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The Internal Ribosome Entry Site (IRES) Contained within the RNA-binding Motif Protein 3 (Rbm3) mRNA Is Composed of Functionally Distinct Elements

2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês

10.1074/jbc.m303495200

1083-351X

Stephen A. Chappell, Vincent P. Mauro,

RNA Research and Splicing

Although the internal ribosome entry sites (IRESes) of viral mRNAs are highly structured and comprise several hundred nucleotides, there is a variety of evidence indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. In this study, we performed deletion and mutational analyses of an IRES contained within the 720-nucleotide (nt) 5′ leader of the Rbm3 mRNA and demonstrated that this IRES is highly modular, with at least 9 discrete cis-acting sequences. These cis-acting sequences include a 22-nt IRES module, a 10-nt enhancer, and 2 inhibitory sequences. The 22-nt sequence was shown to function as an IRES when tested in isolation, and we demonstrated that it did not enhance translation by functioning as a transcriptional promoter, enhancer, or splice site. The activities of all 4 cis-acting sequences were further confirmed by their mutation in the context of the full IRES. Interestingly, one of the inhibitory cis-acting sequences is contained within an upstream open reading frame (uORF), and its activity seems to be masked by translation of this uORF. Binding studies revealed that all 4 cis-acting sequences could bind specifically to distinct cytoplasmic proteins. In addition, the 22-nt IRES module was shown to bind specifically to 40 S ribosomal subunits. The results demonstrate that different types of cis-acting sequences mediate or modulate translation of the Rbm3 mRNA and suggest that one of the IRES modules contained within the 5′ leader facilitates translation initiation by binding directly to 40 S ribosomal subunits. Although the internal ribosome entry sites (IRESes) of viral mRNAs are highly structured and comprise several hundred nucleotides, there is a variety of evidence indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. In this study, we performed deletion and mutational analyses of an IRES contained within the 720-nucleotide (nt) 5′ leader of the Rbm3 mRNA and demonstrated that this IRES is highly modular, with at least 9 discrete cis-acting sequences. These cis-acting sequences include a 22-nt IRES module, a 10-nt enhancer, and 2 inhibitory sequences. The 22-nt sequence was shown to function as an IRES when tested in isolation, and we demonstrated that it did not enhance translation by functioning as a transcriptional promoter, enhancer, or splice site. The activities of all 4 cis-acting sequences were further confirmed by their mutation in the context of the full IRES. Interestingly, one of the inhibitory cis-acting sequences is contained within an upstream open reading frame (uORF), and its activity seems to be masked by translation of this uORF. Binding studies revealed that all 4 cis-acting sequences could bind specifically to distinct cytoplasmic proteins. In addition, the 22-nt IRES module was shown to bind specifically to 40 S ribosomal subunits. The results demonstrate that different types of cis-acting sequences mediate or modulate translation of the Rbm3 mRNA and suggest that one of the IRES modules contained within the 5′ leader facilitates translation initiation by binding directly to 40 S ribosomal subunits. Translation of eukaryotic mRNAs begins with recruitment of the translation machinery at either the 5′-m7G cap structure or an internal ribosome entry site (IRES) 1The abbreviations used are: IRES, internal ribosome entry site; nt, nucleotide; uORF, upstream open reading frame; SV40, simian virus 40; EMCV, encephalomyocarditis virus; RNP, ribonucleoprotein; CAT, chloramphenicol actyltransferase.1The abbreviations used are: IRES, internal ribosome entry site; nt, nucleotide; uORF, upstream open reading frame; SV40, simian virus 40; EMCV, encephalomyocarditis virus; RNP, ribonucleoprotein; CAT, chloramphenicol actyltransferase. (reviewed in Refs. 1Pestova T.V. Kolupaeva V.G. Lomakin I.B. Pilipenko E.V. Shatsky I.N. Agol V.I. Hellen C.U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7029-7036Crossref PubMed Scopus (599) Google Scholar and 2Vagner S. Galy B. Pyronnet S. EMBO Rep. 2001; 2: 893-898Crossref PubMed Scopus (236) Google Scholar). The cap-dependent mechanism for initiating translation is generally thought to be more common; however, the number of mRNAs reported to initiate translation internally is growing (3Bonnal S. Boutonnet C. Prado-Lourenco L. Vagner S. Nucleic Acids Res. 2003; 31: 427-428Crossref PubMed Scopus (76) Google Scholar). Internal initiation seems to facilitate the translation of particular viral and cellular mRNAs under conditions or subcellular locations that render the cap-dependent mechanism less efficient, for example, during the G2/M phase of the cell cycle, under conditions of mild hypothermia, or in dendrites (4Pyronnet S. Pradayrol L. Sonenberg N. Mol. 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Springer-Verlag, Berlin1995: 203Google Scholar), various lines of evidence indicate that they comprise a diverse group of sequences that may recruit the translation machinery by a variety of mechanisms (1Pestova T.V. Kolupaeva V.G. Lomakin I.B. Pilipenko E.V. Shatsky I.N. Agol V.I. Hellen C.U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7029-7036Crossref PubMed Scopus (599) Google Scholar, 2Vagner S. Galy B. Pyronnet S. EMBO Rep. 2001; 2: 893-898Crossref PubMed Scopus (236) Google Scholar). Accumulated data also suggest that there are intrinsic differences between viral and cellular IRESes. Picornavirus IRESes, for example, seem to be highly structured, well defined functional entities of up to several hundred nucleotides. These IRESes have been categorized on the basis of sequence and structural similarities, and some of these features have been shown to be functionally important (9Jackson R.J. Kaminski A. RNA (N. Y.). 1995; 1: 985-1000PubMed Google Scholar). In contrast, cellular IRESes do not seem to contain conserved secondary structures, and their sequences are not obviously similar to each other or to viral IRESes. A feature noted in some cellular but not in viral IRESes is that they seem to be composed of shorter elements that can function independently when tested in isolation (10Chappell S.A. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1536-1541Crossref PubMed Scopus (206) Google Scholar, 11Zhou W. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1531-1536Crossref PubMed Scopus (59) Google Scholar, 12Mauro V.P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12031-12036Crossref PubMed Scopus (196) Google Scholar). The modular nature of some cellular IRESes is suggested by numerous observations. For example, it has been difficult to define discrete 5′ and 3′ boundaries for particular cellular IRESes. Moreover, the activity of some cellular IRESes seems to be contained within two or more nonoverlapping fragments (e.g. Refs. 13Yang Q. Sarnow P. Nucleic Acids Res. 1997; 25: 2800-2807Crossref PubMed Scopus (86) Google Scholar, 14Stoneley M. Paulin F.E.M. Le Quesne J.P.C. Chappell S.A. Willis A.E. Oncogene. 1998; 16: 423-428Crossref PubMed Scopus (284) Google Scholar, 15Huez I. Creancier L. Audigier S. Gensac M.-C. Prats A.-C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (244) Google Scholar, 16Gan W. La Celle M. Rhoads R.E. J. Biol. Chem. 1998; 273: 5006-5012Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17Bernstein J. Sella O. Le S.-Y. Elroy-Stein O. J. Biol. Chem. 1997; 272: 9356-9362Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In our analysis of the Gtx 5′ leader, we identified four nonoverlapping fragments that functioned as IRESes (10Chappell S.A. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1536-1541Crossref PubMed Scopus (206) Google Scholar), and the activity of one of these fragments was contained within a 9-nt segment. Other short nucleotide sequences that function as IRESes include two cis-acting sequences selected from libraries of random nucleotides (18Owens G.C. Chappell S.A. Mauro V.P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1471-1476Crossref PubMed Scopus (48) Google Scholar) and short GA-rich repeats, which were shown to function as IRESes in both plants and animals (19Dorokhov Y.L. Skulachev M.V. Ivanov P.A. Zvereva S.D. Tjulkina L.G. Merits A. Gleba Y.Y. Hohn T. Atabekov J.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5301-5306Crossref PubMed Scopus (108) Google Scholar). The 9-nt Gtx IRES module is complementary to a segment of 18 S rRNA, and we previously showed that this sequence could bind directly to 40 S ribosomal subunits by base pairing to its rRNA complement (20Hu M.C.-Y. Tranque P. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1339-1344Crossref PubMed Scopus (67) Google Scholar). In other studies, we noted that many cellular mRNAs contain segments with complementarity to rRNA (21Mauro V.P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 422-427Crossref PubMed Scopus (55) Google Scholar) and showed that numerous short complementary matches also occur in both cellular and synthetic IRESes (6Chappell S.A. Owens G.C. Mauro V.P. J. Biol. Chem. 2001; 276: 36917-36922Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Zhou W. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1531-1536Crossref PubMed Scopus (59) Google Scholar, 18Owens G.C. Chappell S.A. Mauro V.P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1471-1476Crossref PubMed Scopus (48) Google Scholar). Other studies showed that complementary mRNA sequences could bind to ribosomes and affect the translation of the host mRNA (e.g. Refs. 22Tranque P. Hu M.C.-Y. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12238-12243Crossref PubMed Scopus (56) Google Scholar and 23Verrier S.B. Jean-Jean O. RNA (N. Y.). 2000; 6: 584-597Crossref PubMed Scopus (21) Google Scholar). These observations prompted the ribosome filter hypothesis, which postulates that the ribosomal subunits themselves are regulatory elements that modulate patterns of protein expression by reducing the translation of some mRNAs while enhancing that of others (12Mauro V.P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12031-12036Crossref PubMed Scopus (196) Google Scholar). In addition to recruiting ribosomes directly, we expect that some IRES elements might recruit ribosomes indirectly by binding to initiation factors or other trans-factors. In a previous study, we noted that a 720-nt 5′ leader of the cold stress-induced Rbm3 mRNA, which encodes a putative RNA-binding protein, contains 13 uORFs (6Chappell S.A. Owens G.C. Mauro V.P. J. Biol. Chem. 2001; 276: 36917-36922Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Although uORFs should block translation of the main ORF (24Meijer H.A. Thomas A.A. Biochem. J. 2002; 367: 1-11Crossref PubMed Scopus (253) Google Scholar, 25Geballe A.P. Sachs M.S. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 595-614Google Scholar), translation of a reporter mRNA containing the Rbm3 5′ leader was relatively efficient. The identification of an IRES within this 5′ leader suggested a mechanism by which the uORFs were bypassed. In our initial characterization of this IRES, its activity was confirmed by using a number of criteria, both in cells and in cell-free lysates. The notion that short nucleotide motifs may be key elements controlling the activity of some cellular IRESes is further developed in the present study. DNA Constructs—Reporter constructs were based on vectors that use the simian virus 40 (SV40) promoter to transcribe a dicistronic mRNA that encodes Renilla and Photinus luciferases as the first and second cistrons, respectively. These vectors were kindly provided by Dr. Anne E. Willis (14Stoneley M. Paulin F.E.M. Le Quesne J.P.C. Chappell S.A. Willis A.E. Oncogene. 1998; 16: 423-428Crossref PubMed Scopus (284) Google Scholar), who subsequently renamed them pRF and phpRF (hairpin). For consistency with our previous publications, we use the alternative nomenclature of RP and RPh, respectively. Fragments of the Rbm3 5′ leader were generated by PCR amplification using Pfu DNA polymerase with either the full-length Rbm3 5′ leader or oligonucleotides as templates and cloned into the intercistronic region of the RP or RPh vectors. Amplification oligonucleotides for cloning into RP contained EcoRI and NcoI restriction sites, whereas oligonucleotide templates for cloning into RPh contained SpeI and EcoRI. The 5′-nucleotides flanking the Rbm3 sequences in the 5′ deletion series are ACUAGU. In some deletions, the 5′-nucleotide of the Rbm3 sequence is U and overlaps with the 3′-nucleotide from the 5′-flanking sequence. Oligonucleotides containing various combinations of the AclI, HindIII, and NcoI restriction sites were used to introduce mutations into the full-length Rbm3 5′ leader by replacement of wild-type sequences. To generate promoterless dicistronic constructs, the SV40 promoter was excised using BglII and BlnI restriction sites. Particular fragments and mutations of the Rbm3 5′ leader were also tested in a monocistronic context 5′ of the Photinus luciferase cistron. mRNA levels in these constructs were estimated by using a second cistron which encodes a synthetic Renilla luciferase protein (R′, Promega) expressed via the IRES of the encephalomyocarditis virus (EMCV) (P(EMCV)R′). This construct was modified from the P(EMCV)-CAT construct (6Chappell S.A. Owens G.C. Mauro V.P. J. Biol. Chem. 2001; 276: 36917-36922Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) by replacement of the second cistron (CAT) with the synthetic Renilla luciferase. Transfection and Cell-free Analyses—Reporter constructs (0.5 μg) were transfected into cells (1 × 105) using FuGENE 6 (Roche Applied Science). Cell lines were mouse neuroblastoma N2a, rat glial tumor C6, mouse fibroblast NIH 3T3 (3T3), and human neuroblastoma SK-N-SH (SK). Transfection efficiencies were normalized by cotransfection with 0.2 μg of a LacZ reporter gene construct (pCMVβ, CLONTECH). Cells were harvested 24 h after transfection, and reporter gene activities were determined (10Chappell S.A. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1536-1541Crossref PubMed Scopus (206) Google Scholar). Note that, in this paper, the Photinus:Renilla luciferase expression ratio is referred to as IRES activity, even for putative cis-acting sequences. For cell-free translation studies, capped dicistronic mRNAs were transcribed by using the mMESSAGE mMACHINE T7 transcription kit (Ambion, Austin, TX). Translation reactions were performed by using 0.5 μg of mRNA in the presence or absence of 0.2 mm cap analogue (m7G(5′)ppp(5′)G, Roche Diagnostics) in C6 cell-free lysates (6Chappell S.A. Owens G.C. Mauro V.P. J. Biol. Chem. 2001; 276: 36917-36922Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 20Hu M.C.-Y. Tranque P. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1339-1344Crossref PubMed Scopus (67) Google Scholar). mRNA integrity and size after translation were determined by Northern blot analyses (26Stoneley M. Chappell S.A. Jopling C.L. Dickens M. MacFarlane M. Willis A.E. Mol. Cell. Biol. 2000; 20: 1162-1169Crossref PubMed Scopus (194) Google Scholar) using a Photinus luciferase riboprobe. Electrophoretic Gel Mobility Shift and Nitrocellulose Filter Binding Analyses—RNA oligonucleotides (Dharmacon Research Inc.) were 5′-end-labeled using [γ-32P]ATP with T4 polynucleotide kinase and tested in electrophoretic mobility gel shift assays based on the method previously described (27Paulin F.E.M. Chappell S.A. Willis A.E. Nucleic Acids Res. 1998; 26: 3097-3103Crossref PubMed Scopus (24) Google Scholar) using cytoplasmic extracts prepared from N2a, C6, or 3T3 cells (20Hu M.C.-Y. Tranque P. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1339-1344Crossref PubMed Scopus (67) Google Scholar). Labeled RNA oligonucleotides corresponding to cis-acting sequences with complementarity to 18 S rRNA were tested for their ability to bind to puromycin-dissociated N2a ribosomes and purified C6 40 S ribosomal subunits (28Blobel G. Sabatini D. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 390-394Crossref PubMed Scopus (381) Google Scholar) in nitrocellulose filter binding assays (based on Ref. 29Wong I. Lohman T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5428-5432Crossref PubMed Scopus (361) Google Scholar) using 1.25 mm MgCl2 in the binding buffer. Nonspecific competitor RNA (SI/SIII) was based on mouse β-globin 5′ leader sequences and poly(A) (10Chappell S.A. Edelman G.M. Mauro V.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1536-1541Crossref PubMed Scopus (206) Google Scholar). The relative amounts of probe retained on nitrocellulose membranes was quantified with a Phosphor-Imager (Amersham Biosciences). The Rbm3 IRES Contains Numerous Segments That Function Independently and Contribute to Overall Activity—To determine whether the Rbm3 IRES contains a discrete 5′ boundary, the full-length Rbm3 5′ leader and sequential deletions of ∼100 nucleotides from its 5′ end were tested in dicistronic constructs in four cell lines (Fig. 1). In the cell lines tested, the IRES activity of the Rbm3 5′ leader was up to ∼50-fold over the background level of activity obtained with the parent construct RP. Sequential deletions of this 5′ leader progressively reduced IRES activity and indicated that the Rbm3 IRES does not contain a discrete 5′ boundary; this suggests that IRES activity is derived from the summation of a series of shorter functional elements that are distributed throughout the Rbm3 5′ leader rather than from a single, well defined functional entity. This suggested modular composition was further investigated by arbitrarily fragmenting the 720-nt 5′ leader into seven segments (Fig. 1, I–VII) and testing these for IRES activity. Six of the fragments had IRES activity at least 2-fold over background in at least one cell line. In addition, the activities of two of the fragments (V and VII) varied in the different cell lines. Both were active in SK cells and inactive in C6 and 3T3 cells; however, in N2a cells, fragment V was active and VII was inactive. Fragment III was the most active segment in all four cell lines, and in two cell lines (N2a and SK) it was more active than the full IRES. In a previous publication, we showed that the 5′ leader of the Rbm3 mRNA functioned as an IRES in a cell-free lysate and that the activities obtained were not caused by the production of monocistronic mRNAs corresponding to the second cistron (6Chappell S.A. Owens G.C. Mauro V.P. J. Biol. Chem. 2001; 276: 36917-36922Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In this study, we noted that the activity of one of the 100-nt fragments (III) was greater than that of the full-length 5′ leader in N2a and SK cells. We sought to fine-map the cis-acting elements contained within this fragment; however, before proceeding, we performed an additional control. To ensure that the enhanced activity obtained with the construct containing fragment III was not caused by promoter activity leading to the production of monocistronic Photinus luciferase mRNAs, we deleted the SV40 promoter from the Rbm3(III)/RP construct (Fig. 2A). After transfection into N2a cells, the activities of both cistrons were reduced to a background level compared with the activities obtained from the Rbm3(III)/RP construct containing the SV40 promoter, demonstrating that the activities obtained from fragment III were not caused by promoter activity. To begin to identify cis-acting sequences within fragment III, it was deleted and fragmented at intervals of ∼20 nucleotides (Fig. 2B). As in our original characterization of the Rbm3 IRES, we included a hairpin structure in the 5′ leader of these constructs to minimize the contribution of the first cistron through reinitiation or leaky scanning. In these experiments, one of the 20-nt segments (III-g) had a level of IRES activity ∼5-fold over the background level of activity obtained with the parent construct RPh, but its activity was enhanced dramatically by nucleotide sequences located immediately 3′ of it (Fig. 2B, compare fragments III-g to III-c), even though the 3′ sequences themselves (III-d) had no detectable IRES activity. These results may indicate that fragment III-g contains a truncated IRES module; alternatively, the nucleotides 3′ of III-g contain an independent cis-acting sequence that enhances the activity of the IRES module. A 100-nt Fragment Contains Four cis-Acting Sequences That Affect IRES Activity—Sequential deletion of fragment III in steps of 1–5 nucleotides in a 5′ to 3′ direction revealed two activities (Fig. 3A, constructs d1–d14). The first, observed with deletion d4, resulted in an almost complete loss of IRES activity, although activity was recovered by deletion of an additional 4 nucleotides. Deletion d4 destroyed the putative initiation codon of a 15-nt uORF; the effects of this uORF on translation are addressed later. The second activity, observed with deletion d8, resulted in diminished IRES activity and localized the 5′ boundary of a putative IRES module to nucleotide U–286. Deletion of the next five nucleotides (d10) decreased IRES activity further, and activity was completely lost with deletion d11 (nucleotide U–277). The 3′ boundary of the cis-acting sequence defined by the 5′ deletions d8–d11 was determined by mutating 3′ sequences to adenosine. The use of mutations rather than deletions avoids any changes in activity related to the spacing of cis-acting sequences relative to the downstream cistron. Sequential 3′ to 5′ mutations of fragment d6 (Fig. 3A, m1–m12) localized the 3′ boundary of a putative IRES module to nucleotide A–265 (m2), with a level of activity ∼8-fold over background. We refer to the segment defined by nucleotides U–286 to A–265 as the putative 22-nt IRES module. Mutation of fragment III also revealed two other activities. An inhibitory sequence is contained between nucleotides C–228 and U–242, and mutation of these nucleotides increased IRES activity from ∼40-fold to ∼90-fold over background (Fig. 3A, compare d6 to m10). The 3′ boundary of a second cis-acting sequence was localized 16 nucleotides 3′ of the putative 22-nt IRES module to G–249 (m8). These 16 nucleotides enhanced IRES activity more than 9-fold (Fig. 3A, compare m2 to m8), even though they did not have detectable IRES activity by themselves (d14). To address whether these 16 nucleotides represent the 3′ end of a putative IRES module or contain a discrete enhancer element, nucleotides located immediately 3′ of the putative IRES module were sequentially mutated to adenosine (m13–m19). Mutation of the first 7 nucleotides (m13–m16) diminished IRES activity somewhat from ∼40-fold to ∼30-fold over background; however, activity was still enhanced relative to that of the putative IRES module (Fig. 3A, compare m16 to m2). These results suggest the existence of a discrete enhancer element with a 5′ boundary at nucleotide G–257 (m16). To investigate this possibility further, the spacing between the putative IRES module and enhancer sequences was varied by using poly(A). Mutation of the 7 nucleotides between the putative IRES module and enhancer sequences (m20) resulted in a level of IRES activity comparable with that of m16, even though these constructs differ in their 3′ sequences: m20 contains a stretch of poly(A), and m16 contains Rbm3 sequences. When the putative IRES module was spaced 9, 11, or 19 nucleotides from the sequences with enhancer activity (m21–m23, respectively), IRES activity was still enhanced relative to the activity of the putative IRES module alone (m2 or m19), indicating that the IRES and enhancer activities are separable. However, these activities were not completely independent, as the enhancer did not function when tested 5′ of the putative IRES module (data not shown). Therefore, we refer to this segment as the 3′ enhancer element. The 22-nt Putative IRES Module Functions Independently—To control for the possibility that deletions and mutations generated an IRES element (for example, by juxtaposition of intercistronic and Rbm3 sequences), we tested the putative 22-nt IRES module in isolation and observed that this sequence also seemed to function as an IRES. Its activity was 115-fold over background in N2a cells (Fig. 3A, 22-nt), 86-fold in C6 cells, 95-fold in 3T3 cells, and 46-fold in SK cells. The activity of the 22-nt IRES module is up to ∼15-fold greater than that of the EMCV IRES. In addition, these activities were greater than those observed for the full-length IRES or those expected from the results of the deletion and mutational analyses. The results suggest that the activity of the putative 22-nt IRES module may be affected by flanking sequences or by its spacing relative to the Photinus luciferase initiation codon, both of which differ in these particular constructs. In summary, deletions and mutations of fragment III identified four cis-acting sequences that affected its translation, which together are schematically represented in Fig. 3B. The 22-nt Putative IRES Module Is Confirmed to Be an IRES by Various Criteria—To validate that the 22-nt cis-acting sequence functions as an IRES, we excluded other possibilities that might yield similar results, i.e. the possibility that this sequence functions as a transcriptional promoter, enhancer, or splice site that leads to the production of monocistronic second cistron mRNAs. The possibility of promoter or enhancer activities was considered unlikely because the 22-nt cis-acting sequence accounts for the activity of fragment III, and fragment III was shown not to function as a promoter in Fig. 2A. However, to further investigate this possibility, we also showed that the activity of the 22-nt cis-acting sequence depended on the production of the dicistronic mRNA (data not shown). In addition, we tested this sequence in a cell-free translation reaction (Fig. 4), which also addressed the possibility that the putative 22-nt IRES module might generate monocistronic Photinus luciferase mRNAs by functioning as a splice site that results in excision of the first cistron. For these experiments, dicistronic mRNAs were transcribed and capped in vitro and translated in cell-free lysates that lack nuclear splicing factors. The 22-nt cis-acting sequence increased translation of the second cistron relative to a control mRNA (RP) lacking these sequences by ∼70-fold. This translation was cap-independent because it was not blocked by the presence of a cap analogue, which decreased the translation of the first cistron by ∼80% (Fig. 4A). These results also indicate that the 22-nt sequence does not facilitate translation of the second cistron by mechanisms that depend on translation of the first cistron (e.g. reinitiation). In fact, when translation of the first cistron was blocked, second cistron expression actually increased by ∼1.8-fold. Nuclease hypersensitivity or splicing activities in these lysates were monitored by Northern analysis of the dicistronic reporter mRNAs after in vitro translation (Fig. 4B). The results showed no obvious differences in either mRNA integrity or size after 60 min of incubation. Taken together, the observations from the transfection and cell-free studies confirm that the 22-nt cis-acting sequence functions as an IRES. Translation of a uORF Appears to Mask an Inhibitory cisActing Sequence—The presence of a fourth cis-sequence, located upstream of the IRES module, was suggested from deletion d4 (Fig. 3A). This deletion destroyed a putative initiation codon for a 15-nt uORF and led to an almost complete loss of IRES activity. However, deletion of the next 4 nucleotides (d5) restored activity, suggesting that translation of this uORF was not required for activity but rather masked an inhibitory sequence that overlaps the first 5 nucleotides. To investigate this possibility, this uORF was disrupted by mutating its initiation codon from AUG to UUG, CUG, AAG, or AUA (Fig. 5A, m24–m27, respectively) and tested in the intercistronic region of a dicistronic mRNA in N2a cells. In this context, all four mutations inhibited IRES activity by ∼60–80%, consistent with the notion that translation of the uORF masks an inhibitory activity. However, using dicistronic mRNAs, it is not possible to distinguish between a loss of IRES activity and an inhibition of translation, because the second cistron is translated at a background level in the absence of an IRES. To distinguish between these possibilities, we tested the IRES sequences in the 5′ leader of a monocistronic mRNA, which is translated at a high level even in the absence of the IRES (Fig. 5B, m28–m31). In this context, fragment III increased translation by ∼30% relative to the parent cons