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Originally published In Press as doi:10.1074/jbc.M405885200 on August 31, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47419-47430, November 12, 2004
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Structurally Distinct Elements Mediate Internal Ribosome Entry within the 5'-Noncoding Region of a Voltage-gated Potassium Channel mRNA*

Gwendolyn M. Jang{ddagger}§, Louis E.-C. Leong{ddagger}, Lily T. Hoang{ddagger}, Ping H. Wang||, George A. Gutman{ddagger}, and Bert L. Semler{ddagger}**

From the Departments of {ddagger}Microbiology and Molecular Genetics and ||Medicine, College of Medicine, University of California, Irvine, California 92697

Received for publication, May 26, 2004 , and in revised form, August 31, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ~1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream ~1.0 kb and the downstream ~0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most ~0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops {Psi} and {Omega}) in the presence and absence of the upstream ~1.0 kb. Although the systematic deletion of sequences within the 3'-most ~0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Potassium channels are ubiquitously expressed in nature, facilitating diverse cellular processes in response to intra- and extracellular changes (1). Voltage-gated potassium (Kv)1 channels modulate action potentials in electrically excitable cells and facilitate cell proliferation, cell volume regulation, and secretion in non-electrically excitable cells. Kv channel genes encode a single {alpha}-subunit that forms potassium-selective pores through the assembly of tetramers. Physiological stimulation during stress regulates transcription through the release of hormones and neurotransmitters (2). Similar changes are also observed in response to pharmacological agents and with the onset of pathological conditions. Post-translational modifications, interactions with auxiliary {beta}-subunits, and heterotetramerization between subfamily members may alter channel properties, promote cell-surface expression, and/or modify channel localization (1, 3).

Many mammalian Kv channel mRNA species contain extensive 5'- and 3'-noncoding regions (NCRs) (4). The NCRs of many eukaryotic mRNAs have been shown to regulate gene expression by affecting mRNA localization, mRNA stability, and translation. Although the roles of the 5'- and 3'-NCRs in Kv channel expression have yet to be investigated in detail, the presence of unusually long 5'-NCRs in this gene family raised the possibility that such sequences may mediate and perhaps modulate translation.

Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, forms rapidly activating and inactivating A-type channels (5–7) and has been determined to have cell type- and species-specific patterns of expression (8–10). Two distinct Kv1.4 mRNA species, ~3.5 and ~4.5 kb in length, are expressed in mouse brain and cardiac tissue (11). The ~1.2-kb 5'-NCR, common to both mRNA species, contains 18 upstream AUG codons and a distinct polypyrimidine tract (4). Dicistronic assays in cell culture and in vitro indicate that the 5'-NCR of mouse Kv1.4 (mKv1.4) mRNAs mediates internal ribosome entry (4).

Translation initiation of most eukaryotic mRNAs is mediated through the cap- and end-dependent mechanism of ribosome scanning (12). However, atypical features within the 5'-NCR, including extensive length, stable secondary structure, and multiple upstream initiation codons, have been shown to inhibit ribosome scanning (13). Additionally, cellular conditions unfavorable to conventional cellular protein synthesis necessitate the existence of alternative mechanisms of translation initiation.

Internal ribosome entry, initially described for two small (+)-strand RNA viruses (14, 15), has since been identified in other (+)-strand RNA viruses (16) as well as in {gamma}-herpesviruses (17–19). Since the identification of the first cellular internal ribosome entry site (IRES) (20), cellular IRES elements have been shown to facilitate protein expression when cap-dependent translation is down-regulated during picornaviral infection (21, 22), mitosis (G2/M phase) (23, 24), apoptosis (25, 26), differentiation (27), and cellular stress (28–34). The study of cellular IRES elements has revealed the significance of internal ribosome entry in cellular gene expression and has underscored important aspects in the analysis of IRES elements (35).

In this work, two different subsets of mKv1.4 5'-NCR sequences are shown to independently mediate internal ribosome entry. These sequences demonstrated cell type-specific activities distinct from those mediated by a viral IRES. RNA structure probing indicated that the 3'-most ~0.2 kb forms defined structural elements in the presence and absence of the upstream ~1.0 kb. Deletion of sequences within the 3' ~0.2 kb resulted in distinct changes in expression mediated by the 3'-most ~0.2 kb (referred to as 0.2) and the ~1.2-kb mKv1.4 5'-NCR (referred to as 1.2), initially indicating that specific RNA elements may positively or negatively affect expression. Secondary structure probing suggested that local RNA folding is partially conserved with the introduction of various deletions within the 3' ~0.2 kb. Therefore, these findings suggest that interactions between functionally/structurally distinct RNA elements collectively promote internal ribosome entry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HeLa cells were cultured in minimal essential medium supplemented with 8% newborn calf serum, 1% nonessential amino acids, 2 mM glutamine, and 1% antibiotics/antimycotics. Human neuroblastoma cell lines SK-N-SH and NLF were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum and 1% antibiotics/antimycotics. African green monkey kidney cell lines CV1 and COS and the mouse muscle myoblast cell line C2F3 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics/antimycotics. Primary rat cardiac myocytes (PrCM) (36) were cultured in Dulbecco's modified Eagle's medium supplemented with 10–20% fetal bovine serum and 1% antibiotics/antimycotics. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. All cell culture reagents were obtained from Invitrogen.

Cloning of Plasmid Constructs and PCR Deletion Mutagenesis—The monocistronic firefly luciferase reporter construct p1209luc was constructed by inserting mKv1.4 5'-NCR cDNA sequences into pGEMluc.2 mKv1.4 5'-NCR cDNA sequences, excised from the chloramphenicol acetyltransferase-luciferase dicistronic construct (4) with HindIII and NcoI, lack the 5'-terminal 13 nucleotides. p229luc was subsequently generated by removing the 5'~1.0 kb from p1209luc using HindIII and XbaI, filling in 5'-overhangs with Klenow fragment, and ligating the resultant blunt-ended plasmid vector with T4 DNA ligase. The 3' ~0.2-kb 5'-NCR cDNA sequence within p1209luc and p229luc differs from the original published sequence (4) by two nucleotides (nucleotide 1196 (C) is deleted, and nucleotide 1197 is an A instead of a C).

Deletions within the 3'-most ~0.2 kb of the mKv1.4 5'-NCR were created using a PCR-based deletion mutagenesis scheme described previously by Imai et al. (37). Briefly, divergent non-overlapping oligonucleotides flanking targeted sequences were used to PCR amplify p229luc or p1209luc with a thermophilic DNA polymerase, Deep Vent® or PfuTurbo (Stratagene). The plasmid template was removed by DpnI digestion, leaving PCR-amplified DNA fragments that were gel-purified, phosphorylated with T4 polynucleotide kinase, and transformed into competent XL1-Blue bacteria. Deletions were verified by restriction digestion and sequence analysis.

The following oligonucleotides were utilized for deletion mutagenesis: 1703N, 5'-ACCACCATGGAAGACGCCAA-3'; 1732N, 5'-ACAACTGGAAGCAGCCATTT-3'; 1740N, 5'-CTTCTCTTACAACTGGAAGC-3'; 1800N, 5'-TAAGGCTTCCAAACTTACCT-3'; 1840N, 5'-GTTGGACTGAAAATATCCCA-3'; 1880N, 5'-GGAGCATAGGCTGTGCTGAT-3'; 1740X, 5'-AAGAAAGAAAAATAGGGCAG-3'; 1758X, 5'-CAGCTTATTTTCTTACCAAA-3'; 1780X, 5'-AAGGTAAGTTTGGAAGCCTT-3'; 1830X, 5'-TTCAGTCCAACTTTGCATTT-3'; 1880X, 5'-CCTCTTCTCAGAGACTCGGC-3'; 1910X, 5'-AGAGCTTGCCTCGTCTAGAG-3'; and 1920X, 5'-TCGTCTAGAGCTTGTCTCCC-3'.

Deletions at the 3'-end of 0.2 (3'{Delta}), 0.2{Delta}1', 0.2{Delta}pY, 0.2{Delta}1, 0.2{Delta}2, and 0.2{Delta}3, were generated using oligonucleotide pairs 1703N/1740X, 1732N/1758X, 1703N/1780X, 1740N/1780X, and 1740N/1830X, respectively. Deletions at the 5'-end of 0.2 (5'{Delta}), 0.2{Delta}4, 0.2{Delta}5, 0.2{Delta}6, 0.2{Delta}7, 0.2{Delta}8, and 0.2{Delta}9, were generated using oligonucleotide pairs 1800N/1830X, 1800N/1880X, 1840N/1880X, 1840N/1910X, 1880N/1910X, and 1880N/1920X, respectively. 0.2{Delta}10 and 0.2{Delta}11 were generated with oligonucleotide pairs 1740N/1880X and 1740N/1910X, respectively.

Dual-luciferase Dicistronic Constructs—A dual-luciferase plasmid construct, p2luci (a gift from Dr. R. F. Gesteland, University of Utah) (38), was used to generate dicistronic constructs. Firefly luciferase coding region sequences were excised from pGL3 (Promega) using NcoI and XbaI and ligated with the vector fragment of p2luci, which was digested with BamHI, treated with Klenow fragment, and digested with XbaI, resulting in p2luci-fx. p2luci-fx was linearized with SalI, treated with Klenow fragment, and religated to generate pR2TAAF, which encodes two tandem stop codons in-frame with the Renilla luciferase coding region. pRstF was subsequently generated by inserting an inverted repeat between the Renilla and firefly luciferase coding region sequences in pR2TAAF at the BglII restriction site using two ~50-bp fragments excised from the multiple cloning site of pGEM-4z with BamHI-SphI (Promega). Plasmids were examined by restriction digestion.

The full-length ~1.2-kb mKv1.4 5'-NCR was excised from p1209luc with HindIII and NcoI, whereas the 3'~0.2 kb and the 5'~1.0 kb were excised with XbaI and NcoI and with HindIII and XbaI, respectively, and treated with Klenow fragment. Similarly, 3' ~0.2-kb fragments containing deletions (0.2{Delta}) were excised from the appropriate p229{Delta}luc or p1209{Delta}luc constructs using XbaI and NcoI. Fragments were treated with Klenow fragment or T4 DNA polymerase and inserted into Eco47III- or AfeI-linearized pRstF plasmid, which had been treated with calf intestinal alkaline phosphatase. 1.2-kb 5'-NCR deletion sequences (1.2{Delta}) were similarly inserted into pRstF using a three-fragment ligation. The 5' ~1.0 kb was excised from p1209luc with HindIII and XbaI, whereas 3' ~0.2-kb fragments containing deletions were excised from the appropriate p229{Delta}luc constructs using XbaI and NcoI. Only the HindIII and NcoI overhangs were filled in with T4 DNA polymerase, allowing cohesive XbaI ends to preferentially ligate. Plasmids were verified by restriction digestion and sequence analysis.

pRstCVB3F was generated by inserting the 5'-NCR of coxsackievirus B3 (CVB3) into p2luci-fxnk1, a dicistronic construct containing a hairpin and additional intercistronic sequences. p2luci-fx was linearized with NotI, treated with Klenow fragment, and subsequently religated with T4 DNA ligase to generate p2luci-fxnk. p2luci-fxnk1 was generated by inserting a 148-bp fragment, excised from pTrap+12 with StuI and ApaI, between the blunted SalI and ApaI sites of p2luci-fxnk, introducing a hairpin described previously by Vagner et al. (39) and additional restriction sites, including PacI and NotI. pT7CVB+1 was generated by inserting the 5'-NCR of CVB3 into pTrap+1 with restriction sites PacI and NotI. The 5'-NCR of CVB3 was PCR-amplified from pCVB3-0 (40) with CVB3 PacI(+) (5'-CCTTAATTAATTAAAACAGCCTGTGGGTTGA-3') and CVB3 NotI(–) (5'-ATAAGATATGCGGCCGCTCCCATTTTGCTGTATTCAACTTA-3'). CVB3 5'-NCR sequences were removed from pT7CVB+1 using PacI and NotI and inserted into p2lucifxnk to generate p2luci-fxnkC1. Finally, p2luci-fxnkC1 was linearized with BamHI, treated with Klenow fragment, and religated to generate pRstCVB3F.

Dicistronic constructs lacking the SV40 promoter, the chimeric intron, and the T7 promoter were generated by two- or three-fragment ligations. The vector backbone of pRstF was isolated by digesting the plasmid with NcoI and NotI to remove sequences encompassing the SV40 promoter to the end of firefly luciferase. The vector was ligated with the NheI-NotI fragment isolated from pRstF, pRst1.2F, pRst0.2F, or pRst1.0F or with two fragments from pRstCVB3F, generated by digestion with NheI, PacI, and XbaI. Additionally, following restriction digestion with NcoI and NheI, reactions were incubated with T4 DNA polymerase to fill-in 3'-overhangs. Unless otherwise noted, enzymes were obtained from New England Biolabs Inc.

In Vitro Transcription—Dicistronic constructs were digested with NotI or XbaI, and p229luc and p229{Delta}luc constructs were digested with BstBI. Linearized plasmids were subjected to phenol/chloroform extraction and ethanol precipitation. In vitro transcription reactions were performed using 1.0 µg of linearized template (Ambion Inc.) and incubated at 37 °C for 4 h. Reactions were subsequently treated with DNase I at 37 °C for 30 min, subjected to phenol/chloroform extraction, and isolated using RNeasy columns (QIAGEN Inc.).

Transient Transfection—Cells were seeded 24–48 h before transfection, generating monolayers with 50–80% confluency. 35- or 22-mm well plates and 100-mm dishes were used for luciferase assays and total RNA isolation, respectively. Neonatal PrCM, seeded onto 35- or 22-mm well plates, were allowed to grow for 3 days before transfection. A 1:3 ratio of plasmid (micrograms) to FuGENE 6 (microliters) (Roche Applied Science) was complexed in Opti-MEMI (Invitrogen) at room temperature for ≥15 min. Monolayers were initially incubated with transfection complexes for 5 h, rinsed with medium, and incubated for an additional 19 h after the addition of fresh medium. The cytomegalovirus-{beta}-galactosidase reporter construct used in experiments presented in Fig. 2A was generously provided by Judy Jimenez and Dr. Marian L. Waterman (University of California, Irvine, CA).



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  		FIG. 2.
A, dicistronic constructs with or with out the SV40 promoter (SV40P) were transiently cotransfected into HeLa cells with a cytomegalovirus-{beta}-galactosidase reporter construct for 24 h and assayed for firefly luciferase (Fluc) and {beta}-galactosidase ({beta}-Gal) activities. B, in vitro transcribed dicistronic RNAs synthesized from plasmid constructs described in the legend to Fig. 1A were transiently transfected into HeLa cells for 15 h and assayed for luciferase activities. All transfections were performed in triplicate, and firefly luciferase/Renilla luciferase and firefly luciferase/{beta}-galactosidase ratios were calculated for each sample. The average value is represented as -fold above background levels, with expression measured in the absence of insert. Standard deviations were calculated from ratios obtained from triplicate samples. C, shown are the results from Northern blot analysis of total RNA from transiently transfected HeLa cells 5 and 15 h post-transfection. Dicistronic RNAs were detected using random hexamer-primed 32P-labeled probe against firefly luciferase coding region sequences, and the blot was subsequently reprobed for {beta}-actin to determine relative RNA loading. M, mock transfected; CV, CVB3.

 
For transient RNA transfections, cells were seeded onto 35-mm well plates ≥24 h prior to transfection, generating monolayers with 80–90% confluency. 2 µg of uncapped, in vitro transcribed RNA was transfected per well. A 6:1 ratio of Transmessenger transfection reagent (microliters) (QIAGEN Inc.) to RNA (micrograms) was added, and transfection complexes were incubated at room temperature for 10 min and combined with Opti-MEMI. Monolayers were rinsed twice with 2.0 ml of Opti-MEMI prior to the addition of transfection complexes and incubated for 1 h at 37 °C. Cells were subsequently rinsed with Dulbecco's modified Eagle's medium and incubated with serum-containing medium for an additional 14 h.

Dual-luciferase Assays—Monolayers were washed with 1x phosphate-buffered saline and lysed with 1x passive lysis buffer (Promega) ~24 h post-transfection. Cell lysates were subjected to two freeze/thaw cycles at –70 and 30 °C, respectively, and assayed for luciferase activities using the Dual-Luciferase reporter assay system (Promega). Firefly and Renilla luciferase activities were sequentially assessed by initially adding lysate to 100 µl of firefly luciferase substrate, measuring luminescence for 10 s, adding 100 µl of Renilla luciferase substrate to the same sample, and measuring luminescence for an additional 10 s. Assays were performed using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) or a SIRIUS luminometer (Berthold Detection Systems). Transfections were performed in triplicate. Firefly luciferase/Renilla luciferase and firefly luciferase/{beta}-galactosidase ratios were calculated for each sample, and the average value is represented as -fold above background levels, with expression measured in the absence of insert. Dicistronic assays were independently performed at least twice in all cells and cell lines used.

Isolation of Total RNA and Oligo(dT) Selection—Total RNA was isolated with TriReagent (Molecular Research Center, Inc.) and subjected to oligo(dT) selection using the Micro-FastTrack mRNA isolation kit (Invitrogen) and DNase I digestion (Worthington). Following DNase I digestion, oligo(dT)-selected RNAs were subjected to phenol/chloroform extraction and ethanol precipitation and subsequently resuspended in diethyl pyrocarbonate-treated H2O.

Northern Blot Analysis—2 µg of oligo(dT)-selected RNA was denatured with glyoxal/Me2SO (1 M deionized glyoxal, 50% (v/v) deionized Me2SO, and 25 mM sodium phosphate) at 50 °C for 60 min and separated on a 10 mM sodium phosphate-containing 1.0% agarose gel with continuous buffer circulation at 5 V/cm (~100 V). RNA was transferred to a GeneScreen Plus hybridization transfer membrane (PerkinElmer Life Sciences) by upward capillary transfer with 20x SSC for 20–24 h. Membranes were briefly rinsed with 2x SSC; UV light-irradiated; incubated in 20 mM Tris (pH 8.0) for ~30 min at room temperature; prehybridized with 10% (w/v) dextran sulfate, 0.3 M NaCl, and 1% SDS at 65 °C for 3 h; and hybridized overnight with random hexamer-primed 32P-labeled probes against firefly luciferase coding region sequences in the prehybridization buffer at 55 °C. Following hybridization, membranes were initially washed with 2x SSC and 0.1% SDS at ambient temperature; subsequently washed with 0.2x SSC and 0.1% SDS at ambient temperature, 37 °C, and 42 °C; and exposed to a PhosphorImager screen. The membranes were subsequently reprobed for {beta}-actin to determine relative RNA loading. Images were quantified using Quantity One Version 4.3.0 (Bio-Rad).

Random Primer Labeling of DNA Fragments and 5'-End Labeling of Oligonucleotides—Random hexamer priming reactions were performed based on methods described previously (41, 42). An ~1.7-kb fragment encoding the firefly luciferase coding region was excised from pRstF using NcoI and NotI, and an ~1.8-kb fragment was excised from a plasmid containing human {beta}-actin cDNA sequences (43). Gel-purified DNA fragments were denatured and incubated with random hexamer primer (New England Biolabs Inc.), [{alpha}-32P]dATP, and Klenow fragment at 37 °C for 30 min. Unincorporated [{alpha}-32P]dATP was removed using Chroma Spin-30 diethyl pyrocarbonate-treated H2O columns (Clontech).

9 pmol (~60 ng) of oligonucleotide (Fluc (5'-TCCATCTTCCAGCGGATAGA-3'), 1740X, or 1780X) was incubated in 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, and 5 mM dithiothreitol with 50 µCi (6000 Ci/mmol) of [{gamma}-32P]ATP and 20 units of T4 polynucleotide kinase in a final volume of 50 µl for 60 min at 37 °C. Unincorporated [{gamma}-32P]ATP was removed using Chroma Spin-10 TE columns (Clontech).

Enzymatic Structure Probing by Primer Extension—RNA structure probing was carried out as a modification of methods described previously (44–46). In vitro transcribed RNA (0.5 µg) was combined with 40 µg of yeast tRNA in 0.7x buffer containing 30 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 270 mM KCl under reducing conditions (18 mM 2-mercaptoethanol) in a final volume of 40 µl. RNAs were sequentially incubated at 68 and 37 °C for 5 min each and then at room temperature for 5–10 min before the addition of RNase V1 (Pierce) or RNase T1, PhyM, or CL3 (Industrial Research Ltd.) at room temperature for 5–20 min (as described in the figure legends). Reactions were halted by the addition of 160 µl of stop solution (final concentrations of 0.3 M NaOAc, 10 mM EDTA, and 0.3% SDS), subjected to phenol/chloroform extraction, and ethanol-precipitated in the presence of glycogen. RNA pellets were initially resuspended in a final volume of 15.5 µl with 106 cpm 32P-5'-end-labeled oligonucleotide and sequentially incubated at 70 °C and on ice for 10 min each. RNAs were incubated at 42 °C for 40–60 min with 10 units of avian myeloblastosis virus reverse transcriptase (PerkinElmer Life Sciences) in 25 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM dithiothreitol, 5 mM MgCl2, 1 mM each dNTP, and 40 units of RNasin (Promega). After the addition of 180 µl of stop solution (final concentrations of 0.3 M NaOAc, 10 mM EDTA, and 0.3% SDS), reactions were subjected to phenol/chloroform extraction and ethanol-precipitation with glycogen. Samples were resuspended in 7.2 µl of Tris-HCl (pH 8.0), 1 mM EDTA, and 0.1 µg/µl RNase A and incubated at 37 °C for 30 min. Finally, 4.8 µl of formamide loading buffer was added to a final volume of 12 µl. 2.5–3.0 µl of each sample was resolved on a 7 M urea and 8% polyacrylamide gel with appropriate sequencing ladders.

Secondary Structure Predictions—Secondary structure predictions were generated for mKv1.4 5'-NCR sequences using mfold (Version 3.1) by Turner and co-workers (47, 48) with the latest energy parameters under default conditions (www.bioinfo.rpi.edu/applications/mfold/old/rna/).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The 5'-NCR of mKv1.4 mRNA Species Mediates Internal Ribosome Entry in HeLa Cells—The ~1.2-kb mKv1.4 5'-NCR (referred to as 1.2; previously reported to mediate internal ribosome entry) (4), the 3'-most ~0.2 kb (referred to as 0.2), and the 5'-most ~1.0 kb (referred to as 1.0) were examined for IRES activity in HeLa cells utilizing a dual-luciferase dicistronic construct (Fig. 1A). The 3'~0.2-kb sequence described in this study contains the 3'-most 208 nucleotides originally examined (4) as well as an additional 22 upstream nucleotides. The 5'-NCR of CVB3, encoding a type 1 IRES element (49, 50), was employed as a positive control. Following transient DNA transfection, the full-length mKv1.4 5'-NCR (1.2) mediated the highest level of expression, ~174-fold above background levels, whereas 0.2 and 1.0 independently exhibited substantial (albeit lower) levels of expression, ~40-fold above background levels (Fig. 1B). The CVB3 5'-NCR mediated intermediate levels of expression, ~90-fold above background levels (Fig. 1C). The ability of the 3'-most ~0.2 kb as well as the 5'-most ~1.0 kb to separately promote internal initiation indicates a substantial level of complexity involved in IRES-mediated translation for this transcript.



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  		FIG. 1.
mKv1.4 5'-NCR sequences mediate internal ribosome entry in the context of dicistronic RNAs. A, dicistronic constructs encoding Renilla and firefly luciferases as the upstream and downstream cistrons, respectively, were employed to assess IRES function within the 5'-NCR from Kv1.4 mRNA species expressed in mouse brain tissue. Plasmid constructs contained the SV40 promoter (SV40pm), a chimeric intron to promote nuclear export, the T7 promoter (T7pm) upstream of Renilla luciferase, and the SV40 polyadenylation signal (SV40pA) downstream of firefly luciferase. An inverted repeat or hairpin was inserted within the intercistronic sequence (ICS). B, dicistronic constructs encoding mKv1.4 5'-NCR sequences, the full-length ~1.2 kb (1.2), the 3'-most ~0.2 kb (0.2), and the 5'-most ~1.0 kb (1.0), within the intercistronic sequence were transiently transfected into a human cervical carcinoma cell line (HeLa), two human neuroblastoma cell lines (SK-N-SH and NLF), a mouse muscle myoblast cell line (C2F3), PrCM, and two African green monkey kidney cell lines (CV1 and COS). C, a dicistronic construct encoding the CVB3 5'-NCR was also transfected in parallel as a positive control for IRES activity. Constructs were transfected in triplicate, and cell lysates were isolated 24 h post-transfection and assayed for luciferase activity. Firefly luciferase/Renilla luciferase ratios were calculated for each sample, and the average value is represented as -fold above background levels, with expression in the absence of insert. Standard deviations were calculated for firefly luciferase/Renilla luciferase ratios obtained from triplicate assays. D, total RNA was isolated from HeLa cells transiently transfected with dicistronic constructs 14 h post-transfection and subjected to oligo(dT) selection and DNase I digestion. 2 µg oligo(dT)-selected RNA was initially analyzed by Northern blot analysis using random hexamer-primed 32P-labeled probe against the firefly luciferase coding sequences and subsequently reprobed for {beta}-actin to determine relative RNA loading. RNA was isolated from untransfected HeLa cells (NT; lane 1) and from cells transiently transfected with dicistronic constructs containing no insert (lane 3) or mKv1.4 (lanes 4–6) or CVB3 (lane 2) 5'-NCR sequences. Smaller RNA species are denoted by single and double asterisks. Slower migrating bands observed in lanes 3–6 may be attributed to incomplete DNase I digestion of transiently transfected plasmid DNA.

 
Full-length dicistronic RNAs were detected 14 h post-transfection in transiently transfected HeLa cells by Northern blot analysis (Fig. 1D, lanes 2–6). However, a smaller species, ~2.0 kb (double asterisks), was detected when 1.2 or 0.2 was encoded within the intercistronic sequence (Fig. 1D, lanes 4 and 5), whereas two additional species, ~3.0 kb (single asterisk) and ~2.2 kb (double asterisks), were detected with CVB3 (lane 2). The intensities of the smaller RNA species were ~10–20% of their respective dicistronic RNAs for CVB3 and 1.2 and 60–75% for 0.2 (Fig. 1D, lanes 2, 4, and 5). The presence of smaller RNA species suggests the involvement of cryptic promoter activity or RNA splicing. When the SV40 promoter was removed from dicistronic constructs, firefly luciferase expression (normalized to {beta}-galactosidase activity from a cotransfected reporter plasmid) was similar to background levels in HeLa cells (Fig. 2A) as well as in CV1 and SK-N-SH cells (data not shown), indicating that mKv1.4 5'-NCR cDNA sequences do not mediate cryptic promoter activity. The smallest RNA species observed (Fig. 1D, lanes 2, 4, and 5) lacked the majority of Renilla luciferase sequences (data not shown) and may possibly represent a splicing product between the donor site of the chimeric intron and a cryptic acceptor site within the 3' ~0.2 kb of the mKv1.4 5'-NCR. When the chimeric intron was removed from dicistronic constructs, firefly luciferase expression, although decreased overall, was still 8–24-fold above background levels for 1.2, 0.2, and 1.0 (data not shown).

To eliminate possible contributions to firefly luciferase expression by splicing products, in vitro transcribed dicistronic RNAs were transfected directly into HeLa cells (Fig. 2B). Both 1.2 and 1.0 demonstrated expression 20- and 14-fold above background levels, unequivocally demonstrating IRES activity. The 0.2 IRES construct generated lower levels of firefly luciferase activity compared with dicistronic RNAs encoding 1.2 or 1.0, ~2.5-fold above background levels (Fig. 2B). Similar results were observed in CV1 and COS cells (data not shown). Dicistronic transcripts were detected 5 and 15 h post-transfection (Fig. 2C). Although the levels of RNA decreased over time, smaller RNA species were not detected, indicating that these RNAs are not subject to specific cleavage.

The 5'-NCR of mKv1.4 mRNA Differentially Mediates IRES Activity in Cell Culture—mKv1.4 mRNA species are natively expressed in brain, heart, and skeletal muscle (11, 51); therefore, it was of particular interest to examine IRES activity in cells and cell lines derived from these tissues. IRES activity was assessed in two human neuroblastoma cell lines (SK-N-SH and NLF), neonatal PrCM, a mouse muscle myoblast cell line (C2F3), and two African green monkey kidney cell lines (CV1 and COS). The results parallel those observed in HeLa cells, with 1.2 mediating the highest level of expression and 0.2 and 1.0 independently mediating lower levels of expression (Fig. 1B). In NLF, C2F3, and COS cells, the levels of expression mediated by 0.2 or 1.0 were ≤2-fold above background levels (Fig. 1B), indicating the lack of significant IRES activity. mKv1.4 5'-NCR sequences (1.2, 0.2, and 1.0) mediated higher levels of expression in HeLa and CV1 cells compared with the human neuroblastoma cell lines (Fig. 1B), whereas CVB3 promoted similar levels of expression in all four cell lines (Fig. 1B). In transient RNA transfections, 1.2-mediated expression was ~20-fold above background levels in HeLa and CV1 cells, but only ~13-fold in COS cells (Fig. 2B) (data not shown). In contrast, CVB3 promoted expression >200-fold above background levels in these three cell lines (Fig. 2B) (data not shown). This suggests that mKv1.4 5'-NCR sequences mediate cell type-dependent IRES activities distinct from CVB3 5'-NCR sequences.

Deletion Analysis of the 3'-Most ~0.2 kb Encoded within the mKv1.4 5'-NCR—To investigate the importance of specific RNA elements, overlapping deletions within the 3' ~0.2 kb were generated in the context of 0.2 (0.2{Delta}) and 1.2 (1.2{Delta}) (Fig. 3A). Deletion-containing sequences (0.2/1.2{Delta}1'–{Delta}11) were examined for IRES activity in HeLa cells (Fig. 3B) and subsequently in PrCM and SK-N-SH cells (Fig. 3C) (data not shown).



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  		FIG. 3.
Deletion analysis of the mKv1.4 5'-NCR. A, shown is a schematic representation of deletions within the 3' ~0.2 kb of the mKv1.4 5'-NCR. Deletions spanning the 3' ~0.2 kb of the mKv1.4 5'-NCR were introduced into the 3' ~0.2-kb (0.2{Delta}) and full-length ~1.2-kb (1.2{Delta})5'-NCRs and inserted into the intercistronic sequence of the dual-luciferase dicistronic construct (Fig. 1A). In addition to deletions of contiguous overlapping sequences ({Delta}1'–{Delta}11), a 29-nucleotide sequence containing a polypyrimidine tract (nucleotides 1154–1173) upstream of the start codon was specifically deleted ({Delta}pY). Deletions are roughly grouped as 3'-deletions ({Delta}1', {Delta}pY, {Delta}1, {Delta}2, and {Delta}3) and 5'-deletions ({Delta}4, {Delta}5, {Delta}6, {Delta}7, and {Delta}8) based on position and expression compared with wild-type sequences. {Delta}9 differs from {Delta}8 by the deletion of an additional 10 upstream nucleotides, and {Delta}10 and {Delta}11 represent the most extreme deletions, removing 139 and 169 nucleotides from the original 234 nucleotides, respectively. B and C, dicistronic constructs containing mKv1.4 5'-NCR deletion-containing sequences within the intercistronic sequence were transiently transfected into HeLa cells and PrCM, respectively, for 24 h and assayed for luciferase activity. The upper four columns represent identical results from dicistronic constructs encoding no insert or wild-type mKv1.4 5'-NCR sequences 1.2, 0.2, and 1.0 (Fig. 1A) and are set relative to appropriate wild-type sequences 0.2 and 1.2 (indicated by asterisks) in the left and right panels, respectively. Expression mediated by deletion sequences 0.2{Delta} (left panels) and 1.2{Delta} (right panels) is represented as -fold relative to corresponding wild-type sequences 0.2 and 1.2, respectively.

 
3'-Deletions ({Delta}1', {Delta}pY, {Delta}1, {Delta}2, and {Delta}3) that partially or completely removed a 20-nucleotide polypyrimidine tract decreased 0.2 expression 12.5–50-fold (Fig. 3B, left panel) and 1.2 expression 11–100-fold in HeLa cells (Fig. 3B, right panel). The effect observed in 1.2 is surprising since 1.0 independently demonstrated IRES activity (Figs. 1B and 2B). In contrast, 5'-deletions ({Delta}4, {Delta}5, {Delta}6, {Delta}7, and {Delta}8) increased 0.2 expression ≥2.3–3.9-fold (Fig. 3B, left panel), but had little effect on 1.2 expression in HeLa cells (right panel). Interestingly, {Delta}9, which differs from {Delta}8 only by the deletion of an additional 10 upstream nucleotides, slightly decreased 0.2 expression. The most extreme deletions, {Delta}10 and {Delta}11, containing only ~98 and ~68 nucleotides of the original 3' ~0.2 kb, respectively, decreased the signal generated by 0.2 to background levels of expression (Fig. 3B, left panel), whereas 1.2, closer to the 5' ~1.0-kb construct in sequence with these deletions, mediated expression levels similar to 1.0 (right panel). In PrCM, 5'-deletions increased 0.2 expression ≥3–4-fold, equal to or greater than wild-type 1.2 (Fig. 3C, left panel), whereas deletions introduced into 1.2 minimally affected expression (right panel). Deletion of the polypyrimidine tract also decreased 0.2- and 1.2-mediated expression to background levels in PrCM (Fig. 3C). Similar results were observed in SK-N-SH cells (data not shown).

Secondary Structure Analysis of mKv1.4 5'-NCR Sequences—To investigate the relationship between RNA structure and IRES function, enzymatic structure probing and the RNA folding algorithm mfold were used to examine mKv1.4 5'-NCR sequences. In vitro transcribed RNAs were subjected to partial digestion using RNases with specificities for paired (RNase V1) and unpaired (RNases T1, PhyM, and CL3) nucleotides. Nucleotide-specific cleavage was detected by primer extension and subsequent analysis on a sequencing gel. Since RNA cleavage occurs 3' of the nucleotide, reverse-transcribed products are one nucleotide shorter than the corresponding fragment on the DNA sequencing ladder.

The 3'-terminal 200 nucleotides of the mKv1.4 5'-NCR had previously been predicted to form an extended helix flanked by three distinct stem-loop structures (4). Using constraints determined by enzymatic structuring probing (Fig. 4), 0.2 was predicted to form the structure shown in Fig. 5A. Although encompassing additional upstream nucleotides, the structure for 0.2 includes elements in common with those previously predicted for the 3'-terminal 200 nucleotides.



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  		FIG. 4.
Enzymatic structure probing of mKv1.4 5'-NCR sequences using primer extension. 0.5 µg of in vitro transcribed RNA was subjected to mock digestion for 10 and 20 min (lanes 1 and 6, respectively) or partial RNase digestion with 18.2 units of RNase V1 (V1) for 10 and 5 min (lanes 4 and 5, respectively), 0.025 and 0.017 units of RNase T1 (T1) for 20 min (lanes 2 and 3, respectively), 1.0 unit of RNase PhyM(P) for 20 and 15 min (lanes 7 and 8, respectively), and 0.04 and 0.02 units of RNase CL3 (C) for 20 min (lanes 9 and 10, respectively) at room temperature in the presence of 40 µg of tRNA. Primer extension reactions were performed using 32P-5'-end-labeled oligonucleotides (~106 cpm). Samples were subsequently resolved on a 7 M urea and 8% acrylamide sequencing gel. A–D, the 3'-most 229 nucleotides (0.2) were analyzed using oligonucleotide 1740X (A and B, 2.5 h and 3 h, 40 min, respectively) and the firefly luciferase oligonucleotide (C and D, 2.5 h and 3 h, 40 min, respectively). E, the full-length ~1.2-kb 5'-NCR was subjected to partial RNase digestion and subsequent primer extension with 32P-5'-end-labeled oligonucleotide 1740X. mKv1.4 5'-NCR sequences were preceded by 10 additional nucleotides and followed by 169 nucleotides of the firefly luciferase coding region.

 



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  		FIG. 5.
mfold secondary structure predictions for 0.2 and 0.2{Delta}. Using the RNA folding algorithm mfold (Version 3.1) and constraints determined from enzymatic structure probing, mKv1.4 5'-NCR sequences 0.2 (A), 0.2{Delta}pY (B), 0.2{Delta}2 (C), 0.2{Delta}4 (D), 0.2{Delta}8 (E), and 0.2{Delta}9 (F) were predicted to form the structures shown above. Arrowheads denote cleavage by single strand-specific RNases T1, PhyM, and/or CL3, whereas arrows indicate cleavage by RNase V1, the double strand-specific RNase. The intensity of RNase cleavage was subjectively assigned as greater or lesser and is denoted by closed and open arrowheads or arrows, respectively. Nucleotides composing the polypyrimidine tract (nucleotides 1154–1173) are shaded for 0.2. Altered structures predicted to form within 0.2{Delta} sequences are denoted with a prime. In 0.2, a potential pseudoknot (PK) interaction between neighboring single-stranded regions (nucleotides 1138–1143 and 1151–1157) is indicated by arrows. The numbering of the nucleotides is relative to the full-length 1.2-kb mKv1.4 5'-NCR.

 
The polypyrimidine tract (nucleotides 1154–1173) had been predicted to participate in the formation of an extended helical structural (4). Nucleotides 1162, 1165, 1167, 1168, and 1170 were susceptible to RNase V1 cleavage (Fig. 4D, lanes 4 and 5), correlating with base pair formation; however, nucleotides 1165–1170 are positioned within a stem-loop (Fig. 5A), not an extended helix as previously predicted. Strong RNase PhyM and/or CL3 cleavage at nucleotides 1173 and 1175 indicated the presence of unpaired residues (Fig. 4D, lanes 7–10). These data are consistent with the formation of a stem-loop, designated stem-loop Z (nucleotides 1165–1186), within the 3'-end of the polypyrimidine tract (Fig. 5A).

Nucleotides 1155–1157 within the 5'-end of the polypyrimidine tract were sensitive to RNase PhyM digestion (Fig. 4D, lanes 7 and 8). Surrounding nucleotides 1150 and 1162 were sensitive to RNase V1, whereas nucleotide 1158 was susceptible to both RNases V1 and PhyM (Fig. 4, C and D, lanes 4, 5, 7, and 8). Upstream nucleotides 989 and 990, predicted to base pair with nucleotides 1147 and 1148, were susceptible to RNase V1 (Fig. 4A, lanes 4 and 5). These data suggest the presence of a single-stranded U-rich loop (nucleotides 1151–1157) linking two regions of helical structure.

Of the three stem-loop structures that had been previously predicted by Negulescu et al. (4) to form within the 3' 200 nucleotides, the two stem-loop structures positioned upstream of the polypyrimidine tract were corroborated by structure probing: the proximal stem-loop structure (nucleotides 1119–1137) and the distal stem-loop structure (nucleotides 1048–1106); however, the latter structure was only partially corroborated. Strong RNase V1 cleavage at nucleotides 1119 and 1120 and weaker cleavage at adjacent nucleotides 1121–1123 and on the complementary strand (nucleotides 1133–1135) were consistent with stem formation of the proximal stem-loop structure (Fig. 4C, lanes 4 and 5), whereas strong RNase PhyM cleavage at nucleotides 1125–1129 was indicative of a highly exposed single-stranded region, consistent with the loop of the proximal stem-loop structure (lanes 7 and 8). The intensity of the bands suggested that this stem-loop, designated stem-loop Y (Fig. 5A), is highly exposed, perhaps forming a putative protein-binding site. Identical RNase cleavage patterns were also observed for 1.2 as well as 1.2{Delta}pY, 1.2{Delta}4, 0.2{Delta}pY, 0.2{Delta}4, 0.2{Delta}6, 0.2{Delta}8, and 0.2{Delta}9 (data not shown), and stem-loop Y was predicted to form in 0.2{Delta}pY, 0.2{Delta}4, 0.2{Delta}6, 0.2{Delta}8, and 0.2{Delta}9 (Fig. 5, B–E) (data not shown).

Nucleotides 1073–1086, predicted to form the loop of the distal stem-loop structure (nucleotides 1050–1106), were sensitive to single strand-specific RNases. Specifically, nucleotide 1074 was sensitive to RNase T1/PhyM, whereas nucleotides 1075–1080 were susceptible to RNase PhyM/CL3 (Fig. 4B, lanes 2, 3, and 7–10). Upstream nucleotides 1071 and 1072 were susceptible to RNase V1, whereas nucleotide 1073 was susceptible to both RNase V1 and PhyM cleavage (Fig. 4B, lanes 4, 5, 7, and 8). Downstream nucleotides 1092 and 1093 were also sensitive to RNase V1 cleavage (Fig. 4B, lanes 4 and 5). These data corroborate the formation of a large loop, designated loop {Omega} (Fig. 5A); however, although adjacent nucleotides were paired, structure probing did not clearly support the formation of an extended helix as previously predicted.

Nucleotides 1002–1039 were predicted to form an additional stem-loop structure, designated stem-loop X (Fig. 5A). RNase V1 cleavage at nucleotides 1035–1037 and 1024–1026 was consistent with helix formation at the base of the structure and within the terminal stem-loop, respectively; however, only low level RNase CL3 cleavage at nucleotide 1030 was consistent with the presence of an internal loop. Nucleotides 1019–1020 within the loop were equally susceptible to low level cleavage by RNases V1, T1, and PhyM. Although a loop was not directly supported by structure probing, susceptibility to RNases V1, T1, and PhyM may indicate the formation of transient higher order RNA interactions or multiple RNA conformations within this domain.

Nucleotides 991–1001, preceding stem-loop X, were predicted to form an internal loop, designated loop {Psi} (Fig. 5A). Nucleotides 997–998 were highly sensitive to RNase T1/PhyM cleavage, whereas flanking nucleotides 995, 996, and 999 were susceptible to RNase PhyM cleavage (Fig. 4A, lanes 2, 3, 7, and 8). The intensity of these bands, especially nucleotides 997 and 998, suggests that this loop is highly exposed.

Local RNA Structures Are Conserved in the Absence of Upstream and Adjoining Sequences—Enzymatic structure probing of 1.2 demonstrated that RNase cleavage within nucleotides 978–1207 was fundamentally conserved with that observed for 0.2 (Fig. 4, compare A, B, and E) (data not shown) and indicated that the upstream ~1.0 kb does not significantly contribute to the formation of RNA interactions within the 3' ~0.2 kb. The conservation of local RNA structure also suggested the potential modular character of the mKv1.4 IRES. This idea is underscored by the ability of non-overlapping regions to independently mediate internal ribosome entry (Figs. 1B and 2B). Furthermore, RNase cleavage within mKv1.4 5'-NCR sequences was partially maintained in the presence of various deletions within the 3' ~0.2 kb (data not shown), and 0.2{Delta}pY, 0.2{Delta}2, 0.2{Delta}4, 0.2{Delta}6, 0.2{Delta}8, and 0.2{Delta}9 were predicted to form structures similar or identical to 0.2 (Fig. 5) (data not shown).

Alterations in Secondary Structure Formation—The systematic deletion of sequences within 0.2 resulted in distinct changes in IRES activity (Fig. 3). To investigate the impact of the deletions on local RNA interactions and folding, a subset of RNA sequences harboring deletions within the 3' ~0.2 kb was examined by enzymatic structure probing. The general pattern of RNase cleavage was not drastically altered with the introduction of these deletions (data not shown), suggesting that RNA folding is partially conserved. Indeed, defined RNase cleavage patterns were consistently observed, substantiating the formation of specific structural elements, including stem-loop Y and loop {Psi} (see Figs. 7 and 8) (data not shown). As expected, sequences adjacent to the deletion displayed variable differences in RNase cleavage (data not shown); however, defined regions distal to the deletion were also notably altered by the loss of sequences.



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  		FIG. 7.
Enzymatic structure probing within the polypyrimidine tract. A, 0.2; B, 0.2{Delta}2; C, 0.2{Delta}4; D, 0.2{Delta}8. RNAs were subjected to partial RNase digestion (as described in the legend to Fig. 4) and primer extension using 32P-5'-end-labeled oligonucleotides 1740X (for 0.2, 0.2{Delta}4, and 0.2{Delta}8) and 1780X (for 0.2{Delta}2). T1, RNase T1; V1, RNase V1; P, RNase PhyM; C, RNase CL3.

 



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  		FIG. 8.
Enzymatic structure probing of sequences forming loop {Psi}. A, 0.2; B, 0.2{Delta}8; C, 0.2{Delta}9. RNAs were subjected to partial RNase digestion (as described in the legend to Fig. 4) and primer extension using 32P-5'-end-labeled oligonucleotide 1740X. T1, RNase T1; V1, RNase V1; P, RNase PhyM; C, RNase CL3.

 
Nucleotide-specific cleavage within stem-loop X was altered by the deletion of downstream sequences (Fig. 6). 0.2{Delta}pY, 0.2{Delta}2, and 0.2{Delta}4 demonstrated increased RNase PhyM cleavage at nucleotides 1018–1020 (Fig. 6, A–D, compare lanes 7 and 8). Increased RNase PhyM cleavage was also observed at nucleotides 1023 and 1025–1027 in 0.2{Delta}pY and, to a lesser extent, in 0.2{Delta}2 (Fig. 6, B and C, lanes 7 and 8). Accordingly, nucleotides 1024–1026 were no longer significantly susceptible to RNase V1 cleavage in 0.2{Delta}pY and 0.2{Delta}2, whereas only nucleotide 1024 remained distinctly susceptible to RNase V1 in 0.2{Delta}4 (Fig. 6, A–D, lanes 4 and 5). These data suggest that stem-loop X may directly interact with downstream sequences. Alternatively, stem-loop formation may be influenced by distal sequences as indicated by the prediction of alternative stem-loop X structures, designated stem-loop X', in 0.2{Delta}pY, 0.2{Delta}2, and 0.2{Delta}4 (Fig. 5, B–D). Interestingly, nucleotides 1013–1026 within 0.2{Delta}4 were predicted to form a GNRA tetraloop (Fig. 5D). In 0.2{Delta}6, juxtaposition of stem-loop X and loop {Psi} sequences was predicted to generate a novel stem-loop structure (data not shown). Nucleotide 1020 was sensitive to RNase V1 cleavage, whereas nucleotides 1023, 1025, and 1026 were cleaved by RNase PhyM (Fig. 6E, lanes 4, 5, 7, and 8).



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  		FIG. 6.
Enzymatic structure probing of sequences forming stem-loop X. A, 0.2; B, 0.2{Delta}pY; C, 0.2{Delta}2; D, 0.2{Delta}4; E, 0.2{Delta}6. RNAs were subjected to partial RNase digestion (as described in the legend to Fig. 4) and primer extension using 32P-5'-end-labeled oligonucleotides 1740X (for 0.2, 0.2{Delta}4, and 0.2{Delta}6) and 1780X (for 0.2{Delta}Y and 0.2{Delta}2). T1, RNase T1; V1, RNase V1 P;, RNase PhyM; C, RNase CL3.

 
Whereas deletion of sequences within the polypyrimidine tract affected stem-loop X, conversely, disruption of stem-loop X altered RNase cleavage within the polypyrimidine tract. Deletions {Delta}6 and {Delta}8/{Delta}9, which extend into stem-loop X, altered RNase cleavage within the polypyrimidine tract (Fig. 7D) (data not shown). In 0.2{Delta}8, nucleotides 1158, 1161, 1162, and 1165 were equally susceptible to RNase V1 and PhyM cleavage (Fig. 7D), indicating the possible formation of more than one structure in this region of the RNA. Nucleotides 1168 and 1170 remained susceptible only to RNase V1 cleavage, whereas adjacent nucleotides 1169 and 1171 displayed weak RNase V1 and CL3 cleavage. Only nucleotides 1156 and 1157 were distinctly unpaired, as indicated by RNase PhyM cleavage (Fig. 7D, lanes 7 and 8). 0.2{Delta}6 and 0.2{Delta}9 demonstrated similar patterns of RNase cleavage (data not shown). In contrast, although 0.2{Delta}4 displayed altered cleavage within stem-loop X, RNase cleavage within the polypyrimidine tract remained similar to that of 0.2 (Fig. 7, compare A and C). Similarly, when the upstream portion of the polypyrimidine tract (nucleotides 1154–1165) was removed in 0.2{Delta}2, the remaining downstream nucleotides retained a pattern of RNase cleavage comparable with that of 0.2 (Fig. 8, compare A and B). Collectively, these data suggest that stem-loop X and the polypyrimidine tract interact; however, mfold predictions indicate that these two regions contribute to the formation of discrete structures in 0.2 (Fig. 5A). Alternatively, the formation of stem-loop X may indirectly stabilize RNA interactions within the polypyrimidine tract.

Although displaying identical cleavage patterns throughout most of the sequence (data not shown), 0.2{Delta}8 and 0.2{Delta}9, which differ by 10 nucleotides (nucleotides 987–996), demonstrated opposing changes in IRES activity (Fig. 3). These 10 nucleotides contribute to the formation of loop {Psi} in 0.2 (Fig. 5A). Within loop {Psi}, strong RNase T1/PhyM cleavage at nucleotides 997 and 998 was apparent in 0.2 (Fig. 8A, lanes 2, 3, 7, and 8) and was highly conserved in 1.2, 1.2{Delta}pY, 1.2{Delta}2, 1.2{Delta}4, 0.2{Delta}pY, 0.2{Delta}2, 0.2{Delta}4, and 0.2{Delta}6 (data not shown). However, in 0.2{Delta}8, nucleotides 997 and 998 were deleted. Instead, two upstream guanosine residues (nucleotides 987 and 988) were strongly susceptible to RNase T1/PhyM (Fig. 8B, lanes 2, 3, 7, and 8). In 0.2{Delta}9, nucleotides 1026 and 1027, which border the deletion, were less susceptible to RNase T1/PhyM cleavage than the corresponding nucleotides in 0.2 and 0.2{Delta}8 (Fig. 8C, lanes 2, 3, 7, and 8). Although a comparable single-stranded region was predicted to form within 0.2{Delta}9 (Fig. 5F), cleavage of adjacent nucleotides suggested that RNA folding within this region differs from the corresponding structures formed by 0.2 and 0.2{Delta}8 (Fig. 8C). Therefore, differences in 0.2{Delta}8- and 0.2{Delta}9-mediated expression may be attributed to the ability of sequences within 0.2{Delta}8 to form a functionally/structurally equivalent element that compensates for the disruption of loop {Psi} (Fig. 5E).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we used dual-luciferase dicistronic constructs to investigate internal ribosome entry mediated by mKv1.4 5'-NCR sequences in several mammalian cell lines (Fig. 1B). Although the expression of dicistronic RNAs was confirmed by Northern blot analysis, submolar amounts of smaller RNA species, possibly generated through cryptic promoter activity or RNA splicing, were also detected (Fig. 1D). In the absence of the SV40 promoter, firefly luciferase expression was similar to background levels (Fig. 2A), demonstrating that mKv1.4 5'-NCR cDNA sequences do not mediate promoter activity. Moreover, transfection of in vitro transcribed dicistronic RNAs directly into cultured cells clearly demonstrated that 1.2 and 1.0 constructs and, to a lesser extent, 0.2 mediated internal ribosome entry (Fig. 2B). In vitro translation using HeLa S10 cytoplasmic extract as well as rabbit reticulocyte lysate also demonstrated that 1.2 and 1.0 mediated IRES activity (data not shown); however, compared with CVB3, mKv1.4 IRES activity is much lower in vitro than in transfected cells in culture.

IRES activity had previously been attributed to the 3'-most 208 nucleotides (4). In this study, we have shown that the upstream ~1.0 kb can independently mediate internal ribosome entry (Figs. 1B and 2B). The significance of functionally independent IRES elements within mKv1.4 5'-NCR sequences is not known, but their presence in the 5'-NCR of this mRNA may provide an additional level of translational regulation via RNA-RNA interactions between distal elements or RNA-protein interactions. Noncontiguous, non-overlapping sequences have also been demonstrated to mediate internal ribosome entry within other cellular IRES elements (52–54). Chappell et al. (33) proposed that IRES elements are composed of distinct functional modules individually capable of promoting internal ribosome entry. Although mKv1.4 5'-NCR sequences have not been determined to be composed of many discrete IRES elements, deletion analysis supports the idea that functionally/structurally distinct regions interact to mediate IRES activity.

The presence of two non-overlapping IRES elements within the 5'-NCR of vascular endothelial growth factor mRNA had been correlated with the expression of distinct protein isoforms (53, 55). Although only a single polypeptide is known to be expressed from mKv1.4 mRNA species, the authentic start codon is preceded by 18 upstream AUG codons. Several are in good Kozak consensus (56) and may potentially initiate the expression of upstream open reading frames; however, their significance has yet to be examined. In the proto-oncogene c-myc, expression of c-Myc proteins and an upstream open reading frame (MYCHEX1) is mediated through independent IRES elements (57–59). The significance of the MYCHEX1 protein is unknown; however, Nanbru et al. (59) speculated that translation of the MYCHEX1 open reading frame, which overlaps the c-Myc IRES, may alter RNA structure and affect IRES-mediated expression of c-Myc proteins. Similarly, during amino acid deprivation, translation of an upstream open reading frame was hypothesized to up-regulate IRES-mediated expression of the cationic amino acid transporter Cat-1 by altering RNA structure (60).

The 3'~0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two single-stranded regions (loops {Psi} and {Omega}) (Fig. 5A). The formation of stem-loop Y and loop {Omega} was consistent with the structure previously predicted for the 3' 200 nucleotides (4). Furthermore, the full-length 5'-NCR (1.2) demonstrated similar patterns of RNase cleavage within the 3' ~0.2 kb, indicating that RNA folding within 0.2 is independent of upstream sequences. Strong cleavage within stem-loop Y and loop {Psi} suggests that these regions are exposed and may potentially facilitate RNA-protein interactions.

Structure-function analyses of mKv1.4 5'-NCR sequences indicated that the formation of complex RNA interactions contributes to IRES activity. Significant decreases in 0.2- and 1.2-mediated expression in the absence of the complete polypyrimidine tract initially indicated the importance of this motif (Fig. 3). Deletion of polypyrimidine tract sequences correlated with changes in RNase cleavage within sequences forming stem-loop X in 0.2 (Fig. 6). Conversely, when stem-loop X was directly disrupted, as in 0.2{Delta}6, 0.2{Delta}8, and 0.2{Delta}9, nucleotides within the polypyrimidine tract no longer clearly displayed strand-specific RNase cleavage (Fig. 7D) (data not shown). However, in contrast to 0.2{Delta}pY and 0.2{Delta}2, 0.2{Delta}6 and 0.2{Delta}8 demonstrated increased expression, whereas 0.2{Delta}9 was slightly decreased (Fig. 3). These data strongly suggest an interaction between stem-loop X and the polypyrimidine tract. Purine-rich sequences within stem-loop X may mediate this interaction (Fig. 5A).

Complementary sequences (nucleotides 1151–1157 within the polypyrimidine tract and upstream nucleotides 1138–1143) were predicted to form single-stranded bulges in 0.2 and may potentially form a pseudoknot (Fig. 5A, denoted PK). Deletion of these sequences in 0.2{Delta}pY and 0.2{Delta}2 correlated with the loss of IRES activity. Although these sequences are present in 0.2{Delta}4, 0.2{Delta}6, 0.2{Delta}8, and 0.2{Delta}9, they were not necessarily predicted to be in the same context as 0.2 (Fig. 5, D–F and data not shown).

When sequences between stem-loop X and the polypyrimidine tract were deleted, as in 0.2{Delta}4, expression increased (Fig. 3); however, RNase cleavage within stem-loop X, but not the polypyrimidine tract, changed (Figs. 6D and 7C). An alternative stem-loop structure corresponding to stem-loop X, designated stem-loop X', in 0.2 was predicted to include a GNRA tetraloop, a highly stable RNA motif (Fig. 5D). Therefore, stem-loop X is likely to be a significant structural element, forming a focal point for higher order RNA interactions. The significance of the polypyrimidine tract is less clear in the context of 1.2 considering that the data reported here demonstrate that the 1.0 construct can independently mediate internal ribosome entry (Figs. 1B and 2B). When additional sequences upstream of the polypyrimidine tract were also removed, as in 1.2{Delta}10 and 1.2{Delta}11, the expression levels were similar to those of 1.0 (Fig. 3B). Therefore, in the absence of a complete polypyrimidine tract, putative interacting sequences located upstream of the polypyrimidine tract may potentially facilitate the repression of 1.0-mediated IRES activity. Conversely, when these sequences were deleted, 0.2-mediated expression increased, whereas 1.2-mediated expression was minimally affected (Fig. 3).

Our data show that the mKv1.4 IRES is able to mediate translation initiation in cells derived from brain, heart, and skeletal muscle. These cell types are derived from tissues in which Kv1.4 mRNA expression has been shown to occur, suggesting that the IRES elements derived from such mRNAs are active in the "appropriate" cell types. Dicistronic assays in transfected cells in culture also demonstrated that mKv1.4 and CVB3 5'-NCR sequences displayed distinct cell type-specific IRES activities (Fig. 1B). Moreover, although the deletion of sequences within 0.2 similarly affected 0.2- and 1.2-mediated expression in three cell types, increases in expression were greater in PrCM and SK-N-SH cells than in HeLa cells (Fig. 3) (data not shown). Differences in the levels of mKv1.4 5'-NCR-mediated IRES activity may be attributed to the expression of cell-specific trans-acting factors.

Preliminary experiments suggest that 0.2 and 1.0 interact with a unique set of proteins.3 The 3' ~0.2 kb specifically interacted with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)4; however, the functional significance of this interaction has yet to be determined. In addition to its role in glycolysis, GAPDH has been linked to many other cellular processes (61). GAPDH has been reported to destabilize RNA duplex formation within the hepatitis A virus IRES and may modulate IRES activity in conjunction with polypyrimidine tract-binding protein (62, 63), a cellular protein required for other IRES elements (64–66). Secondary structure analysis supports the formation of single-stranded AU-rich regions within the polypyrimidine tract, stem-loop Y, and loop {Omega} (Fig. 5A). GAPDH has also been reported to selectively bind AU- and U-rich RNA sequences (67); however, specific sites of interaction between GAPDH and mKv1.4 5'-NCR sequences have yet to be determined. Ongoing studies are aimed at identifying sites of functional RNA-protein complexes within the mKv1.4 5'-NCR. Such structures may form nucleation sites for formation of IRES-dependent translation initiation complexes, perhaps in a cell-specific manner.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service Grant AI26765 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Predoctoral trainee supported by United States Public Health Service Training Grant GM07311. Back

Present address: Molecular Probes, Inc., Eugene, OR 97402. Back

** To whom correspondence should be addressed. Tel.: 949-824-7573; Fax: 949-824-2694; E-mail: blsemler{at}uci.edu.

1 The abbreviations used are: Kv, voltage-gated potassium; NCRs, noncoding regions; mKv1.4, mouse Kv1.4; IRES, internal ribosome entry site; PrCM, primary rat cardiac myocytes; CVB3, coxsackievirus B3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

2 L. E.-C. Leong and B. L. Semler, unpublished data. Back

3 L. E.-C. Leong, D. Negulescu, G. A. Gutman, and B. L. Semler, unpublished data. Back

4 L. E.-C. Leong, G. A. Gutman, and B. L. Semler, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Brandon Walter, Christopher Cornell, and Kristin Bedard for critical comments on the manuscript and Hung Nguyen for assistance in cell culture and DNA sequencing. We are grateful to MyPhuong Tran for contributions to generation of the initial dual-luciferase dicistronic constructs.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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