGAGGTCTAAGC-30 ), sense primer for Ib and Id; PR-I (50 -GGCTTCATCAAGAATCTC TTCCAGCTTCTCTG-30 ), antisense primer for all Dda3-I isoforms; PF-II (50 -AAGGATGGAGGTGAA GACTGACCGACTG-30 ), sense primer for all Dda3-II
Fig. 2. Schematic representation for the 50 heterogeneity of mouse Dda3 transcripts. Exons (E1–E4) and introns (IN1–IN3) are shown as hatched and open rectangles, respectively. Regions of the antisense RNA probes (Ia and IIa to IIc) used for RNase protection assay are indicated with their sizes in nucleotides (nt) shown in parentheses. Positions of the antisense primers (PE1 and PE2) used for primer extension assay are depicted by arrows. Two of the alternative splice donor sites located in intron 1 and exon 1 (detailed sequences are shown in Fig. 3) are indicated by asterisks.
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isoforms; and PR-II (50 -GCCCTCCGAAGTCCAAG GTCTCGTCC-30 ), antisense primer for all Dda3-II isoforms. We also used the sense (50 -GGACCGACTA GGTAAGAAGAGCACG-30 ) and antisense (50 -CAAG TTCTGTACATCAGCTGTCCTCCTGGC-30 ) primers, both derived from the sequence of exon 8, to quantitate the expression levels of all Dda3 isoforms. For determining the p53 levels, the sense (50 -CGAA GACTGGATGACTGCCATGGAGGAGTC-30 ) and antisense (50 -ATGCAGAGGCAGTCAGTCTGAGT CAGG-30 ) primers were used. Quantitation of p21WAF1 expression in MEF cells was carried out using the sense (50 -CATGTCCAATCCTGGTGATGTCCGAC-30 ) and antisense (50 -AAGACACACAGAGTGAGGGCTAA GG-30 ) primers. As an internal control, expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed using the sense (50 -TGAAGGTCGGTG TGAACGGATTTGGC-30 ) and antisense (50 -CAT GTAGGCCATGAGGTCCACCAC-30 ) primers. PCR was performed using 0.2 mM of each dNTP, 5 U Titanium Taq DNA polymerase (Clontech), with 0.4 lM of each primer. PCR was carried out for 30 s at 94 °C, 30 s at 68 °C, and 1 min at 72 °C. The PCR products were separated by electrophoresis on 1.6% agarose gels and visualized after staining with ethidium bromide.
Results Cloning and characterization of the alternatively spliced forms of mouse Dda3 We have previously cloned the mouse Dda3 cDNA (GenBank Accession No. AF156598), hereafter designated Dda3-I, from IW32 erythroleukemia cells stably expressing the temperature-sensitive mutant p53 (IW32tsp53) by 50 -RACE using RACE-30 R as a primer (Fig. 1B) [19]. Along the course of that study, a cDNA clone, designated Dda3-II, containing a 50 untranslated region (UTR) distinct from Dda3-I was also identified (Fig. 1A). Comparison of the Dda3-II 50 -UTR to the mouse Dda3 genome (GenBank Accession No. AF322890) [21] revealed that the Dda3-II 50 -UTR was derived from intron 1 (starting at position )279 relative to the translation start site) (Fig. 1A). Based on these findings, it appeared that in addition to the 50 nucleotide(s) of exon 1, transcription of Dda3 gene can also initiate at the 50 -end of intron 1. To confirm the results from 50 -RACE, RT-PCR was performed using two sense primers, Dda3-I-50 F and Dda3-II-50 F (Fig. 1A), corresponding to regions of exon 1 and intron 1 of Dda3, respectively, and an antisense primer, Dda3-30 R, which is complementary to the 30 -end region of exon 8 (Fig. 1B). As shown in Fig. 1C, the Dda3-I-specific primer amplified a DNA fragment of approximately 1.7-kb in size, and Dda3-II-specific pri-
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mer produced fragments with sizes of around 1.9 kb. By cloning and sequencing of these cDNA fragments, we were able to identify four Dda3-I transcripts (designated Dda3-Ia, -Ib, -Ic, and -Id) and three Dda3-II transcripts (designated Dda3-IIa, -IIb, and -IIc). Comparison of these cDNA sequences with the Dda3 genomic sequence (GenBank Accession No. AF322890) [21] indicated that transcripts within each subgroup were the products of differential splicing, as indicated in Fig. 2. Dda3-Ia (GenBank Accession No. AF156598) was produced by usage of the previously reported 50 splice donor site positioned at )340 to )339 (Fig. 3) [21], whereas usage of the donor site at )371 to )370 (Fig. 3), internally located in exon 1 (Fig. 2, asterisk), resulted in the generation of Dda3-Ib; inclusion of intron 2 converted Dda3-Ia and -Ib to Dda3-Ic and -Id, respectively (Fig. 2). The transcripts of Dda3-II subgroup started at intron 1, inclusion of intron 2 resulted in the production of Dda3-IIa, whereas the normal splicing out of intron 2 led to the generation of Dda3-IIb (Fig. 2). Dda3-IIc was produced by using the alternative 50 splice-donor site at the 30 region of intron 1 (asterisk, Fig. 2; nucleotides )51 to )50, Fig. 3), that spliced out a segment containing the last 20 nucleotides of intron 1 plus the entire regions of exon 2 and intron 2 (Fig. 2). Sequence analysis of the newly identified Dda3 isoforms indicated that only Dda3-Ia, -Ib, and -IIb retained the complete open reading frame (ORF) capable of encoding the DDA3 protein of 329 amino acids. The intron 2 containing transcripts Dda3-Ic, -Id, and -IIa contained an in-frame stop codon ‘‘TGA’’ at positions 52–54 (Fig. 3, bold italic), resulting in premature termination of translation within intron 2. Dda3-IIc isoform lacked the entire exon 2, and is also unable to encode the Dda3 protein. Although an internal ORF of 100 amino acids was predicted for all the non-coding transcripts, we were unable to detect this alternative ORF in the human DDA3 (GenBank Accession No. AF223000) [21], suggesting that the alternative ORF is probably non-functional. Verification of the expression of Dda3-I isoforms and mapping of their transcription start sites To verify that Dda3-I cDNA isoforms we isolated were expressed in cells and to map their transcription initiation sites, we concomitantly performed the primer extension assay and RNase protection analysis. A synthetic oligonucleotide (PE1, Figs. 2 and 3) complementary to nucleotides 125–147 was used as an antisense primer for primer extension assay; for RNase protection analysis, a 174-nt antisense RNA probe (Ia probe, Fig. 2), encompassing the 30 -terminus 84 nt of exon 1, the entire 50 nt of exon 2, and the 50 -end 40 nt of exon 3, was synthesized by in vitro transcription with PE1 as primer. Because PE1 was used for both the primer
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Fig. 3. Part of the DNA sequence of Dda3 gene encompassing the upstream genomic region to exon 3. Exons and introns are indicated by upper- and lowercase letters, respectively, with exons underlined. The putative CCAAT- and GC-box elements are boxed as indicated and the major transcriptional start sites identified by primer extension and RNase protection analyses are indicated by arrowheads. Two of the alternative splice donor sites are boxed. The occurrence of the stop codon (tga) in intron 2-containing variants Ic, Id, and IIa is shown in italic bold letters. Nucleotides are numbered relative to the translation start codon which is designated as +1.
extension assay and antisense RNA probe synthesis, identical extension products and the protected fragments were therefore expected for any given transcription initiation site. The RNA templates for both assays were derived from IW32-tsp53 cells cultured at 32 °C for 0 and 4 h. As shown in Fig. 4, multiple extension products (Fig. 4A) and protection fragments (Fig. 4B) were obtained and their intensities were significantly elevated with RNA templates prepared from IW32tsp53 cells cultured at 32 °C for 4 h. These results were consistent with our previous report that Dda3 was a transcriptional target of p53 [19]. By comparing the results from primer extension (Fig. 4A) to those from RNase protection (Fig. 4B), we have identified seven RNase protection fragments that were identical in size to the primer extension bands designated as Ia-2 (164 nt), Ia-3 (149–152 nt), Ia-4 (136 nt), and Ia-5 (125 nt). Analysis of these results revealed that the transcription of Dda3 gene could be initiated at multiple sites located at nucleotides )414, )402 to )399, )386, and )375 relative to the translation start codon ATG (Fig. 3). In addition, a weak extension band Ia-1 (180 nt) was also detected that represented the farthest tran-
scription start site at )430 (Figs. 3 and 4A). Interestingly, analysis of the Dda3 sequence did not show the presence of a TATA-box in the upstream genomic region, instead, a CCAAT-box and two tandem GC-box elements, which is the potential binding site for SP1, were found near the identified transcription initiation sites (Fig. 3). Examination of the primer extension products of Dda3-I also revealed the presence of alternatively spliced transcripts that utilized transcription start sites at nucleotides )402 to )399 (Fig. 4A). These include Ib3 that were 118–121 nt in size, equivalent to the length of Ia-3 minus 31 nt from the 30 -end of exon 1; Id-3 that were 206–209 nt in size, corresponding to the size of Ib-3 plus that of intron 2 (88 nt); and Ic-3 that were 237– 240 nt in size, equivalent to the size of Ia-3 plus intron 2 (88 nt). The presence of some of these splice variants was further confirmed by the results from RNase protection analysis. Protected fragments of 109–112 nt in size were obtained that correlated to the intron 2 containing transcript Ic; and the 90 nt band may result from protection by the E2/E3 region of the transcripts Ib and IIb (Fig. 4B). Taken together, these results demonstrated
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Fig. 4. Identification of Dda3-I mRNA variants and mapping of their transcription initiation sites by primer extension and RNase protection analyses. (A) Primer extension analysis. A 23-nt end-labeled antisense primer (PE1) complementary to the sequence from +125 to +147 (Fig. 3) was annealed to 10 lg of the poly(A)þ RNA prepared from IW32-tsp53 cells incubated at 32 °C for 0 (lane 2) or 4 h (lane 3) and extended upstream with reverse transcriptase. A reaction containing 10 lg of yeast RNA was included as a negative control (lane 1). The extension products were separated on a 6% (w/v) polyacrylamide gel containing 8.3 M urea and visualized by autoradiography. The extension bands on lane 3 that derived from Dda3Ia, -Ib, -Ic, and -Id mRNAs are depicted by arrows with their sizes shown in parenthesis. The DNA ladders shown on the left lanes were sequencing results from Dda3-Ia cDNA, whose 50 -end begins at )424, using PE1 as primer. Enlargement of the sequence ()424 to )372) is shown on the left of the sequencing ladders and the nucleotides corresponding to the mapped transcriptional start sites are indicated by arrow. (B) RNase protection analysis. The antisense Ia probe (174 nt, Fig. 2) was synthesized by in vitro transcription as described in the ‘‘Materials and methods.’’ Total RNA (10 lg) from IW32-tsp53 cells with (lane 2) or without (lane 1) incubation at 32 °C for 4 h was hybridized to the Ia probe, treated with RNase, and separated by electrophoresis on a 6% (w/v) polyacrylamide sequencing gel. The sizes of the protected fragments as shown in parentheses were calculated from a sequencing ladder run in parallel. The RNase protection analysis for b-actin mRNA was also included as an internal control for equal loading.
that the four alternatively spliced Dda3-I mRNA isoforms we have identified whose transcription initiated at multiple sites scattered throughout exon 1 are indeed expressed in IW32 cells. Validation of the expression of Dda3-II isoforms and mapping of their transcription start sites To validate the expression of the cloned Dda3-II transcripts in cells, RNase protection assay was performed using three isoform-specific antisense RNA
probes IIa (411 nt), IIb (323 nt), and IIc (253 nt) (Fig. 2) with RNAs isolated from IW32-tsp53 cells cultured at 32 °C for 0 or 4 h. The 411-nt antisense IIa probe protected mRNA fragments of 411, 283, and 213 nt in length, consistent with the expected protection sizes of Dda3-IIa, -IIb, and -IIc (Fig. 5). Using the 323-nt antisense IIb probe, protected fragments of 323, 283, and 213 nucleotides were obtained, which were derived, respectively, from Dda3-IIb, -IIa, and -IIc; in addition, the 90-nt protected fragment was derived from Dda3-Ia and -Ib (Fig. 5). As expected, the 253-nt antisense IIc probe
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Fig. 5. Identification of Dda3-II mRNA variants by RNase protection assay. The antisense IIa (411 nt), IIb (323 nt), and IIc (253 nt) probes (shown in Fig. 2) were synthesized by in vitro transcription as described in the ‘‘Materials and methods.’’ Total RNA (10 lg) from IW32-tsp53 cells cultured at 32 °C for 0 or 4 h was hybridized to the 32 P-labeled probes, reacted with RNase, and separated on 6% (w/v) polyacrylamide sequencing gels. The transcripts responsible for the protected bands are indicated by arrows on the right, with protected sizes calculated from a sequencing ladder run in parallel shown in parentheses. Protected fragments for b-actin were shown as an internal control for equal loading.
protected a 253-nt fragment from Dda3-IIc and a 213-nt fragment from Dda3-IIa and -IIb (Fig. 5). Similar to the Dda3-I mRNA isoforms, the levels of all these Dda3-II transcripts were significantly induced by wild-type tsp53Val-135 expression. To map the transcription start sites, primer extension analysis was performed using the antisense primer PE2 (Figs. 2 and 3) complementary to nucleotides )111 to )138 of the Dda3 intron 1; the poly(A)þ RNAs derived from IW32-tsp53 cells cultured at 32 °C for 0 or 4 h were used as templates. Four prominent extension fragments of 187, 186, 178, and 177 nt in length were generated from template derived from IW32-tsp53 cells cultured at 32 °C for 4 h (Fig. 6, lane 3), but not from the same cells
Fig. 6. Mapping of the transcriptional start sites for Dda3-II mRNA isoforms by primer extension analysis. A 28-nt end-labeled antisense primer (PE2) complementary to the sequence from )138 to )111 (shown in Fig. 3) was annealed to 10 lg of poly(A)þ RNA from IW32tsp53 cells cultured at 38 °C (lane 2) or shifted to 32 °C for 4 h (lane 3) and extended upstream with reverse transcriptase. A reaction containing 10 lg yeast RNA was included as a negative control (lane 1). The extension products were analyzed on a 6% (w/v) polyacrylamide sequencing gel and visualized by autoradiography. The DNA ladders shown on the right are sequencing results from Dda3 genomic DNA containing intron 1 using PE2 as primer. The transcription start sites are indicated by arrows in the enlargement of the corresponding sequence shown to the right of the sequencing ladders. The sizes of 32 Plabeled /X174 HinfI DNA markers are shown on the left.
cultured at 38 °C (Fig. 6, lane 2). These results demonstrated that the transcription initiation sites for Dda3-II transcripts start at )297, )296, )288, and )287 (Fig. 3) and their expression is tightly correlated with the presence of functional p53 (Fig. 6). Expression of the mouse Dda3 transcripts in various tissues and developmental stage embryos We next examined the tissue distribution and developmental stage-specific expression of the Dda3 transcripts by PCR using cDNAs prepared from different mouse tissues and embryos of various developmental stages. Specific primers as described below and in Fig. 7A were used to determine the level of each mRNA isoform. For Dda3-1a and -Ic, the sense primer PF-Ia, corresponding to the 30 -end 25 nucleotides of exon 1
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Fig. 7. Tissue- and developmental stage-specific expression of the mouse Dda3 mRNA isoforms. (A) Schematic representation of the partial genomic structure of mouse Dda3 gene and the primers used for PCR. Exons (E1–E4) and introns (IN1–IN3) are shown as hatched and open rectangles, respectively. Two of the alternative splice donor sites (detailed sequences in Fig. 3) are indicated by asterisks and the PCR primers are depicted by arrows. Nucleotide sequences of the primers are shown in the ‘‘Materials and methods.’’ The sequence of the 30-nt PF-Ib primer is shown, which is complementary to the 22-nt sequence 50 upstream of the alternative splice site of exon 1 and the 8-nt 50 -end sequence of exon 2. (B) Determination of the levels of mouse Dda3 mRNA isoforms in tissues and embryos during various developmental stages by PCR. PCRs were performed with cDNAs (0.25 ng) prepared from various mouse tissues and embryos at different developmental stages (Clontech) using primer pairs shown in (A). The isoform-specific primers amplified fragments of Ia (299 bp), Ib (294 bp), Ic (387 bp), Id (384 bp), IIa (416 bp), IIb (328 bp), and IIc (258 bp), respectively, whereas primers complementary to exon 8 produced a fragment of 490 bp from all forms of Dda3 transcripts. Lanes are: M, 100-bp ladder markers; H, heart; B, brain; Sp, spleen; Lu, lung; Li, liver; Sk, skeletal muscle; K, kidney; T, testis; E7–E17, day-7 to day-17 embryos; and N, no template control. Results from PCR analysis on p53 expression at different developmental stages of embryos were also shown. Analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as an internal control. Expression of GAPDH was more abundant in heart and skeletal muscle.
()365 to )341), and an antisense primer PR-I complementary to the central region of exon 4 were used; for Dda3-Ib and -Id, the sense primer PF-Ib, corresponding to the 22 nucleotides upstream of the internal splice donor site ()371 and )370) of exon 1 plus the first 8 nucleotides of exon 2, and an antisense primer PR-I were used; and for Dda3-IIa, -IIb, and -IIc, the sense primer PF-II, corresponding to nucleotides )267 to )240 of intron 1, and the antisense primer PR-II complementary to the nucleotides +124 to +149 of exon 3 were used. To determine the expression level of total Dda3, primers that amplify a region in exon 8 were used. As shown in Fig. 7B using exon 8-specific primers, the expression of Dda3 was abundant in the brain, spleen,
and lung, and, to a lesser extent, in the heart, kidney, and testis. On the other hand, liver and skeletal muscle expressed very little Dda3. Among the Dda3-I subgroup transcripts, Dda3-Ic and -Ia were more abundant and were expressed in most tissues, in contrast, Dda3-II expression was absent in the brain and liver (Fig. 7B). These results suggest that the expression of Dda3 transcripts is regulated in a tissue-specific manner. It has been shown by Komarova et al. [26] that the transactivation activity and mRNA expression of p53 are under developmental regulation. We examined the expression of Dda3 transcripts in embryos of various stages, and showed that total Dda3 expression was detectable in day-7 embryos, increased significantly from
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day 11 to 15, and decreased at day 17 (Fig. 7B). The expression of Dda3-I and II isoforms essentially followed this pattern except that Dda3-Ib and -Id as well as all Dda3-II isoforms were not detectable in day-7 embryos. Using the p53-specific primers, we demonstrated that the expression of p53 mRNA during embryonic development closely correlated with that of the Dda3 transcripts (Fig. 7B), suggesting that Dda3 expression is tightly regulated by p53 during development.
Expression of the Dda3 mRNA isoforms is induced by DNA damage in a p53-dependent manner We next examined if the expression of Dda3 transcripts was regulated by p53 in cells undergoing DNA damage using the mouse embryonic fibroblast cells (MEF) with or without wild-type p53 expression. Levels of p21WAF1 mRNA were determined by RT-PCR as a positive control for p53-induced target. As shown in Fig. 8, transcripts of p21WAF1 and total Dda3 levels were significantly elevated in wild-type MEF cells (p53þ=þ ) undergoing UV- and adriamycin-induced DNA damage; but not in p53-knockout MEF (p53= ) cells under the same treatments, suggesting stress-induced activation of these transcripts is p53 dependent. It is noteworthy that UV irradiation, when compared to adriamycin treatment, had a lesser effect on Dda3-I mRNA induction (Fig. 8), and in the absence of p53, it suppressed the expression of all forms of Dda3 transcripts, including Dda3-I and Dda3-II (Fig. 8). On the other hand, induction of Dda3-IIa and -IIb transcripts appeared more prominent by UV irradiation than by adriamycin treatment, whereas level of Dda3-IIc transcript was down-regulated by UV irradiation (Fig. 8). These results demonstrated that Dda3 mRNA isoforms are regulated distinctively by stress signals in a p53-dependent manner.
Discussion
Fig. 8. p53-dependent induction of Dda3 transcripts by DNA-damaging agents. RT-PCR analysis was performed using isoform-specific primer pairs to determine the levels of the Dda3 transcripts in UVirradiated (UV) or adriamycin-treated (AD) wild-type (p53þ=þ ) and p53-knockout (p53= ) MEF cells. Levels of total Dda3 mRNA were determined using exon 8-specific primers. Cells were irradiated with 60 J/m2 of UV for 10 h or treated with 400 ng/ml adriamycin for 16 h, total RNA was prepared and used for cDNA synthesis. As a positive control of p53 transcriptional target, levels of p21WAF1 were determined using specific primers as described in the Materials and methods. The GAPDH mRNA level was shown to assure equal amounts of RNAs used for RT-PCR analysis. Numbers on the right are PCR amplification cycles for the indicated isoforms.
We have demonstrated the presence of multiple mouse Dda3 transcripts with 50 -heterogeneity, and shown that they are the consequence of alternative splicing in conjunction with transcription initiation at multiple sites scattered throughout exon 1 and the 50 region of intron 1. In addition to the previously reported isoform Dda3-Ia [19], two newly identified transcripts Dda3-Ib and -IIb also retain the complete ORF of Dda3. On the contrary, this ORF is disrupted in the rest of transcripts due to premature termination resulting from intron 2 inclusion or exon 2 exclusion. All of these identified Dda3 transcripts are expressed in a tissuespecific manner and are developmentally regulated, moreover, their expressions are differentially affected by different stress signals. We have mapped the transcription start sites and shown that Dda3 transcripts can be classified into two subgroups that use transcription initiation sites located in exon 1 and intron 1, respectively, suggesting the existence of two promoters for Dda3 gene. This hypothesis is supported by the finding of distinctive expression patterns of these subgroups in some tissues examined. For example, whereas the expression of subgroup I transcripts is observed in the brain, the subgroup II transcripts are absent from the brain, indicating that the
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transcription initiation of these two subgroups may be differentially regulated in some tissues. We have searched the human expressed sequence tag (EST) database of the NCBI and encountered two EST cDNA clones that starts at nucleotides 27 and 44 of intron 1 (GenBank Accession Nos. BX644686 and BM014528, respectively), suggesting that the use of intron 1 as transcription initiation is conserved in mouse and human. Multiple transcription initiation sites are features typical of constitutively expressed housekeeping genes or proto-oncogenes [27–29]. These genes have characteristics of a high GC-rich upstream promoter that lacks the apparent CAAT or TATA motif, and the presence of SP1 binding sequence preceding the transcription initiation sites [27–29]. Examination of the 200-nt upstream sequence of Dda3 gene indicates that this region lacks a consensus TATA box and is rich in GC content (61%); moreover, two putative SP1-like GC boxes contiguous to the transcription initiation sites are detected (Fig. 3). Whether these SP1-like elements are involved in controlling the transcription of Dda3 gene remains to be elucidated. It has been shown that the length and nucleotide composition of the 50 -UTR of a gene may affect its interaction with protein factors involved in the regulation of mRNA transports, turnover and protein translation [30,31]. The coding isoforms Dda3-Ia, -Ib, and -IIb all contain an intact DDA3 ORF, however, they vary in the sequence of the 50 -UTR; Dda3-Ib has a shorter 50 -UTR as compared to Dda3-Ia, in contrast, Dda3-IIb contains intron 1, but not exon 1, as its 50 UTR. The possibilities that these transcripts are translated with different efficiencies or possess distinct stability or are localized to discrete intracellular compartments cannot be excluded. There is growing evidence that non-coding mRNAs constitute a major population of expressed transcripts in mammalian cells [32]. The non-coding RNAs have been implicated in a variety of cellular processes including control of chromosome architecture, RNA processing and modification, mRNA turnover and translation, and even transcription and alternative splicing [22–25]. The functional significance of coexistence of non-coding Dda3 mRNAs among the coding isoforms is not clear, it is possible that by changing the relative ratios of coding to non-coding transcripts, the expression levels of Dda3 may be modulated. In this sense, differential initiation of transcription and alternative splicing provide alternative pathways for regulating the expression of a gene. We have shown that the extent of induction of the Dda3 isoforms differs in cells subjected to distinct DNAdamaging agents, suggesting that both promoter and splicing mechanisms of the Dda3 gene are under differential regulation by various cellular stresses. It is worth noting that the levels of the non-coding Dda3 isoforms are subjected to regulation by DNA damage. For ex-
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ample, the expression of the non-coding IIa and IIc transcripts responded in a p53-dependent manner to UV irradiation and adriamycin treatment (Fig. 8), which cause different types of DNA damage and trigger distinct damaging signals. Dda3-IIa was induced to a greater extent by UV irradiation than by adriamycin, unlike the subgroup I transcripts that showed opposite response; on the other hand, Dda3-IIc was suppressed by UV treatment (Fig. 8). The findings that these noncoding Dda3 mRNAs respond distinctively to various cellular stresses raise the possibility that as RNA regulatory molecules, they may play roles in stress-signaling.
Acknowledgments This work is supported by Grant NHRI-EX929124BI from National Health Research Institute, Taiwan, Republic of China.
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