BCL-2 Translation Is Mediated via Internal Ribosome Entry during Cell Stress
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m402727200
ISSN1083-351X
AutoresKyle W. Sherrill, Marshall P. Byrd, Marc E. Van Eden, Richard E. Lloyd,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe cellular response to stress involves a rapid inhibition of cap-dependent translation via multiple mechanisms, yet some translation persists. This residual translation may include proteins critical to the cellular stress response. BCL-2 is a key inhibitor of intrinsic apoptotic signaling. Its primary transcript contains a 1.45-kb 5′-untranslated region (UTR) including 10 upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Thus, we hypothesized that this 5′-UTR may contain an internal ribosome entry site (IRES) that facilitates BCL-2 translation, particularly during cell stress. Here we show that the BCL-2 5′-UTR demonstrated IRES activity both when translated in vitro and also when m7G-capped and polyadenylated mRNA was transiently transfected into 293T cells. The activity of this IRES in unstressed cells was ∼6% the strength of the hepatitis C virus IRES but was induced 3–6-fold in a dose-dependent manner following short term treatment with either etoposide or sodium arsenite. Thus, the IRES-mediated translation of BCL-2 may enable the cell to replenish levels of this critical protein during cell stress, when cap-dependent translation is repressed, thereby maintaining the balance between pro- and anti-apoptotic BCL-2 family members in the cell and preventing unwarranted induction of apoptosis. The cellular response to stress involves a rapid inhibition of cap-dependent translation via multiple mechanisms, yet some translation persists. This residual translation may include proteins critical to the cellular stress response. BCL-2 is a key inhibitor of intrinsic apoptotic signaling. Its primary transcript contains a 1.45-kb 5′-untranslated region (UTR) including 10 upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Thus, we hypothesized that this 5′-UTR may contain an internal ribosome entry site (IRES) that facilitates BCL-2 translation, particularly during cell stress. Here we show that the BCL-2 5′-UTR demonstrated IRES activity both when translated in vitro and also when m7G-capped and polyadenylated mRNA was transiently transfected into 293T cells. The activity of this IRES in unstressed cells was ∼6% the strength of the hepatitis C virus IRES but was induced 3–6-fold in a dose-dependent manner following short term treatment with either etoposide or sodium arsenite. Thus, the IRES-mediated translation of BCL-2 may enable the cell to replenish levels of this critical protein during cell stress, when cap-dependent translation is repressed, thereby maintaining the balance between pro- and anti-apoptotic BCL-2 family members in the cell and preventing unwarranted induction of apoptosis. The BCL-2 protein is the prototype member of a conserved superfamily of proteins that regulate the cellular response to various intrinsic stresses, including damage to DNA, the endoplasmic reticulum, or Golgi apparatus (1Borner C. Mol. Immunol. 2003; 39: 615-647Crossref PubMed Scopus (620) Google Scholar). BCL-2 prevents apoptosis in response to these stresses through a variety of mechanisms, many of which are now clearly defined. Its predominant isoform (BCL-2α) contains a C-terminal transmembrane domain and localizes to membranes of the mitochondria, endoplasmic reticulum, and nuclear envelope (2Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). The anti-apoptotic members of the BCL-2 family, including BCL-2, all contain highly homologous regions (BH domains 1–3), which together form a hydrophobic groove required for anti-apoptotic functionality (3Hanada M. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11969Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 4Borner C. Olivier R. Martinou I. Mattmann C. Tschopp J. Martinou J.C. Biochem. Cell Biol. 1994; 72: 463-469Crossref PubMed Scopus (26) Google Scholar). BCL-2 forms heterodimers with a variety of pro-apoptotic proteins, thereby sequestering these proteins to prevent the onset of apoptosis (5Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). 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With such diverse roles in regulating apoptosis, it is not surprising that the expression of BCL-2 is regulated at multiple levels. First, the transcription of BCL-2 mRNA is transcribed from two different promoters, and its mRNA stability is regulated by an AU-rich element in the 3′-UTR 1The abbreviations used are: UTR, untranslated region; IRES, internal ribosome entry site; ORF, open reading frame; uORF, upstream ORF; RNAi, RNA interference; RT, reverse transcriptase; Na Ars, sodium arsenite; Etopo, etoposide; HCV, hepatitis C virus; CMV, cytomegalovirus; Rluc, Renilla luciferase; FLuc, firefly luciferase; RRL, rabbit reticulocyte lysates. (15Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar). In addition, the phosphorylation state of BCL-2 (16Ruvolo P.P. Deng X. May W.S. 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This 5′-UTR contains a 219-bp alternatively spliced intron that spans the region from 286 to 505 bases upstream from the translation start site. The splicing frequency of this intron varies among B-cell lines, although both spliced and un-spliced forms are often simultaneously expressed (25Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (457) Google Scholar). The BCL-2 5′-UTR region is highly conserved between human, mouse, rat, and even chicken, suggesting a potential regulatory role for this region (30Eguchi Y. Ewert D.L. Tsujimoto Y. Nucleic Acids Res. 1992; 20: 4187-4192Crossref PubMed Scopus (104) Google Scholar, 31Negrini M. Silini E. Kozak C. Tsujimoto Y. Croce C.M. Cell. 1987; 49: 455-463Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 32Sato T. Irie S. Krajewski S. Reed J.C. Gene (Amst.). 1994; 140: 291-292Crossref PubMed Scopus (95) Google Scholar). The vast majority of cellular mRNAs initiate translation via m7G cap-dependent recruitment of the 40 S ribosomal subunit to the 5′ end of a mRNA, followed by linear 5′–3′ scanning to the first AUG codon in proper context (33Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). However, during induced cellular stress this cap-dependent translation is rapidly inhibited by multiple mechanisms (34Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 35Sheikh M.S. Fornace Jr., A.J. Oncogene. 1999; 18: 6121-6128Crossref PubMed Scopus (103) Google Scholar). Even so, a portion of cellular translation persists that is thought to occur via a cap-independent recruitment of ribosomes directly onto certain mRNAs containing internal ribosome entry site (IRES) elements (35Sheikh M.S. Fornace Jr., A.J. Oncogene. 1999; 18: 6121-6128Crossref PubMed Scopus (103) Google Scholar, 36Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (805) Google Scholar). IRES-mediated translational initiation was initially described as a mechanism that enables certain viruses to translate effectively viral proteins despite an inhibition of cap-dependent translation in the infected cell (37Jang S.K. Krausslich H.G. Nicklin M.J. Duke G.M. Palmenberg A.C. Wimmer E. J. Virol. 1988; 62: 2636-2643Crossref PubMed Google Scholar, 38Pelletier J. Sonenberg N. Nature. 1988; 334: 320-325Crossref PubMed Scopus (1395) Google Scholar). In eukaryotes, IRES-mediated translation initiation has been most often observed for mRNAs that possess unusually long and thermodynamically stable 5′-UTRs with multiple potential uORFs, features that can dramatically inhibit scanning-dependent translation initiation (36Hellen C.U. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (805) Google Scholar). The predominant mRNA transcript coding for BCL-2 possesses both of these characteristics, containing a 1.45-kb 5′-UTR with 10 upstream AUGs. Thus, we examined the 5′-UTR of BCL-2 for its ability to mediate translational initiation via an unconventional, cap-independent mechanism. Through transfection of m7G-capped and polyadenylated reporter mRNAs, we demonstrate that BCL-2 expression is regulated via a stress-inducible IRES located within its 5′-UTR. This IRES mediated little reporter gene translation in unstressed cells, yet was induced 3–6-fold during stress induced by treatment with either sodium arsenite or the chemotherapeutic agent etoposide. Thus, IRES-mediated translation of BCL-2 may enable the cell to replenish levels of this critical protein during periods of cell stress, when little cap-dependent translation occurs. Materials—Radiolabeled nucleotides [α-32P]CTP and [35S]Met-Cys (Tran35S-label) were from ICN Biomedicals, Inc. (Irvine, CA). Oligonucleotide primer synthesis was by Integrated DNA Technologies (Coralville, IA). Cell Culture—Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 100 units/ml penicillin, 10 μg/ml streptomycin, and 10% fetal bovine serum (Hyclone, Logan UT). Growth was at 37 °C in 5.0% CO2 at 95% relative humidity, and cells were thinned 1:5 every 2–3 days at <85% confluency. Construct Assembly—pRL-HL was a gift from S. Lemon (University of Texas Medical Branch, Galveston, TX), and its construction was described previously (39Honda M. Kaneko S. Matsushita E. Kobayashi K. Abell G.A. Lemon S.M. Gastroenterology. 2000; 118: 152-162Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). A unique SacII site was introduced into pRL-HL upstream from FLuc to facilitate insertion of sequences between NotI and SacII sites, and the construct was named pRL-HCV-FL. The BCL-2 5′-UTR was amplified via RT-PCR from total RNA of HeLa S3 cells. Gene-specific primers amplified a region from bases –1146 to +3 of the BCL-2 cDNA (relative to the translational start site) and added NotI (5′ end) and SacII (3′ end) sites. This fragment was inserted into a NotI/SacII-digested pRL-HCV-FL backbone to form pRL-BCL2-FL. The original BCL-2 start codon was retained and inserted in-frame with FLuc, creating a FLuc ORF with six extra N-terminal codons. pRL-FL was created by removing the HCV IRES region between NotI and SacII sites. pRL-revH-FL was constructed via PCR of the HCV sequence utilizing primers that swapped the NotI and SacII sites. This fragment was then ligated into the NotI/SacII-cut backbone of pRL-HCV-FL. phpRL-BCL-2-FL included a 143-bp region that forms a stable RNA hairpin (ΔG = –60 kcal/mol) inserted at a unique NheI site located immediately upstream from RLuc. p(ΔCMV)RL-BCL-2-FL was created using long distance PCR and the 5′-phosphorylated primers 5′-cagccgcggagaactagtggatcccccggg-3′ and 5′-ctagcggccgcttggtgttacgtttggtttttctttg-3′, which allowed amplification of all but the CMV promoter region. The PCR product was then blunt-ligated with T4 ligase. pBCL-2-FL was similarly constructed using the primers 5′-gacatccactttgcctttctctcca-3′ and 5′-gtgggttacatcgaactggatctca-3′. Construction of phpB-CL-2-FL utilized primers 5′-cgttgagcgagttctcaaaagtgaacaataattctagagcgg-3 and 5′-ggtggctagcttataaaagcagtgg-3′. All clones were verified by both restriction digest and sequencing. In Vitro Transcription and Translation—Constructs were linearized with either AgeI or XhoI (pRL-revH-FL and pRL-HCV-FL). In vitro transcription was performed using purified T7 polymerase in a typical 100-μl reaction. m7G(5′)ppp(5′)G cap analog (m7G) (Ambion, Austin, TX) was included at a ratio of 4:1 versus rGTP. Translations were performed in typical fashion using nuclease-treated RRL (Promega) and 10 μCi of Tran35S-Label Met/Cys (ICN Biomedicals, Inc.). Reactions were resolved via 12% SDS-PAGE, dried, and then exposed to Kodak Biomax film (Rochester, NY). RNAi Directed against RLuc—The RNAi vector pBS-RLi was utilized to elicit a small interfering RNA response targeting the RLuc coding region. This vector contains a 50-bp RNA hairpin downstream from the murine U6 promoter, and its construction has been described previously (40Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar). pBS-RLi was transiently co-transfected into cells along with dicistronic reporter constructs at a 1:1 ratio (0.5 μg each) using FuGENE 6 transfection reagent (Roche Applied Science). The empty vector pBS-ApaI was used as control. 293T cells were plated at 4.0 × 105 cells/well on 12-well plates 16 h prior to transfection. Cells were harvested 72 h after transfection, and luciferase activity was measured using the dual luciferase assay kit (Promega) on a Sirius model luminometer (Berthold Detection Systems). RT-PCR of Transfected BCL-2 DNA Constructs—Total RNA was Trizol-extracted (Sigma) from 293T cells that had been transiently transfected with dicistronic DNA test constructs. RNA was DNase I-treated for 15 min at room temperature, and RT-PCR was then performed by using an upstream primer just downstream from the CMV promoter translation initiation site, 5′-cagatcactagaagctttattgcg-3′, and a downstream primer just inside the FLuc ORF, 5′-tctcttcatagccttatgcagttgc-3′. Control PCRs using DNA as template were not DNase I-treated. Transient Transfection of DNA and mRNA—DNA constructs were transiently transfected into cells using FuGENE 6 reagent (Roche Applied Science). Cells were plated in 12-well plates at 1.25 × 105 cells/well 16 h prior to transfection. The ratio of FuGENE/DNA was 6 μl:2 μg, and transfections were performed in Dulbecco's modified Eagle's medium + 10% fetal bovine serum. Cells were lysed 8 h post-transfection and assayed for both RLuc and FLuc activities. Transient transfection of in vitro transcribed mRNA was achieved by first cloning a 35-bp poly(A)-containing sequence into all reporter constructs at the unique ApaI site immediately downstream from FLuc. This sequence included a unique AgeI site at its 3′ end. Each construct was linearized by digesting with AgeI (except for pRL-HCV-FL and pRL-revH-FL, which were linearized with XhoI), followed by phenol/CHCL3/isoamyl extraction and isopropyl alcohol precipitation. Transcription was performed as described above. Cells were plated at 1.25 × 105 cells/well in 12-well plates and attached overnight. Transfection proceeded using 1.5 μg/well mRNA with 8 μl of DMRIE C reagent (Invitrogen). pSV40-Bgal DNA was co-transfected with monocistronic RNAs at 0.1 μg/well to control for transfection efficiency. Serum was re-added 3 h post-transfection. Luciferase analysis was as described above. β-Galactosidase was quantified using the Beta-Glo™ Assay Reagent (Promega). Treatment with various stress-inducing agents was maintained during transfection. Etopo and Na Ars were both obtained from Sigma. RNA Analysis—For in vivo assays, additional wells were simultaneously transfected and treated identically for analysis of post-transfection reporter mRNA integrity. Six hours post-transfection, total RNA was harvested with 300 μl of Trizol reagent (Sigma). Purified total RNA was denatured using established glyoxal/Me2SO methodology and 2.5 μg of RNA/well run in a 1% NaHPO4-buffered gel. RNA was then transferred to Hybond N+ nylon membrane (Amersham Biosciences) and exposed to Kodak X-OMAT™ film. Northern analysis to assess in vivo expression of pRL-BCL2-FL was performed by transferring RNA to nylon (as described above) and then pre-hybridized with 10 ml of Ultra Hyb™ solution for 1 h (Ambion). A 489-bp FLuc-specific riboprobe was transcribed in vitro using SP6 polymerase, along with 10 μCi of [α-32P]CTP. 1 × 106 cpm of riboprobe was hybridized for 12 h at 68 °C, followed by washing in 2× SCC and autoradiography. Immunoblotting for BCL2—Cells were lysed in RIPA buffer, and 40 μg of total protein was mixed 1:1 with 4× SDS-PAGE sample buffer, boiled 5 min, and then resolved via 12% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride, blocked 1 h in TBST + 3% milk, then probed with 1:1000 anti-BCL-2 monoclonal antibody (clone SC-7382, Santa Cruz Biotechnology) overnight at 4 °C, followed by a horseradish peroxidase-conjugated anti-mouse secondary F(ab′)2 fragment (Jackson ImmunoResearch). Normalization for total protein loading was accomplished by using a monoclonal antibody specific for α-tubulin (Sigma). The predominant BCL-2 promoter (P1) produces a 5′-UTR of 1.45 kb that contains numerous structural features that likely regulate BCL-2 translation. For example, an extremely stable secondary structure (ΔG = –530 kcal/mol) as well as multiple potential uORFs would be expected to largely inhibit conventional ribosomal scanning (Fig. 1A) (41Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10315) Google Scholar). Indeed, a uORF that spans –119 to –84 bp upstream from the start codon has been reported to inhibit significantly BCL-2 translation, presumably by causing a fraction of scanning ribosomes to translate the uORF, and then disengage from the mRNA (29Harigai M. Miyashita T. Hanada M. Reed J.C. Oncogene. 1996; 12: 1369-1374PubMed Google Scholar). The region of the BCL-2 5′-UTR that was examined for this study extended 1146 bp upstream from the start codon and included the alternatively spliced 219-bp intron (Fig. 1B). This sequence was inserted into several reporter constructs in order to assess potential IRES activity, including a dicistronic construct that placed the BCL-2 5′-UTR between an upstream RLuc reporter gene and a downstream FLuc gene (Fig. 2A). The upstream RLuc cistron was immediately followed by two "inframe" stop codons that inhibit ribosomal reinitiation. This dicistronic DNA reporter assay is commonly utilized to assess potential IRES activity because cap-dependent translation of RLuc not only restricts cap-dependent translation of the downstream FLuc but also acts as an internal control to measure relative increases in FLuc expression. A positive control for IRES activity included a 426-bp region containing the HCV IRES between the reporter genes (pRL-HCV-FL), whereas the negative control contained the HCV IRES in an inverted orientation (pRL-revH-FL). For in vitro assays, the negative control (pRL-FL) contained a 27-bp spacer sequence between the cistrons. The 5′-UTR of BCL-2 Functions as an IRES in Vitro—As an initial test for IRES activity, test constructs containing the 5′-UTR of BCL-2 were transcribed in vitro in the presence of 7-methyl-G(5′)ppp(5′)guanosine cap analog (m7G). Equimolar amounts of transcripts were then used to program translation in nuclease-treated rabbit reticulocyte lysates (RRL) (Fig. 2B). The empty vector control (Fig. 2B, pRL-FL, lane 1) efficiently translated the upstream RLuc cistron, yet little translation of the second cistron (Fluc) was observed. Similar to previous reports, insertion of the HCV IRES (Fig. 2B, pRL-HCV-FL, lane 2) stimulated expression of downstream FLuc, indicative of IRES-mediated translation. Likewise, expression of FLuc also increased downstream from the BCL-2 5′-UTR (pRL-BCL2-FL, lane 5), suggesting that this region supports cap-independent translation, and may contain an IRES. Insertion of a sequence that forms a stable RNA hairpin structure (ΔG = –60 kcal/mol) upstream of RLuc (Fig. 2B, phpRL-BCL2-FL, lane 6) inhibited cap-dependent translation by ∼98%, yet significant expression of the second cistron (FLuc) persisted. This indicated that increased translation of FLuc mediated by the BCL-2 5′-UTR was not due to an increase in ribosomal re-initiation. Similar results were obtained by using monocistronic RNAs. Translation of the monocistronic pBCL2-FL (Fig. 2B, lane 3) demonstrated that the BCL-2 5′-UTR supported ample translation of FLuc, although at a significantly lower level than constructs containing RLuc preceded by only a short leader sequence (Fig. 2B, lanes 1, 2, and 5) or FLuc alone (data not shown). Insertion of the stable RNA hairpin structure in front of the BCL-2 5′-UTR (Fig. 2B, phpBCL2-FL, lane 4) inhibited its ability to mediate FLuc translation by ∼65% but far less than the 98% inhibition of RLuc translation observed when this hairpin was inserted into phpRL-BCL2-FL. Together, these data strongly indicated that the 5′-UTR of BCL-2 could support cap-independent translation, possibly through IRES-mediated recruitment. In Vivo Assessment of BCL-2 IRES Function in Cells Transfected with DNA Constructs—We sought to confirm our in vitro results by transfecting DNA constructs containing the BCL-2 5′-UTR into 293T kidney cells. Following transfection, a similar increase in FLuc:RLuc ratio above background was observed for both pRL-BCL2-FL and pRL-HCV-FL (Fig. 3A), suggesting that the BCL-2 5′-UTR contained an IRES of comparable strength to the HCV IRES. HeLa S3 and HepG2 cells were likewise transfected with these constructs, and similar results were obtained (results not shown). To test whether this apparent BCL-2 IRES activity was the result of monocistronic transcripts being generated from a promoter within the BCL-2 5′-UTR, a DNA construct lacking the cytomegalovirus (CMV) promoter was created [p(ΔCMV)RL-BCL2-FL]. Upon its transfection into 293T cells, no RLuc or FLuc activity was detected, demonstrating negligible promoter activity (Fig. 3A, bottom panel). Next, we utilized several procedures to rigorously evaluate the transcripts produced from pRL-BCL2-FL in vivo. First, we used a new RNAi method to test for spliced transcripts derived from the dicistronic vector (54Breitschopf K. Haendeler J. Malchow P. Zeiher A.M. Dimmeler S. Mol. Cell. Biol. 2000; 20: 1886-1896Crossref PubMed Scopus (290) Google Scholar). An RNAi vector targeted against RLuc was transfected into cells, resulting in silencing of RLuc expression by ∼80% (results not shown), in accordance with previous observations (40Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar). RLuc-directed RNAi in the cells containing pRL-HCV-FL resulted in equal inhibition of RLuc and FLuc, suggesting the presence of only a single dicistronic transcript (Fig. 3B). However, aberrantly spliced monocistronic transcripts coding only for FLuc would not be subject to the same RLuc-specific RNAi silencing. When the RNAi vector was co-transfected with pRL-BCL2-FL, FLuc translation decreased but was 2.3-fold greater than RLuc, suggesting the presence of monocistronic FLuc transcripts (Fig. 3B). Similar results were obtained using a construct containing the XIAP IRES (pRL-XIAP-FL), which was recently demonstrated to contain a splice acceptor (Fig. 3B) (40Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar). To confirm potential splicing, nonquantitative RT-PCR was performed on total RNA isolated from 293T cells that had been transiently transfected with pRL-BCL2-FL (Fig. 3C). PCR primers were selected to amplify the region between the transcription start site of the CMV promoter to the FLuc ORF, such that control DNA PCR fragments for pRL-HCV-FL (1.9 kb) and pRL-BCL2-FL (2.4 kb) were expected (Fig. 3C, lanes 2 and 5). RT-PCR products were anticipated to be ∼200 bp smaller due to splicing of the chimeric intron (Fig. 3C, lanes 4 and 6). pRL-HCV-FL-transfected cells produced an RT-PCR product of 1.7 kb, indicating an intact dicistronic transcript. However, cells transfected with pRL-BCL2-FL produced not only a 2.2-kb PCR product representing a dicistronic transcript (visible only upon overexposure, data not shown), but also a smaller product of 350 bp (Fig. 3C). Sequencing showed that the smaller 350-bp product represented a monocistronic FLuc transcript with a 290-bp 5′-UTR. Finally, Northern analysis confirmed the presence of both transcripts (Fig. 3D). Although the dicistronic transcript (∼4.7 kb) was the predominant species, a significant portion of FLuc expression had resulted from the production of the smaller, monocistronic transcript. Therefore, these data showed that pRL-BCL2-FL generated monocistronic transcripts containing only FLuc in vivo, and thus IRES function within the BCL-2 5′-UTR could not be accurately quantified via DNA transfection. Although Northern analysis confirmed the presence of two different transcripts in this instance, the more sensitive techniques described here can detect low level aberrant splicing that is below the sensitivity threshold of Northern blotting (40Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar). The BCL-2 5′-UTR Functions as an IRES in Vivo—We chose to assess potential BCL-2 IRES activity by directly transfecting RNA. All DNA constructs were modified such that in vitro transcription would create transcripts containing a 35-bp poly(A) tail (see "Experimental Procedures"). These constructs (Fig. 4A) were transcribed in vitro in the presence of the m7G cap analog, and the resulting trans
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