factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus

Factorless Ribosome Assembly on the InternalRibosome Entry Site of Cricket Paralysis Virus

Eric Jan and Peter Sarnow*

Department of Microbiologyand Immunology, StanfordUniversity School of Medicine299 Campus Drive, StanfordCA 94305, USA

The cricket paralysis virus (CrPV), a member of the CrPV-like virusfamily, contains a single positive-stranded RNA genome that encodestwo non-overlapping open reading frames separated by a short intergenicregion (IGR). The CrPV IGR contains an internal ribosomal entry site(IRES) that directs the expression of structural proteins. Unlike previouslydescribed IRESs, the IGR IRES initiates translation by recruiting 80 S ribo-somes in the absence of initiator Met-tRNAi or any canonical initiation fac-tors, from a GCU alanine codon located in the A-site of the ribosome.Here, we have shown that a variety of mutations, designed to disruptindividually three pseudoknot (PK) structures and alter highly conservednucleotides among the CrPV-like viruses, inhibit IGR IRES-mediatedtranslation. By separating the steps of translational initiation into riboso-mal recruitment, ribosomal positioning and ribosomal translocation, wefound that the mutated IRES elements could be grouped into two classes.One class, represented by mutations in PKII and PKIII, bound 40 S sub-units with significantly reduced affinity, suggesting that PKIII and PKIIare involved in the initial recruitment of the ribosome. A second class ofmutations, exemplified by alterations in PKI, did not affect 40 S bindingbut altered the positioning of the ribosome on the IRES, indicating thatPKI is involved in the correct positioning of IRES-associated ribosomes.These results suggest that the IGR IRES has distinct pseudoknot-likestructures that make multiple contacts with the ribosome resulting ininitiation factor-independent recruitment and correct positioning of theribosome on the mRNA.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: internal ribosome entry site; translation; RNA structure; cricketparalysis virus; translocation*Corresponding author

Introduction

Translation initiation in most eukaryotic mRNAsproceeds by a cap-dependent mechanism in which40 S ribosomal subunits are recruited to the capped50 end of the mRNA which is bound to the capbinding protein complex eIF4F.1 Factor eIF4F con-sists of the cap binding protein eIF4E, eIF4G, theadapter protein, eIF4G, and eIF4A, which belongsto the DEAD box class of RNA-dependentATPases.1 When tethered to the 50 cap, the 4G com-ponent of eIF4F mediates recruitment of 40 S sub-

units that carry eukaryotic initiation factor eIF3,eIF2-GTP and initiator Met-tRNAi. RNA-bound40 S subunits then scan the mRNA until an appro-priate start codon is encountered at which theGTPase activity of eIF2-GTP is activated by eIF5,resulting in the release of eIF2-GDP from the 40 Ssubunit and subsequent joining of the 60 S subunit,leading to the formation of an 80 S ribosome. This80 S ribosome contains the initiator Met-tRNAi cor-rectly positioned in the ribosomal P-site. Afteraccepting the first aminoacylated-tRNA in the ribo-somal A-site, peptide bond formation between thetRNA-attached amino acid residues ensues andelongation commences.

An alternate mode of translational initiation,internal ribosome entry (review2), allows therecruitment of 40 S subunits by internal ribosomeentry sites (IRES), which are highly structuredRNA elements located in the 50 untranslatedregions of certain viral and cellular mRNAs.

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:[email protected]

Abbreviations used: CrPV, cricket paralysis virus; IGR,intergenic region; eIF, eukaryotic initiation factor; PK,pseudoknot; IRES, internal ribosomal entry site; DMS,dimethyl sulfate; HCV, hepatitis C virus; RRL, rabbitreticulocyte lysate; PSIV, Plautia stali intestine virus.

doi:10.1016/S0022-2836(02)01099-9 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 324, 889–902

Although 40 S subunit recruitment by IRESelements is independent of eIF4E, in most casesother translation initiation factors are needed. Forexample, the poliovirus IRES requires eIF4A, eIF2-GTP/met-tRNAi, eIF3 and at least the C-terminalfragment of eIF4G to recruit 40 S ribosomes.2 Incontrast, the hepatitis C virus (HCV) and theclassic swine fever virus IRES can bind 40 S sub-units directly, but still require eIF2-GTP/Met-tRNAi for proper 40 S positioning of the startAUG codon in the ribosomal P-site, and eIF3 for60 S joining.3 Recent reports have shown that theHCV IRES makes multiple RNA–protein contactswith the 40 S subunit.4 – 8 Furthermore, associationof the 40 S subunit with the HCV IRES inducesconformational changes in the 40 S subunit,9

suggesting that IRES RNA elements have a directactive role in ribosome activity through RNA–protein interactions.

Recently, unusually divergent IRES elementshave been discovered in the cricket paralysisvirus-like (CrPV-like) virus family.10,11 For example,the CrPV contains a single positive-stranded RNAgenome which encodes two non-overlapping read-ing frames, each initiated by an IRES.11 Surpris-ingly, it was shown that the intergenic region(IGR) IRES elements located in both CrPV andPlautia stali intestine virus (PSIV), another memberof the CrPV-like viruses, did not require initiatorMet-tRNAi for translational initiation.10,12 In thecase of the CrPV IGR IRES, the ribosomal P-site isoccupied instead by a CCU triplet, which is base-paired with 50 upstream sequences present in a

conserved secondary structure element (see Figure1). Furthermore, the ribosomal A-site is occupiedby the neighboring GCU triplet that encodesalanine, the amino-terminal amino acid of thestructural precursor protein.12 Pairing between thesequences in the P-site and 50 IRES sequencesplays an essential role in IRES-mediated trans-lation, because mutations that disrupt the resultingpseudoknot (PK) structure abolished IGR IRESactivity, whereas compensatory mutations rescuedIRES activity.10,11

Like the HCV IRES, the CrPV IGR IRES can bind40 S subunits directly. However, unlike the HCVIRES, addition of 60 S subunits to IGR IRES–40 Scomplexes results in the formation of functional80 S ribosomes, indicating that the IGR IRES canassemble 80 S ribosomes in an unprecedentedmanner without the aid of any of the canonicaleIFs.12 Finally, toeprinting analyses in translation-competent extracts revealed that the IGR IRES canmediate a pseudo-translocation event, wherebythe ribosome performs the first translocation eventwithout forming a peptide bond. The chemistryby which this pseudo-translocation event occurs isunknown.

Alignment of the IGRs of the CrPV-like virusesrevealed that many nucleotides are conserved.Using mutational and compensatory mutationalanalysis, Nakashima and colleagues proposed atertiary model of the IGR IRES in PSIV, which canbe used as a working model for the IGRs of theother CrPV-like viruses.13 The model predictsseveral stem-loop structures that, through pairing

Figure 1. The predicted CrPVIGR IRES structure. Shown is aschematic diagram of the predictedstructure of the IGR IRES. Blue let-tering indicates nucleotides thatform pseudoknot I (PKI), rednucleotides indicate pseudoknot II(PKII) and green nucleotides repre-sent pseudoknot III (PKIII).Magenta-colored nucleotides (nt6107–6881) indicate stem-loop 1(SL1). Helical regions are indicatedby a black dash between nucleo-tides. Toeprints of bound 40 S and80 S on the wild-type IGR IRES areshown by the arrows and indicatedby toeprint A and B. The exactpositions of the toeprints are indi-cated in parentheses. The locationof the triplet codon in the P andA-sites of the ribosome can beinferred by the position of the toe-prints. P and A refer to the tripletsequences that occupy the riboso-mal P and A-sites of 40 S and80 S-bound wild-type IGR IRES.Underlined sequences representthe first two amino acid residues inthe viral capsid protein.

890 Ribosome Assembly on the CrPV IRES

of loop sequences with non-contiguous sequences,form three overlapping PK structures (Figure 1:PKI, PKII and PKIII). Here, we have created apanel of mutations within the IGR IRES in CrPVto identify nucleotides and structural elementsessential for IGR IRES-mediated translation. Wehave identified elements in the IGR IRES that areessential for 40 S binding and for positioning of40 S and 80 S ribosomes in the IGR IRES. Theresults suggest that multiple contacts between theribosome and the IGR IRES have distinct roles inIGR IRES-mediated translation initiation.

Results

The CrPV IGR IRES folds into a triplepseudoknot structure

To test whether the predicted structure of theintergenic IRES (Figure 1) represented the majorityof the molecules in solution, dicistronic RNAs con-taining the IGR IRES in the intercistronic spacerregion were treated with chemical and enzymatic

reagents. Specifically, dimethyl sulfate (DMS),kethoxal and RNase T1 were used to detect single-stranded regions in the IRES. DMS methylates Aresidues at N-1 and C residues at N-3; kethoxalreacts with and forms adducts with N-1 and N-2of G residues.14,15 RNase T1 cleaves 30 to unpairedG residues. To detect helical regions in the IRES,template RNA was treated with cobra venomRNase V1, which cleaves nucleotides in double-stranded or paired nucleotides.14,15 Treateddicistronic RNAs were incubated with oligodeoxy-nucleotides that annealed 120 or 26 nucleotidesdownstream of C6214 in the IGR IRES (Figure 2).After addition of reverse transcriptase, thepositions of modified or cleaved nucleotides in theentire IGR IRES were determined by primer exten-sion analysis (Figure 2(a) and (b)). Here, number-ing refers to the chemically modified nucleotide orto the nucleotide whose 30 phosphodiester bondwas enzymatically cleaved.

Results from chemical and enzymatic treatmentof IRES nucleotides 6027–6156 are shown in Figure2(a) and of IRES nucleotides 6123–6217 can beexamined in Figure 2(b). A summary of the

Figure 2. Chemical andenzymatic probing of the IGR IRES.(a). Dicistronic RNAs containingthe IGR IRES were treated withKethoxal (lane 2), DMS (lane 4),RNase V1 (lane 6) or RNase T1

(lane 8). Lanes 1, 3, 5 and 7 showuntreated RNAs in parallel for eachcondition. Primer extension wasperformed as described in Materialsand Methods, using oligo PrEJ69which anneals 120 nt 30 to C6214.The reaction products were separ-ated on denaturing polyacrylamidegels. Shown are nucleotides 6027–6156 of the IGR IRES. Numberingof the nucleotides are referred fromthe sequence of the IGR IRES in theCrPV cDNA.11 Nucleotides indi-cated to the right of the gel arenucleotides that were modified byKethoxal and DMS or nucleotidesthat were cleaved 30 to it by RNaseT1 and V1. A sequencing ladder ofthe dicistronic construct usingPrEJ69 as a primer is shown on theleft. (b) Same as (a), except primerextension was performed witholigo PrEJ94, which anneals 26 nt 30

to C6214. Shown are nucleotides6123–6217 of the IGR IRES.

Ribosome Assembly on the CrPV IRES 891

positions of the nucleotides that were susceptibleto chemical and enzymatic probing are indicatedin the predicted tertiary model (Figure 3), whichwas based on a predicted structure from the closelyrelated PSIV.13

Specifically, the IGR IRES was modified exten-sively by DMS and kethoxal (Figures 2 and 3).Modifications were distributed in all regions ofthe IRES and, for the most part, were found innucleotides that are predicted to reside in single-stranded regions. However, a few strong kethoxalmodifications were also found in nucleotides pre-dicted to be in helical regions (Figure 3), such asresidues GG6103-4 in PKIII and residues GG6188-9 inPKI. The structure shown in Figure 3 reflects these

observations; nucleotides GG6103-4 in PKIII and inGG6188-9 are marked with asterisks to indicate thatthey are unpaired at equilibrium in the absence ofproteins. Similar to the kethoxal modifications,cleavages in the IGR IRES by RNase T1 occurredmostly at guanosine residues predicted to residein single-stranded regions. Moreover, like thekethoxal modifications, strong RNase T1 cleavageswere also noted in guanosines predicted to residein helical regions, such as GG6103-4 in PKIII andGG6188-9 in PKI.

Cleavages of the IGR IRES by RNase V1 were,generally, in predicted helical regions, supportingthe predicted tertiary structure (Figure 3). How-ever, certain helical regions that were cleaved by

Figure 3. Summary of structuralprobing of the IGR IRES usingchemical and enzymatic techniques.Schematic diagrams of the structureof the IGR IRES are shown. (a) DMSand Kethoxal probing of the IGRIRES. Filled arrowheads indicatenucleotides modified by Kethoxaland open diamonds indicatenucleotides modified by DMS.Large and small symbols representstrong and weak modifications,respectively. Asterisks representnucleotides strongly modified bychemicals that do not fit the pre-dicted structure of the IGR IRES.(b) RNase T1 and V1 probing of theIGR IRES. Open arrowheads indi-cate sites of RNase T1 cleavage,whereas filled diamonds representsites of RNase V1 cleavage. E and Pindicate RNase T1 cleavage sitesthat were enhanced (E) or protected(P) on 40 S and 80 S-bound IGRIRESs.


RNase T1 and modified by kethoxal were alsoaccessible to RNase V1 cleavage. For example,nucleotides GG6188-9 in PKI were cleaved by bothenzymes that target single-stranded and helicalregions. The accessibility of helical regions to bothRNase T1 and V1 has also been reported inprevious structural probings of the HCV IRESand the CSFV IRES.5,6,16 However, nucleotidesUU6105-6,which link PKIII and SL1 (Figures 2(b)and 3(b)) and which are predicted to be in asingle-stranded region, were cleaved weakly byRNase V1, suggesting that these nucleotides areinteracting with other regions in the IRES. Takentogether, these findings suggest that, while thetriple-PK structure is valid, certain parts of theIGR IRES are not static but in a dynamicequilibrium.

Enzymatic probing of ribosome–IRES complexes

Toeprinting analyses have revealed that 40 Ssubunits make at least two contacts with the IGRIRES.12 Specifically, the 30 leading edges of 40 S sub-units could be mapped to nucleotides AA6161-2,designated as site B in Figure 1, and at nucleotidesCA6226-7, designated as site A in Figure 1, on theIGR IRES.12 These findings suggested that 40 Ssubunits bind PKI and PKIII in the IRES. Toexamine any changes in the RNase T1 enzymaticreactivity of the 40 S-bound IGR IRES, free IRESand 40 S–IRES complexes were treated withRNase T1 and the susceptibilities of guanosineresidues to nuclease attack were compared. Theonly G residues protected from RNase T1 cleavagewere G6144 and GG6103-4 in PKIII (Figure 4(a) and(b), lane 3). However, 40 S subunits enhancedRNase T1 cleavage at G6096, located in a single-stranded region between PKII and PKIII (Figure4(b), lane 3). Incubation of the IGR IRES with puri-

fied 60 S subunits, which do not bind to the IRES(not shown), did not significantly affect the nucle-ase cleavage pattern (Figure 4(a) and (b), lane 5),indicating that the protection of RNase T1 clea-vages was specific for the 40 S–IRES complexes.Together with the finding that binding of 40 S sub-units to the IRES reveal a toeprint at AA6161-2 atPKIII,12 these results suggest that 40 S subunitsbind directly to PKIII.

The addition of 60 S subunits to 40 S–IRES com-plexes results in the formation of 80 S–IREScomplexes, which can be detected in sucrosegradients12 or in gel shift assays (E.J. & P.S., unpub-lished results). However, the pattern of RNase T1

cleavage at G6144 and GG6103-4 did not change in80 S–IRES complexes as compared to 40 S–IREScomplexes (Figure 4(a) and (b), lane 4). Similarly,enhanced RNase T1 cleavage at nucleotide G6096

was also observed in 80 S–IRES complexes (Figure4(b), lane 4). These results suggest that bindingsites in PKIII remain intact in the 80 S–IRES com-plex. Surprisingly, nucleotides in PKI, whichoccupy the ribosomal P and A sites,12 and those inother regions of the IRES were not differentiallyaffected by RNase T1 in free, 40 S–IRES or 80 S–IRES complexes. This result indicates that free PKIand ribosome-bound PKI have similar structuralfeatures, as judged by enzymatic probing.

Generation and structural characterization ofIGR IRES elements with mutations in thepseudoknot structures

To investigate the roles of PKI, PKII and PKIII in40 S binding and subsequent IRES function, severalmutations were introduced into the IRES to alterthe predicted helical regions (Figure 5(a)).Changing CAC6148-50 to GUG (DPKIII) was pre-dicted to disrupt PKIII, mutating CCUCUC6165-70 toGGUGUG (DPKII) was predicted to alter PKII and

Figure 4. RNase T1 footprintingof 40 S–IGR IRES and 80 S–IGRIRES complexes. Shown are IGRIRES sequences from 6027–6156 in(a) and from 6123–6217 in (b).Lanes 1 and 6 represents dicistronicRNAs containing the IGR IRES withno treatment. RNAs incubatedalone (lanes 2 and 7), or with 40 S(lanes 3 and 8), or with 80 S (lanes4 and 9), or with 60 S (lanes 5 and10) were treated with RNase T1.Shown to the right of the gels arerelevant RNase T1 cleavage sites onthe IGR IRES that were eithersignificantly enhanced (E) orprotected (P) when bound to 40 Sor 80 S ribosomes. Note that theRNase T1 cleavage sites on the IGR

IRES were not affected significantly when incubated with 60 S subunits (lanes 5 and 10). Primer extension was per-formed using oligo PrEJ94 in (a) or oligo PrEJ69 in (b). See Materials and Methods for oligonucleotide sequences. Thesequence of the IGR IRES is shown to the right.


Figure 5. Location of IGR IRES mutations and their effects on IGR IRES structure. (a) Summary of mutations intro-duced into the IGR IRES. Nucleotides that have been altered are boxed and the exact alterations are indicated in par-entheses. Toeprints of bound 40 S and 80 S on the wild-type IGR IRES are shown by the arrows (A and B).Nucleotides in pseudoknots PKI, PKII and PKIII are shown in blue, red and green, respectively. Nucleotides in stem-loop from nt 6107–6118 are in magenta and denoted as SL1. (b) RNase T1 cleavage sites in wild-type and mutantIRESs. Dicistronic RNAs containing wild-type IGR IRES (lanes 1 and 2) or mutant IRESs CC6214-5GG (lanes 3 and 4) orCAC6148-50GUG (lanes 5 and 6) were incubated with or without RNase T1. Only the cleavage sites that were differentbetween wild-type and mutant IRESs are shown (right of each gel). Primer extension analysis was performed asdescribed in Materials and Methods, using oligo PrEJ69. A sequencing ladder of the dicistronic construct usingPrEJ69 as a primer is shown on the left.


changing CC6214-5 to GG (DPKI) was predicted todisrupt PKI (Figure 5(a)). To determine whetherthe mutations introduced resulted in global mis-folding of the IRES, the structures of the mutatedIRESs were examined by enzymatic probing usingRNase T1 (Figure 5(b)). RNase T1 only affected resi-dues surrounding the introduced mutations. For

example, disruption of PKI (DPKI) by mutatingnucleotides CC6214-5 to GG enhanced RNase T1

cleavages at the mutated residues GG6214-5 but hadno significant effect on cleavage of other residues(Figure 5(b), lane 3 and 4). Similarly, mutant IRESDPKIII, which alters nucleotides CAC6147-50 toGUG enhanced RNase T1 cleavages only at sur-rounding G6150 and at G6138 (Figure 5(b), lanes 5and 6). The enhanced cleavages at G6150 and atG6138 are in agreement with the disruption of thehelical region in PKIII. Because the mutations didnot appear to disrupt the overall structure of theIRES, the mutant IRESs were used to examine thefunctional roles of the individual PK structures.

Functional roles of PKI, PKII and PKIII in IGRIRES-mediated translation

The translational activities of the IGR IRES PKmutations were tested in the rabbit reticulocytelysate (RRL) using dicistronic mRNAs that con-tained the wild-type and mutant IGR IRESelements in the intercistronic spacer region. Thetranslational efficiencies of IRESs with mutationsthat disrupted PKIII (DPKIII), PKI (DPKI) or PKII(DPKII) were all diminished by approximately90% (Figure 6 and Table 1). Compensatorymutations that restored the stem-loop in PKIII bymutating GUG6138-40 to CAC (comp DPKIII) rescuedactivity to approximately 50% of wild-type IRESactivity. Similarly, compensatory mutations thatrestored basepairing in PKI by mutating GG6188-9

to CC (comp DPKI) and in PKII by mutatingGAGAGG6062-67 to CUCUCC (comp DPKII) rescuedtranslational activity to 50% and 25%, respectively,

Figure 6. Translational activity of mutant IRESs inRRL. Dicistronic RNAs containing the wild-type ormutant IGR IRESs were incubated in the RRL. The firstcistron, encoding renilla luciferase, measures cap-dependent translation and the second cistron measuresIGR-IRES mediated translation. Shown is the ratio of fire-fly luciferase to renilla luciferase, normalized to the ratioof a dicistronic RNA containing no IRES in the inter-cistronic spacer region (designated as empty). The renillaluciferase activities did not vary significantly betweenexperiments. Shown are the averages of at least threeindependent experiments. The wild-type and mutantIRESs are indicated below the graph. Mutant IRESs thatcontain compensatory mutations are designated ascomp.

Table 1. Summary of properties of the IGR IRES mutants

Mutant IGRIRESsa

40 S bindingb

KD (nM)Translational activityc

(fold luc ratio)Purified 40 S toeprint

Bd AA6161-2

Purified 40 S toeprintAd CA6226-7

Pseudo-translocatione

WT IGR IRES 24 ^ 6 30 ^ 9 þ þ þDPKI 32 ^ 12 1.3 ^ 0.3 þþ 2 ndcomp DPKI 22 ^ 1 17.7 ^ 3.6 þ þ þDPKIII 93 ^ 39 2.1 ^ .04 2 ^ ndcomp DPKIII 27 ^ 13 17.6 ^ 3.9 þ þ þDPKI/DPKIII .500 2.2 ^ 0.3 2 2 ndDPKII 94 ^ 21 1.5 ^ 0.4 þ 2 ndcomp DPKII 44 ^ 20 5.5 ^ 0.9 þ þ þSL1 D loop 49 ^ 23 2.7 ^ 0.4 ^ þ naPKIII DCAloop

54 ^ 4 1.8 ^ 0.3 ^ þ na

PKIII DAAloop

28 ^ 15 7.0 ^ 1.4 ^ þ na

a WT indicates wild-type.b 40 S binding affinities were performed as described in Materials and Methods. Apparent KD measurements were quantified by

phosphoimager analysis and data plots were fitted to a high affinity one binding site model by Sigma plot. Shown are results fromat least three independent experiments.

c Translational activity of dicistronic RNAs in RRL. Shown is the fold ratio of firefly luciferase to renilla luciferase activities normal-ized to the fold ratio of an empty dicistronic RNA. Values are from at least three independent experiments.

d Presence of toeprints B (AA6161-2) and A (CA6226-7) on wild-type and mutant IGR IRES RNAs when bound to 40 S subunits. “ þ ”indicates a toeprint is present. “ 2 ” indicates no toeprint. “þþ” values indicates enhanced toeprint compared to the toeprint on thewild-type IGR IRES RNA. “ ^ ” indicates a weak toeprint.

e Presence of toeprint TAC6231-3 on wild-type and mutant IGR IRES RNAs in RRL containing cycloheximide. “nd” represents no toe-print is detected indicating lack of ribosome binding to the IRES. “na” indicates that the experiment was not performed.


of wild-type IRES activity (Figure 6). These resultsdemonstrated that the structural integrity of thethree PK structures, but not their precisenucleotide sequences, are essential for IRESactivity.

In addition, we examined the roles of highly con-served residues located in predicted loop regions,in IRES-mediated translation. Mutations at con-served nucleotides AUUU6111-4 (SL1 D Loop) inSL1 and CA6142-3 (PKIII DCA Loop) in PKIII (Figure5(a)) disrupted translational activity of the IRES bygreater than 90% (Figure 6 and Table 1). Changingconserved nucleotides AA6151-2 to UU (PKIII DAALoop) in PKIII reduced translational activity byapproximately 75% (Figure 6 and Table 1). Giventhat the 40 S and 80 S ribosomes protected nucleo-tides in PKIII from RNase T1 cleavage (Figure 4),and that these conserved nucleotides are within orclose to PKIII, it is likely that direct contactsbetween the ribosome and these nucleotides areimportant for IGR IRES-mediated translation.

Roles of the pseudoknot structures inrecruitment 40 S subunits

To investigate which steps in translationinitiation were impaired in the mutated IRESelements, we first examined whether recruitmentof 40 S subunits was affected. We recently devel-oped an agarose gel shift assay 17 to measure40 S–IRES affinities. Incubation of increasingamounts of 40 S subunits with radiolabeled

wild-type IGR IRES produced a slower migrating40 S–IRES complex shift (Figure 7(a)). Little radio-activity was present in the wells, indicating thatthe RNAs did not aggregate significantly underthese conditions (Figure 7(a)). The IGR IRESbound to the 40 S subunit with an apparent KD

value of 24(^6) nM (Figure 7(a) and Table 1). Asimilar KD value was observed when the concen-tration of the IGR IRES RNA was varied or incu-bation times were altered, indicating thatmeasurements were made under equilibrium con-ditions (data not shown). The interaction between40 S subunits and the IGR IRES was specific,because incubation of RNA containing anotherIRES, the EMCV IRES, did not bind to 40 S, andthe CrPV IGR IRES did not form complexes with60 S subunits (data not shown).

Toeprinting analysis12 and mutagenesis (seeabove) have indicated that 40 S subunits can bindto both PKI and PKIII in the IGR IRES. Surpris-ingly, 40 S subunits bound to the DPKI mutantIRES, which abolished IRES-mediated translation(Figure 6), with an apparent KD value(32(^12) nM) that was similar to wild-type (Figure7 and Table 1). The compensatory PKI mutation(comp DPKI), which mutates GG6188-90 to CC andpartially restored translation (Figure 6), displayedsimilar affinity for 40 S (KD ¼ 22(^1) nM) (Table1). Thus, mutated PKI can recruit 40 S subunits.

In contrast, the affinity of 40 S subunits formutant IRES elements DPKIII and DPKII, whichdisrupted the stem-loop in PKIII and base-pairing

Figure 7. Affinity measurementsof 40 S–IGR IRES complexes. (a)Gel mobility shift assays of 40 S–IRES complexes. RadiolabeledRNAs were incubated with increas-ing amounts of purified 40 S sub-unit. The amount of 40 S subunit isindicated at the top. Free RNA isindicated by arrowheads, the 40 S–IRES complex is denoted by a largearrow, and the well of the gel isdenoted with a smaller arrow. Phos-phoimage analyses of the agarosegels are shown. (b) Quantitation of40 S–IGR IRES complex formation.Shown is a representative of fourexperiments using different IRESs.Curve fitting was performed usingSigmaplot.


in PKII, respectively, was reduced (Figure 7 andTable 1). Compensatory mutations that restoredthe base-pairing in the stem-loop in PKIII and thePK in PKII restored 40 S binding (Table 1; compDPKIII and comp DPKII). These results indicatethe reduced 40 S affinity correlated with theobserved loss of IRES activity in these IRESmutants (Table 1).

Interestingly, an IRES containing a doublemutant IRES, DPKI/DPKIII, which disrupts PKIand PKIII, completely abolished 40 S binding aswell as translation activity (Figure 6 and Table 1).The apparent KD value for the double mutant IRESwas not measurable under these experimental con-ditions, indicating that the KD value is greater than500 nM. In addition, in a competition assay,unlabeled mutant DPKI/DPKIII IRES RNAs didnot compete for binding of 40 S subunits fromradiolabeled wild-type IRES molecules, whereas,as expected, unlabeled wild-type IGR IRES RNAswere able to compete (data not shown). On theother hand, IRESs with mutations in conservednucleotides, SL1 D loop, PKIII DCA loop andPKIII DAA loop, did not affect 40 S binding signifi-cantly, but had little IRES activity (Table 1). Theseresults demonstrated that the structure of PKIII isessential for 40 S recruitment with PKI providingadditional stabilization of 40 S binding. Moreover,other conserved parts of the IRES are not essential

for 40 S recruitment, but their integrities areindispensable for subsequent steps in translationinitiation.

Functions of the pseudoknot structures inrecruitment and positioning of 80 S complexes

Because certain mutated IRES elements, such asmutant DPKI IRES, were translationally inactive,but not deficient in 40 S subunit binding, we testedthe next steps in translation initiation, assemblyand positioning of 80 S complexes. Assembled40 S and 80 S complexes block the movement ofreverse transcriptase on an RNA, leading to cDNAarrests or toeprints of the 30-most sequences con-tacted by the ribosome. Two toeprints, termed Aand B were observed at nucleotides CA6226-7 andAA6162-2, respectively, on wild-type IGR IRESRNAs bound to either 40 S or 80 S ribosomes(Figure 8, lanes 2 and 4).12 In contrast, these specificprimer extension arrests were not detected onnaked RNAs or when 60 S subunits were added(Figure 8, lanes 1 and 3).

In mutant DPKIII IRES, the toeprints at both sitesA and B were reduced significantly (Figure 8, lanes6 and 7), in agreement with its reduced affinity of40 S binding (Table 1). The toeprints could berestored by compensatory mutations (Figure 8,lanes 9 and 10; comp DPKIII). In mutant IRESs,

Figure 8. Toeprint analysis of 40 S and 80 S complexes. 40 S subunits, or 40 S and 60 S subunits, were incubated withdicistronic RNAs containing wild-type or mutant IRESs and then analyzed by primer extension analysis. The reactionproducts were separated in denaturing polyacrylamide gels. An autoradiograph of the gel is shown. Wild-type andmutant IGR IRESs are indicated at the top of the Figure comp denotes the mutant IRESs that have compensatorymutations which restore stem-loops and pseudoknot structures. The two major toeprints (A and B) are indicated atright. Reference lanes T, G, C and A depict IGR sequences.


DPKI and DPKII, toeprint A, but not toeprint B wasabolished (Figure 8, lanes 12, 13, 21 and 22). Toe-prints at both A and B sites were restored by theirrespective compensatory mutations (Figure 8,comp DPKI and comp DPKII). The mutant IREScontaining the double mutation, DPKI/D PKIII,which disrupts PKI and PKIII, abolished all toe-prints, which correlates with the lack of 40 S bind-ing (Figure 8, lanes 18 and 19; Table 1).

Finally, mutations of conserved nucleotideslocated in loop regions and bulged regions nearPKIII did not significantly affect toeprint A, butreduced toeprint B. This suggests that thesemutations, like the DPKIII mutations, weaken con-tacts between the ribosome and PKIII (toeprint B),while not affecting contacts between the ribosomeand regions near PKI, giving rise to toeprint A(Figure 8, lanes 27, 28, 30, 31, 33 and 34). Theseobservations suggest that the presence of both toe-prints at A and B correlates with translationalactivity (Table 1). Furthermore, the locations ofmutations in the IRES and their effects on the toe-printing pattern suggest a model in which ribo-somes, initially assembled at PKIII, aresubsequently re-positioned to PKI where trans-lation commences.

Participation of IGR IRES elements in the firstpseudo-translocation step

For the mutant IRES elements, it was possiblethat some of the assembled 80 S–IRES complexeswere non-functional in vitro. Therefore, weexamined the formation of 80 S–IRES complexes

and their first pseudo-translocation step in theRRL.12 Incubation of IGR IRES-containing dicis-tronic luciferase RNA in the RRL revealed a strongtoeprint at site A in the presence of edeine (Figure9, lane 1), which, at this concentration, inhibits thedelivery of an aminoacylated tRNA to the ribo-somal A-site,12,18,19 and was shown to inhibit thefirst pseudo-translocation step in the IGR IRES.12

Upon incubation of RNAs in the RRL in thepresence of cycloheximide, which allows trans-location but inhibits elongation in IGR IRES-mediated translation,12 a strong toeprint wasobserved at TAC6231-33, which is approximately sixnucleotides 30 of the now weaker toeprint at site A(Figure 9, lane 2). Thus, appearance of a toeprintat TAC6231-33 can be used as an indicator that theribosome has undergone an initial translocationstep, in this case, in the absence of formation of apeptide bond.

Mutations that disrupted the structures of PKIIIor PKII (mutant IRESs DPKII or DPKIII) or thedouble mutant DPKI/DPKIII did not produce toe-prints in the RRL (Figure 9, lanes 3, 7 and 8), eventhough the DPKII mutant IRESs still produced toe-print B with purified ribosomal subunits (Figure 8,lanes 21 and 22). Compensatory mutations thatrestored these structures restored both toeprint Aand the downstream toeprint TAC6231-33 (Figure 9,lanes 4, 6 and 9). These results are consistent withthe idea that PKIII and PKII are important for 40 Sand 80 S recruitment.

In contrast, disruption of PKI (mutant IRESDPKI) resulted in loss of toeprints A and TAC6231-33

in RRL (Figure 9, lane 5). However, as was

Figure 9. Toeprint analysis ofribosomal complexes formed in thein RRL. Dicistronic RNAs wereincubated in RRLs in the presenceof edeine (lane 1) or with cyclo-heximide (lanes 2–12) andanalyzed by primer extensionanalysis. The reaction productswere separated in denaturing poly-acrylamide gels. An autoradio-graph of the gel is shown. Themutant IRESs are indicated atthe top of the Figure comp denotesthe mutant IRESs that have com-pensatory mutations which restorestem-loops and pseudoknot struc-tures. The major toeprints (A, Band TAC6231-3) are indicated atright. Reference lanes T, G, C andA depict IGR sequences.


observed in the assembly of IRES–subunit com-plexes from purified components (Figure 8, lanes11–13), toeprint B was still observed (Figure 9,lane 5). Compensatory mutations resulted in weak-ening of toeprint B and appearance of toeprintTAC6231-6233 (Figure 9, lane 6). These results are inagreement with a model whereby ribosomal sub-units first contact PKIII and then re-position to con-tact PKI where translation initiation begins. It isunlikely that ribosomes bound at PKIII facilitatethe recruitment of a second ribosome to PKIbecause complexes larger than 80 S are notobserved for any of these IRES elements in sucrosegradients12 and in gel shift assays (data not shown).

Discussion

Architecture of the CrPV IGR IRES

IGR IRES-mediated translation has the unusualproperty that translation initiation occurs from theA-site of the ribosome in the absence of initiationfactors and initiator Met-tRNAi.

12 Specifically, aCCU triplet, engaged in a PK-like structure,occupies the ribosomal P-site, thereby positioningthe next GCU triplet into the ribosomal A-sitefrom which translation initiation commences.Comparison of the IGRs in members of the CrPV-like virus family revealed many conserved nucleo-tides and three conserved PK-like structures.These features allowed a prediction of a tertiarystructural model for CrPV-like IGR IRES elements20

which, in this study, was largely supported bystructural probing. Mutational analysis of theseconserved regions was performed to identifyelements that are essential for 40 S binding and forIGR IRES-mediated translation. On the basis oftheir properties in electrophoretic mobility shiftand toeprinting assays, the mutated IRESs couldbe grouped into two classes that affected differentaspects of IGR IRES-mediated ribosome recruit-ment and utilization.

One class of IRES mutations, located in PKIIIand PKII, displayed reduced binding affinity for40 S subunits and loss of IRES activity. Forexample, disruption of the base-pairing in thestem-loop of PKIII led to a fourfold decrease in40 S affinity (Table 1). Similarly, toeprint analysesin the RRL showed that assembly of 80 S com-plexes was less efficient with mutants in PKII andPKIII than with wild-type IRESs. The loss of ribo-some binding was not due to an overall disruptionof the IGR IRES structure by the mutations,because only the PK structure in the vicinity of theintroduced mutation was disrupted (Figure 5(b)).This suggests that the PK structures can fold inde-pendently and that the IGR IRES may be quiteflexible.

A second class of mutations, exemplified bychanges in PKI, did not reduce 40 S binding affi-nity but altered the position of the ribosome onthe IRES. Wild-type IGR IRES produced two

primary toeprints, toeprint A at CA6226-7 and toe-print B at AA6161-2 when bound to 40 S or to 80 Sribosomes.12 While the DPKI mutation did notaffect 40 S binding affinity in gel shift assays, theyabolished the 40 S and 80 S at toeprint A (Figure8). Instead, this mutant IRES only displayed toe-print B, which was enhanced. These findingssuggest that the major contacts for the initialrecruitment of 40 S subunits are in domains ofPKIII. In support of this idea, footprinting analysisshowed that nucleotides in PKIII were protectedfrom RNase T1 cleavage in both 40 S–IGR IRESand 80 S–IGR IRES complexes (Figure 4). In con-trast, nucleotides in PKI were not protected andremain accessible to RNase T1 cleavage, indicatingthat the ribosome is not bound tightly to thisregion of the IGR IRES.

Interestingly, the DPKI/DPKIII double mutationabolished the formation of both 40 S–IRES and80 S–IRES complexes in toeprinting analysis(Figures 8 and 9). By gel shift analysis, the appar-ent KD value for 40 S binding was not measurable,indicating a very low affinity for 40 S. Given thatDPKIII reduced 40 S binding by only fourfold andthat DPKI did not affect 40 S binding (Table 1), itwas surprising that the double mutation DPKI/DPKIII abolished all binding. One possibility isthat the double mutation disrupted the overallstructure of the IRES. However, RNase T1 struc-tural probing revealed that the mutations in bothPKI and PKIII only disrupted the structures sur-rounding the mutations (data not shown). Asecond possibility is that upon binding to PKIII,the ribosome undergoes a conformational changeand repositions itself to bind to PKI. This confor-mational change and repositioning of the ribosomecould be facilitated by the binding energy from thePKI–ribosome interaction. In the DPKI mutantIRES, the intact PKIII can still bind to the ribosomewith no loss in affinity. In contrast, in the DPKIIImutant IRES, PKIII can no longer interact with theribosome, therefore the conformational change inthe ribosome, induced by PKI, cannot occur,suggesting that the overall ribosome affinity to themutant DPKIII IRES results from the bindingenergy made by the PKI–ribosome interaction.When both PKI and PKIII are disrupted, thebinding affinity decreases even more. Wepropose that upon binding to PKIII, the ribosomeis re-positioned to PKI so that the GCU alaninecodon is in the ribosomal A-site.

Mutant DPKII IRES also affected the position ofthe ribosome on the IRES. This mutation, like theDPKI mutation, reduced toeprint A near theinitiation codon but not toeprint B, suggesting thatPKII is also important for correct positioning ofthe IRES in the ribosomal P-site. Because thismutation also reduced binding to 40 S, itsuggests that PKII may have dual roles in recruit-ing and positioning the ribosome on the IRES.PKII may serve as a bridge between the majorribosome-binding site in PKIII to position the ribo-some at the initiation codon within PKI.


The overall picture that emerges is that the IGRIRES contains two structural determinants, PKIIIand PKI. These have different roles in the mechan-ism of IGR IRES-mediated translation: PKIII is themajor determinant for ribosome recruitment andPKI is essential for positioning the ribosome at theinitiation GCU alanine codon.

Comparison of IRES elements that can directlyrecruit 40 S subunits

Like the CrPV IGR IRES, the HCV and CSFVIRESs can recruit 40 S subunits in the absence ofinitiation factors. A common feature betweenthese three IRESs is that they contact the 40 S sub-unit at multiple sites. Chemical and enzymaticfootprinting of 40 S–HCV IRES complexesrevealed that multiple contacts are made near thestart AUG codon, which is embedded in a PKstructure.5,16 Interestingly, the IGR IRES also con-tains a PK-like structure near the GCU alaninestart codon. Thus, a general feature of this type ofIRES is that PK structures are important for recruit-ment and positioning of the 40 S at the start codon.Recently, a cryo-electron microscopic image wasobtained of 40 S–HCV IRES complexes, whichshowed that the HCV IRES contacts the ribosomalE-site, which harbors the deacylated tRNA.9 Inanalogy with the HCV IRES, we predict that thePKI occupies the P-site of the ribosome, such thatthe GCU alanine codon is in the A-site of theribosome.12 Given the extensive contacts of theIGR IRES structure with the 40 S, especially inPKIII, it is predicted that PKII and PKIII mayoccupy the E-site of the ribosome.

The IGR IRES is dynamic

Several regions in the CrPV IGR IRES that arepredicted to be helical were found to be accessibleto both RNase T1 and RNase V1. Strong cleavagesby RNases T1 and V1 were observed in the pre-dicted helical region of PKI near the site ofinitiation (Figure 2(b)). Similarly, PK structuresnear the start AUG codon in the HCV and CSFVIRESs have been reported to be accessible to bothenzymes.5,6,16 These observations may indicate thatthe PK structures of these IRESs are not static, butin fact, flexible and dynamic. Previous reportshave suggested that this property may be a generalfeature of RNA PKs.5,6,16,21,22 For example, NMRstudies revealed that the PK of the tRNA-like struc-ture of the turnip yellow mosaic virus has a predis-position to partially unfold and is flexible, whichmay be important for regulating protein binding.22

It has been suggested that flexibility in the PKregion of the HCV IRES may allow the IRES to fitin the mRNA cleft of the ribosome for proper ribo-some attachment to the initiation codon.9 For theIGR IRES, the flexibility of the IRES may allowPKI to fit in the P-site of the ribosome. Alterna-tively, because the IGR IRES undergoes a transloca-tion event in the absence of peptide bond

formation, the dynamic nature of the IRES mayfacilitate the detachment of the ribosome from theIRES during translocation. Identification ofelements in the IGR IRES that modulate thepseudo-translocation event will distinguishbetween these possibilities.

Materials and Methods

DNA constructs

The dicistronic luciferase plasmid and the mono-cistronic luciferase plasmid containing the IGR IREScDNA have been described.11 Mutated IGR IRESs weregenerated using the Quickchange kit (Stratagene).Mutations in the IGR IRES were confirmed bysequencing.

In vitro transcription and translation

For in vitro transcription of dicistronic RNAs, dicis-tronic luciferase plasmids were linearized with Bam HI.For in vitro transcription of monocistronic wild-type andmutated RNAs, plasmids were linearized with Nar I,which cleaves 33 nucleotides downstream of the startATG codon of the firefly luciferase ORF gene. Wild-typeand mutant IRES RNAs were gel purified. The integrityand purity of the RNAs were confirmed using gelanalysis. Uncapped dicistronic RNAs were translated inthe RRL in the presence of 154 mM potassium acetate.11

40 S and 60 S subunit purification

Ribosomal subunits were purified from HeLa cellpellets (National Center for Research Resources) asdescribed.8 HeLa S3 spinner cells were lysed in a TritonX-100 lysis buffer (15 mM Tris–HCl (pH 7.5), 300 mMNaCl, 6 mM MgCl2, 1% (v/v) Triton X-100, 1 mg/mlheparin). Lysates were spun briefly to remove debrisand the supernatant layered on a 30% (w/w) 0.5 M KClsucrose cushion and centrifuged at 100,000g to pelletribosomes. Ribosomes were resuspended in buffer B(20 mM Tris–HCl (pH 7.5), 6 mM magnesium acetate,150 mM KCl, 6.8% (w/v) sucrose, 1 mM DTT), treatedwith puromycin to release ribosomes from mRNA23 andKCl was added to a final concentration of 0.5 M. The dis-sociated ribosomes were then separated on a 10–30%(w/w) sucrose gradient. The 40 S and 60 S peaks weredetected at 260 nm, pooled, concentrated using Centri-con 50 spin concentrators (Amicon) in buffer C (20 mMTris–HCl (pH 7.5), 0.2 mM EDTA, 10 mM KCl, 1 mMMgCl2, 6.8% sucrose) and stored at 280 8C. Western blotanalysis verified the absence of any eIF2 contamination.The purity of 40 S and 60 S was also examined by detect-ing 18 S and 28 S rRNA by ethidium staining. The con-centration of 40 S and 60 S subunits was determined byspectrophotometry, using the conversions 1A260 nm ¼ 50 nM for 40 S and 1 A260 nm ¼ 25 nM for 60 Ssubunits.24

Non-denaturing gel mobility shifts

Conditions for native gel shift assays were adaptedfrom Lorsch & Herschlag.17 The 50 end-labeled RNAs(0.5 nM final concentration) were incubated in buffer E(20 mM Tris (pH 7.5), 100 mM potassium acetate,


2.5 mM magnesium acetate, 0.25 mM spermidine, 2 mMDTT) plus 50 ng/ml of non-competitor RNA at roomtemperature. Non-competitor RNA represents nucleo-tides 880–948 from pcDNA3 vector. Gel shifts were per-formed in 0.5% (w/v) agarose gels in THEM buffer(66 mM Hepes acid, 34 mM Tris acid, 2.5 mM MgCl2,0.1 mM EDTA, final pH 7.5) at room temperature. RNAswere incubated with increasing amounts of 40 S subunitsfrom 0.5 nM to 600 nM. Gel-mobility shifts were quanti-tated by phosphoimager analysis and data was fit for aone binding site between the IGR IRES and 40 S usingSigma plot. Affinity measurements are based upon atleast three independent experiments.

Chemical and enzymatic probing

Ribosomal complexes were assembled on 0.5 pmol ofIGR IRES RNA in 50 ml reactions in buffer E with either100 nM 40 S, 100 nM 60 S or 100 nM 40 S and 100 nM60 S. Reactions were incubated for ten minutes at 30 8C.For chemical probings, 2.5 ml of DMS at a 1:6 dilutionwith ethanol was added or Kethoxal was added to afinal concentration of 1.8 mg/ml. The reactions wereincubated for a further ten minutes at 30 8C, and theRNA was extracted as described.15 For enzymatic prob-ings, free or ribosome-bound IRES RNAs were digestedwith either RNase T1 (Ambion) or RNase V1 (AmershamPharmacia Biotech) at a final RNase T1 concentrations of0.027 unit/ml for free RNA or 0.041 unit/ml for ribo-some-bound RNA, and final RNase V1 concentrationsof 0.022 unit/ml for free RNA or 0.033 unit/ml for riboso-mal-bound RNA. RNAs were extracted with phenol/chloroform and ethanol precipitated as described.15 The50 end-labeled primer PrEJ94 50-GCCTTTCTTTATGTTTT-TGGCG-30, which anneals 26 nucleotides 30 to C6214, orprimer PrEJ69 50-GCCCTGGTTCCTG GAACAATTGCTT-30, which anneals 120 nt 30 to C6214, were annealedto the RNA and extended by AMV reverse transcriptase(Promega) as described.15 cDNA products were analyzedon 6% (w/v) polyacrylamide/8 M urea gel.

Assembly and analysis of ribosomal complexes

Toeprinting analysis of ribosomal complexes usingpurified subunits was performed as described.12 The0.5 mg of dicistronic IGR IRES RNAs were first annealedwith primer PrEJ69 in 40 mM Tris (pH 7.5) and 0.2 mMEDTA by slow cooling from 65 8C to 35 8C. AnnealedRNAs were incubated in buffer E containing either 40 S(final 40 nM), 60 S (40 nM) or 40 S and 60 S subunits(both 40 nM). Ribosomal complexes were analyzed byprimer extension analysis as described25 using AMVreverse transcriptase in the presence of [a-32P]dATP(3000 Ci/mmol; Amersham). Toeprinting analysis inRRL and sucrose gradient analysis were performed asdescribed.12

Acknowledgements

We thank Joan E. Wilson for contributions in thedevelopment of the gel shift assays and toeprintingexperiments. We are grateful to Dan Herschlag formany helpful discussions and to Karla Kirkegaardfor critical reading of the manuscript. This workwas supported by grants from the NIH (GM55979)

(to P.S.) and DRG-1630 of the Damon RunyonCancer Research Foundation (to E.J.).

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Edited by J. Doudna

(Received 21 August 2002; received in revised form 1 October 2002; accepted 3 October 2002)


factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus

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