The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs

Guenther, Ulf-Peter; Weinberg, David E.; Zubradt, Meghan M.; Tedeschi, Frank A.; Stawicki, Brittany N.; Zagore, Leah L.; Brar, Gloria A.; Licatalosi, Donny D.; Bartel, David P.; Weissman, Jonathan S.; Jankowsky, Eckhard

doi:10.1038/s41586-018-0258-0

Letter
Published: 27 June 2018

The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs

Ulf-Peter Guenther¹^na1,
David E. Weinberg^2,3,4^na1,
Meghan M. Zubradt^2,5^na1,
Frank A. Tedeschi¹,
Brittany N. Stawicki¹,
Leah L. Zagore¹,
Gloria A. Brar⁶,
Donny D. Licatalosi¹,
David P. Bartel^3,4,
Jonathan S. Weissman^2,5 &
…
Eckhard Jankowsky^1,7

Nature volume 559, pages 130–134 (2018)Cite this article

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Abstract

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation¹. Mutations in DDX3 are linked to tumorigenesis^2,3,4 and intellectual disability⁵, and the enzyme is targeted by a range of viruses⁶. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p–RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5′ untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5′ untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5′ untranslated regions.

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Main

To systematically analyse how Ded1p influences translation initiation in cells, we first examined how a mutation in the enzyme altered the spectrum of ribosome footprints in cells⁷. We used the ded1-95 mutation (Ded1p^T408I), which reduces the affinity of Ded1p for RNA, diminishes RNA unwinding and confers a temperature-sensitive growth defect to the budding yeast Saccharomyces cerevisiae⁸. The mutation does not affect pre-mRNA splicing or ribosome biogenesis⁹. We performed ribosome profiling on wild-type and ded1-95 strains before and after a temperature shift from 30 °C to 37 °C for 5 min (Extended Data Fig. 1a–h). The short time was chosen to minimize any secondary effects on ribosome footprints arising from broader translation defects.

At 30 °C, wild-type and ded1-95 strains showed virtually indistinguishable RNA expression and translation profiles (Extended Data Fig. 1i, j). After the temperature shift, translation broadly decreased in ded1-95, compared to the wild type (Extended Data Fig. 1k–n). These observations indicate that Ded1p promotes translation initiation for most mRNAs, consistent with previous findings^10,11. However, translation of a subset of mRNAs coding for proteins involved in gluconeogenesis, cell wall synthesis and transcripts encoding histones were less affected by Ded1p than other mRNAs (Extended Data Fig. 2a, b).

The fraction of ribosomes on 5′ untranslated regions (UTRs) markedly increased upon temperature shift in ded1-95, compared to the wild type (Fig. 1a, Extended Data Fig. 3a). The majority of mRNAs showed higher ribosome occupancy of the 5′ UTR in the ded1-95 strain, which correlated with lower translation efficiency of the main open reading frame (ORF) (Fig. 1b, Extended Data Fig. 3b). To examine the link between increased ribosome occupancy in the 5′ UTR and diminished translation of the main ORF, we performed polysome fractionation with northern blot analysis of individual mRNAs. The PSA1 mRNA—the translation efficiency of which is markedly affected by Ded1p—showed a distinct shift from polysomes to monosomes in ded1-95 compared to the wild type, upon temperature shift but not at 30 °C (Fig. 1c, d, Extended Data Fig. 3c). TDH2 mRNA, which is largely unaffected by Ded1p, did not show a comparable shift (Fig. 1c, d). Collectively, these observations suggest that increased ribosome occupancy on 5′ UTRs correlates with binding of the mRNA to only a single ribosome. This notion is consistent with previous reports¹². Ribosome profiling on only the 80S monosome fraction upon temperature shift also showed more footprints on 5′ UTRs in the ded1-95 strain, compared to the wild type (Extended Data Fig. 3d), indicating that ribosome occupancy on 5′ UTRs broadly correlates with the binding of mRNAs to single ribosomes.

**Fig. 1: Defects in Ded1p activate alternative translation initiation sites.**

A large number of sites on 5′ UTRs with increased ribosome footprints in the ded1-95 strain were enriched with near-cognate initiation codons (Fig. 1e–g), which differ from the canonical 5′-AUG-3′ initiation codon by a single nucleotide and can create alternative translation initiation sites (ATISs)¹³. Increased ribosome occupancy on AUG codons in 5′ UTRs was also seen in the ded1-95 strain (Fig. 1f), but only a few of these sites exist in the yeast transcriptome, compared to nine different near-cognate initiation codons, which constitute roughly 14% of all codons¹⁴. Ribosomes can translate from the ded1-95-activated ATISs, as demonstrated by ribosome profiles on small ORFs that start at these ATISs and finish at the respective termination codons, by the lack of ribosome accumulation at ATISs when translation was not arrested and by the periodicity of ribosome footprints starting from ATISs (Extended Data Fig. 3e–l). Collectively, the data indicate that defective Ded1p leads to ATIS activation in 5′ UTRs, which decreases polysome formation on the main ORFs and thereby overall protein production. We conclude that Ded1p function suppresses the use of ATISs.

Although ATIS activation in ded1-95 was extensive, only a subset of all near-cognate initiation codons was used. We detected no preferred length or register of the corresponding small ORFs relative to the main ORFs. However, in the ATISs, near-cognate codons from which translation initiation is most efficient were over-represented, whereas near-cognate codons from which translation initiation is least efficient were underrepresented^15,16 (Extended Data Fig. 4a). These observations show that ATIS activation is influenced by inherent codon preferences of the pre-initiation complex (PIC), although these preferences do not fully explain the ATIS activation pattern (Extended Data Fig. 4b–e).

To better understand this pattern, we examined whether remodelling of mRNA secondary structure by Ded1p is linked to ATIS activation. As an RNA helicase, Ded1p has been implicated in RNA structure remodelling^11,17, but it is not known which mRNA structures Ded1p alters in cells. To delineate the cellular mRNA structures that are remodelled by Ded1p, we used dimethyl sulfate (DMS) probing in vivo¹⁸ and measured changes in mRNA structure in ded1-95 and wild-type strains upon temperature shift (Extended Data Fig. 5a, b). Unwinding of mRNA structure by Ded1p was most pronounced in 5′ UTRs, compared to other mRNA regions (Fig. 2a, Extended Data Fig. 5c). Notably, ded1-95 activated ATISs were generally located 5′ of unwound RNA regions (Fig. 2b, c). Even near-cognate codons for which translation initiation is least efficient were activated, if they were located 5′ of mRNA structure (Fig. 2b). Our observations link the inability of ded1-95 to resolve mRNA structure to ATIS activation, suggesting that Ded1p suppresses ATIS activation by unwinding mRNA structure.

**Fig. 2: mRNA structure unwinding by Ded1p and ATIS activation.**

To investigate how Ded1p physically accomplishes this function, we determined which cellular RNAs bound to wild-type Ded1p using an high-throughput cross-linking-based approach (cross-linking-aided RNA affinity precipitation with sequencing (XL-RAP–seq)) and the individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) technique to map Ded1p-binding sites on these RNAs¹⁹ (Extended Data Fig. 6a–c). Ded1p cross-linked predominantly to mRNAs and ribosomal RNA (Extended Data Fig. 6b), especially to the 40S ribosomal subunit (Fig. 3a), which is part of the PIC that scans 5′ UTRs²⁰. The most frequently cross-linked position maps to helix 16, located at the mRNA entry channel (Fig. 3a). Notable cross-linking was also observed at helix 26, which is located at the mRNA exit site, and in extension segment 6 (around nucleotide 720), which is located in the vicinity of the other cross-link sites on the solvent side of the 40S subunit (Fig. 3a). Ded1p binding to helices 16 and 26 is consistent with reported interactions between Ded1p and eIF3c and the eIF3b–eIF3g–eIF3i sub-complex, that binds near these sites^21,22 (Extended Data Fig. 7a, b). Human DDX3X also binds to helix 16³.

**Fig. 3: Ded1p cross-linking to the 40S ribosomal subunit and to mRNAs.**

Ded1p further cross-linked to virtually all expressed mRNAs, predominantly in 5′ UTRs (Fig. 3b, c). This cross-linking pattern is consistent with the physical contact of Ded1p to the PIC. Aside from a modest preference for A and U, no sequence motifs could be identified in the mRNA cross-linking sites (Extended Data Fig. 7c). However, peaks of Ded1p cross-linking on 5′ UTRs were frequently proximal to ded1-95-activated ATISs (Fig. 3c, d), and unwound mRNA structure was located 3′ of Ded1p cross-linking sites (Fig. 3c, f).

Collectively, the data link Ded1p binding to mRNA, unwound mRNA structure, ATIS location and binding of Ded1p to the PIC. This link is illustrated by a segment of the PSA1 mRNA as an example (Fig. 4a). Ded1p binding is most pronounced 5′ of unwound RNA structure, indicating that Ded1p does not exclusively contact mRNA structure, but also regions that are 5′ of the structure. This finding is consistent with the notion that Ded1p functions in the context of the scanning PIC. The scanning process is slowed by RNA structure^20,23, and a slowed PIC conceivably permits Ded1p to survey the mRNA for structured regions that it then unwinds. Biochemical data show higher functional affinity of Ded1p for unstructured RNA, compared to structured RNA⁸, rationalizing the contacts of Ded1p to unpaired mRNA that is 5′ of unwound mRNA structure, as the helicase travels in a 5′ to 3′ direction with the PIC.

Our data collectively indicate that failure of Ded1p to resolve mRNA structure leads to ATIS activation. To directly probe the link between mRNA unwinding and ATIS activation, we generated a PSA1 mRNA with a mutation in an activated ATIS 5′ of unwound RNA structure. (Fig. 4b). The mutation markedly diminished the sensitivity to Ded1p-deficiency seen with the native PSA1 mRNA (Fig. 4b, c). Alterations in the RNA structure 3′ of the ATIS also decreased sensitivity to Ded1p (Extended Data Fig. 8a, b). Identical observations were made for mutations in an ATIS and the corresponding RNA structure in the ATP5 mRNA (Extended Data Fig. 8c–f). These results show that the effect of Ded1p on translation initiation depends not only on RNA unwinding, but also on proximal ATISs. Without a proximal ATIS, failure of Ded1p to unwind 5′ UTR structures does not abrogate scanning of the PIC and subsequent translation of the main ORF (Extended Data Fig. 9). This finding challenges the notion that cellular 5′ UTR structures alone are insurmountable hindrances for the scanning PIC.

Together, our results suggest the following function for Ded1p on 5′ UTRs (Fig. 4d). The enzyme associates with the PIC in the vicinity of the mRNA entry site of the small ribosomal subunit (Fig. 3a). This site is in close proximity to eIF4G and eIF4A (Extended Data Fig. 7b), both of which bind Ded1p with high affinity and might therefore be important for recruitment and function of Ded1p on the PIC^8,24,25. The density of Ded1p cross-linking sites on 5′ UTRs increases with distance from the 5′ cap (Fig. 3b), suggesting gradual recruitment of Ded1p to the mRNA entry site during the scanning process. This notion is consistent with the reported increase of Ded1p function with greater distance from the 5′ cap and with 5′ UTR length¹¹. The mRNA binding pattern of Ded1p further suggests that Ded1p is targeted to its sites of action through association with the scanning PIC. This is an effective way to deploy the enzyme exactly at sites at which it is needed, even though these sites lack common sequence or defined structure signatures. If Ded1p is missing or defective, mRNA structure persists, the PIC stalls and either dissociates from the mRNA, continues slowed scanning through the structure, or undergoes subunit joining and translation initiation if a near-cognate codon is present (Fig. 4d). Ribosomes initiating on an ATIS block subsequent scanning ribosomes from reaching the canonical initiation site, thereby decreasing translation efficiency for the main ORF (Fig. 1b). Unless an ATIS marks an N-terminal extension of the main ORF, PICs initiating at an ATIS are likely to be deterred from translating the main ORF. PICs encountering 5′ UTR structures without a proximal ATIS also interfere with scanning, but the kinetic pause introduced by PIC stalling, slowed scanning through the structure or a combination thereof is shorter than on an activated ATIS. Slowed PICs will eventually reach the main ORF (Extended Data Fig. 9), and therefore 5′ UTR structure alone affects main ORF translation less in isolation than it does in combination with proximal ATISs. Our model for Ded1p function does not preclude additional roles of the enzyme before the PIC scanning process²⁵. However, the Ded1p function outlined above largely accounts for the observed Ded1p interactions with mRNA, and therefore, additional roles of Ded1p are probably restricted to transient Ded1p–mRNA interactions.

Finally, our data reveal a straightforward mechanism for activation of upstream ORFs. The mRNA structures in the 5′ UTRs represent a large set of riboswitches that are sensitive to Ded1p. Active Ded1p turns the switches off, suppresses ATIS activation and allows efficient translation of the main ORF. Inactivation of the helicase by post-translational modifications¹, by metabolites such as AMP²⁶, by decreased Ded1p levels or by sequestration of Ded1p in RNP granules^25,27 turns the switches on, activating the ATISs and thereby inhibiting translation from the corresponding main ORFs. Certain peptides that are translated from activated ATISs might also have direct biological functions²⁸, but the regulation described here appears to be independent of functional peptides.

This mechanism for activation of upstream ORFs is probably used in biological processes. This notion is supported by several lines of evidence. First, there is a marked increase in sequence conservation in the RNA regions around activated ATIS (Extended Data Fig. 10a). Second, the ded1-95 activated ATIS in the ALA1 transcript produces an N-terminal extension that targets Ala1p to the mitochondria²⁹ (Extended Data Fig. 10b). Third, during meiosis, ATIS activation¹⁵ occurs in a pattern that is highly similar to the ATIS activation pattern seen with ded1-95 upon temperature shift (Fig. 4e, f, Extended Data Fig. 10c). Notably, we find reduced Ded1p levels during meiosis (Fig. 4g, Extended Data Fig. 10d). This link between Ded1p levels and the activation of ATISs proximal to 5′ UTR structures during meiosis suggests a role for the levels of Ded1p in this process. Collectively, our observations show that the regulatory program linking Ded1p to mRNA structure and ATIS activation is used in a physiological cellular process. The results indicate that intricate translation control and activation of upstream ORFs can be based on simple, ubiquitous elements: a helicase, mRNA structure and near-cognate initiation codons.

Methods

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Yeast strains, plasmids and oligonucleotides

Yeast strains used in this study are listed in Supplementary Table 1. Strains were grown at 30 °C unless stated otherwise. Primers, northern blot probes and other DNA oligonucleotides are listed in Supplementary Table 2, RNA oligonucleotides are listed in Supplementary Table 3, and DNA plasmids are listed in Supplementary Table 4.

Generation of a yeast strain expressing Ded1p–HTBH

Construction of plasmid pEJ21 containing the N-terminally haemagglutinin (HA)-tagged Ded1p has previously been described²⁴. pEJ21was then used to generate the plasmid pEJ5. The HA tag was replaced by a sequence containing a HpaI and a SphI site (amplification with primers X1 and X2), generating pEJ1. The His₆-TEV-Biotin (HTB) tag was amplified from pFA6-HTB-kanMX6 plasmid (gift from P. Kaiser) with primers X3 and X4.The resulting PCR product was cloned into pEJ1 via its HpaI and SphI sites yielding pEJ2. A second His₆ tag was introduced by site-directed mutagenesis with primers X5 and X6 generating pEJ3. The C-terminal His₆-TEV-Biotin-His₆ (HTBH) tag was introduced into pEJ4²⁴ by amplification of pEJ3 with primers X8 and X9 and subcloning with PflMI and SpeI into pEJ4, yielding pEJ5. pEJ5 was linearized and used to transform BY4741 by standard lithium acetate transformation yielding yeast strain yEJ2.

Generation of a yeast strain expressing Ded1p–His₆–FLAG₃

Yeast strain yDPB740, containing a C-terminal His₆–FLAG₃ tag on the endogenous DED1 allele, was generated from BY4742 using standard methods. In brief, a homologous recombination template was designed comprising the 40 nucleotides upstream and downstream of the DED1 stop codon flanking the His₆FLAG₃ tag (with stop codon) and kanMX6 drug resistance cassette. This template was generated by amplifying from pFA6a–6×His–3×FLAG–kanMX6 plasmid with primers DW1 and DW2. PCR product was used to transform BY4742 by standard lithium acetate transformation yielding yeast strain yDPB740.

Generation of a yeast strain expressing wild-type, ∆ATIS and secondary structure mutants of PSA1 and ATP5 mRNAs

FLAG-tagged PSA1 and ATP5 strains were generated from the respective cDNAs using standard methods as described above (pEJ14, pEJ15 and pEJ18, pEJ19, respectively). The FLAG-tag was appended at the 3′ terminus of the PSA1 and ATP5 ORF, respectively. For PSA1, mutations in the ATIS in the 5′ UTR (PSA1-∆ATIS, pEJ16, Fig. 4b, c) contained the following changes: (c.-58A>C (c.-58 indicates the 58th nucleotide 3′ of the A of the AUG), c.-56A>C, c.-52T>A). Mutations in the 5′ UTR mRNA structure (PSA1-∆2°, pEJ17, Extended Data Fig. 8a, b), contained the following changes: (c.-39_- 38AG>TC, c.-36_-35TA>AT, c.-32A>T,c.-24_-22AAA>TCT, c.-19_-18AA>CT). For ATP5, mutations in the ATIS in the 5′ UTR (ATP5-∆ATIS, pEJ20, Extended Data Fig. 8e, f) contained the following changes: (c.-126A>C, c.-120_119TT>CC, c.-109A>C). Mutations in the 5′ UTR mRNA structure (ATP5-∆2°, pEJ22, Extended Data Fig. 8e, f), contained the following changes: (c.-102C>A, c.-99C>A, c.-96G>A, c.-83G>A, c.-81C>A, c.-76G>A, c.-74G>A).

Polysome analysis

Polysome analysis using 20U (A₂₆₀) lysate was performed as described³¹, with a lower final concentration of cycloheximide (50 μg ml⁻¹). In brief, after centrifugation through a 15–45% (w/v) sucrose gradient, sixteen fractions were collected at a pump speed of S = 0.9 ml min⁻¹. RNA in each fraction was precipitated by adding two volumes of ice-cold ethanol and incubating overnight at −80 °C. RNA was extracted with phenol–chloroform according to standard protocols. Samples were applied to a 1.4% agarose gel containing 6% (v/v) formaldehyde, and electrophoresis was performed as described³¹. RNA was visualized with ethidium bromide. The amount of 18S rRNA in each fraction of the gradient was quantified with ImageQuant 5.2 software (Molecular Dynamics).

Northern blot analysis

For ribosome association of individual mRNAs, gel electrophoresis was performed after polysome analysis and fractionation as described above. RNA was subsequently transferred to nitrocellulose membranes (AmershamHybond–N, GE Healthcare) and further processed as described³¹. DNA oligonucleotides (Supplementary Table 2; X85, X96, X105, X114) were radiolabelled with PNK according to standard procedures. Probes were incubated with the membranes in hybridization buffer (6× saline sodium citrate (SSC), 0.1% SDS, 10× Denhardt’s reagent) overnight at 42 °C. Membranes were subsequently washed three times with wash buffer (6× SSC, 0.1% SDS). Probe signals were visualized using a Molecular Dynamics Phosphorimager (GE Healthcare) and quantified with ImageQuant 5.2 software (Molecular Dynamics). Normalized signal intensities were compiled for fractions corresponding to monosomes, light and heavy polysomes and averaged from at least three biological replicates.

Western blot analysis

Lysates from yeast strain A14201 at vegetative phase and stage 11 (ndt80 release time course) were prepared as described¹⁵. After loading equal amounts of protein on a 10% NEXT gel, denaturing gel electrophoresis and transfer to a PVDF membrane, western blotting was performed with anti-Ded1p (rabbit; 1:5,000) and anti-hexokinase (rabbit;1:10,000; US Biological) antibodies. Chemiluminescence was quantified by Imagequant software. Hexokinase served as a loading control for normalization.

Ribosome profiling

Yeast cultures (500 ml) were grown at 30 °C in rich medium to mid-log phase (OD_600 nm of approximately 0.4) and divided into two equal volumes. Cycloheximide (final concentration, 50 μg ml⁻¹) was added to one sample. Cells were rapidly collected by centrifugation at 4,000g for 2 min, and snap-frozen on dry ice. One volume of pre-warmed medium (44 °C) was added to the remaining sample, resulting in a temperature of 37 °C for the entire volume. The temperature of 37 °C was verified. The entire sample was immediately moved to a shaking incubator at 37 °C. Five minutes after temperature shift, the yeast culture was treated with cycloheximide and cells were collected as mentioned above. For run-off experiments, yeast cells were collected in the absence of cycloheximide (Extended Data Fig. 3e).

Cell lysis, RNase I treatment (Ambion) and sucrose gradient centrifugation was performed as described³² for 25 units A₂₆₀ per sample. In addition, lysates (approximately 150 μl) were treated with 3.25 μl Turbo DNase I for 1 h at 25 °C. Purification and processing of ribosome-protected fragments were carried out as described³², except that rRNA depletion with the RiboZero kit (Illumina) was omitted. Depletion of rRNA was performed at the level of circularized cDNA, as described⁷ (1 μl of a 5 μM mix of biotinylated DNA oligonucleotides, Supplementary Table 2 (X66–X80) and MyOne Streptavidin C1 DynaBeads (Invitrogen)). PCR amplification and sequencing was performed as described⁷.

Monosome-protected fragments were isolated as described¹². In brief, sucrose gradient centrifugation of lysates was performed and fractions corresponding to monosomes were pooled and treated with 1/10 U RNase I per unit A_260 nm lysate and 0.4U Turbo DNase I per unit A_260 nm lysate for 1 h at 25 °C. Reactions were stopped by phenol–chloroform extraction of RNA. Monosome-protected fragments were processed as described above for ribosome-protected fragments.

The fragmented mRNA control libraries were generated as described³². Sizing, concentration and quality of each DNA library was assessed with the High Sensitivity DNA kit on an Agilent2100 Bioanalyzer system. Up to eight DNA libraries were pooled before performing 50 bp single end read sequencing on an Illumina HiSeq2500 V2 in rapid run mode.

Processing of the ribosome profiling data was performed as described³². In brief, adaptor sequences and ribosomal reads were removed. Remaining reads were mapped to the sacCer3 genome with the TopHat software (parameters set as: --no-novel-juncs -N 2 --read-edit-dist 2 --max-insertion-length 3 --max-deletion-length 3 -g 2 (https://www.yeastgenome.org)). All other parameters were kept at default settings³³. The abundance of mRNAs in ribosome or monosome-protected fragments as well as in the fragmented RNA control libraries were determined using Cufflinks software³³. These values were used to calculate translational efficiencies as described¹⁴. For the calculation of log₂∆TE values we also included a constant factor reflecting the change in the overall size of the mRNA pool, derived from the spike-in of RNA controls (Supplementary Table 3).

P-sites in ribosome-protected fragments (RPFs) were determined using the 13th position from the 5′ end of reads with 28 or 29 nt¹⁴. The fraction of ribosomes on 5′ UTRs was calculated for each mRNA by counting all RPFs on the 5′ UTR (excluding positions −3 to −1), divided by the number of all RPFs mapped to the entire mRNA. mRNAs with 5′ UTRs containing fewer than 10 nt were excluded from the analysis. The centre of ribosome density (CRD) was calculated as described³⁴.The shift in the CRD (∆CRD) in ded1-95 compared to wild-type DED1 upon temperature shift was defined relative to the entire length of the mRNA according to:

$${\rm{\Delta }}CRD=\left(\left(CRD\,ded1-{95}\right)-\left(CRD\,\mathrm{wild}-\mathrm{type}\,DED1\right)\right)/\mathrm{mRNA}\,\mathrm{length}$$

A negative ∆CRD value marks increased ribosome accumulation in the 5′ UTR in ded1-95.

ATIS were identified according to a previously described algorithm¹⁵. In brief, a position is considered an ATIS, (i) if minimal ribosome count value (±1 nt of the nucleotide under consideration) is greater than nine (high-stringency ATIS) or four (medium-stringency ATIS) in all replicates; (ii) if the ratio of ribosome occupancy between two neighbouring nucleotides 5′ to 3′ (position^{n − 1}/positionⁿ) is greater than or equal to three (high-stringency ATIS) in all replicates, or greater or equal of three in one and greater than or equal to 1.75 in the other replicate (medium-stringency ATIS); and (iii) if the normalized ribosome count in ded1-95 cells 5′ after temperature shift to 37 °C is 1.5-fold higher that in wild-type DED1 in all replicates. This algorithm identified 396 high-stringency ATIS and 2,126 medium-stringency ATIS. Near-cognate codons were identified in 259 high-stringency ATIS (65%) and 1,382 medium-stringency ATIS (65%) within a moving window of ±1 nt. Canonical AUG initiation codons were found in 4% high-stringency ATIS, and in 3% medium-stringency ATIS. As a control set, we collected all near-cognate codons on 5′ UTRs of mRNA genes with a 5′ UTR length between 20 and 500 nt. After removal of near-cognate codons in medium-stringency ATIS, we identified 60,666 near-cognate codons.

XL-RAP–seq

Yeast cells containing HTBH-tagged DED1 were grown in rich medium to an OD_600 nm of 1.0–1.5, collected by brief centrifugation at 4,000g, re-suspended in ice-cold water or remaining YPD medium, transferred to a Petri dish, and subjected to UV-light in a Stratalinker (600 mJ cm⁻², 254 nm) on ice. Cells were washed in ice-cold water, sedimented by centrifugation for 5 min at 5,250g, frozen on dry ice and stored at −80 °C.

Frozen cells were lysed in QIA-1M buffer (100 mM NaH₂PO₄ pH 8, 10 mM Tris, 1M NaCl, 8 M Urea, 10 mM imidazole, 0.5% (w/v) IGEPAL, 2.5 mM β -mercaptoethanol, 1 mM PMSF, protease inhibitor cocktail (Roche)) with glass beads six times for 30 s in a Beadbeater system (Biospec products). Glass beads were removed, and lysates were centrifuged at 5,250g for 30 min. Cleared lysates were incubated with Ni²⁺–Agarose (40 μl slurry per g dry pellet weight, pre- equilibrated in buffer QIA-1M; Qiagen) overnight at 4 °C. Ni²⁺-beads were washed in 25 ml of wash buffer 1 (0.3 M NaCl, 10 mM Tris, 100 mM NaH₂PO₄, 8 M Urea, 10 mM imidazole) and sample was eluted with 10 ml elution buffer1 (0.3 M NaCl, 100 mM Tris, 50 mM NaH₂PO₄, 8 M Urea, 500 mM imidazole, 10% (v/v) glycerol). Eluates were then incubated with12.5 μl equilibrated streptavidin-conjugated agarose resin (Pierce Technologies) per g dry pellet weight overnight at 4 °C. Streptavidin beads were washed with 12.5 ml wash buffer 2 (0.3 M NaCl, 100 mM Tris, 8 M Urea, 0.5 mM EDTA)—containing 2% SDS, with wash buffer 2 (12.5 ml) without SDS, and with 1× TEV buffer. Beads were next incubated with 50 U AcTEV (Invitrogen) for 2 h at 4 °C. The sample was eluted with 2 × 0.9 ml TEV elution buffer (300 mM NaOAc pH 6, 8 M Urea, 0.5% NP-40). Eluates were incubated with 175 μl Proteinase K (4 mg ml⁻¹; Roche), and the reaction was stopped by standard phenol–chloroform extraction. Released RNA was precipitated by ethanol precipitation overnight. RNA was re-suspended in 1× Turbo DNase buffer and incubated with 0.4 μl TurboDNase (Ambion) in a final volume of 20 μl for 20 min at 37 °C. Turbo DNase was inactivated according to the manufacturer’s instruction and the RNA was precipitated with ethanol.

cDNA was generated from the RNA sample using the ‘template switch’ activity of M-MLV reverse transcriptase³⁵. Reactions were performed in 20 μl according to the instructions of the manufacturer mix (Invitrogen) with 0.5 μl Superscript II and 0.05 μM final concentration of tailed random hexamer primer (X13, Supplementary Table 2). Reaction conditions were as follows: 20 °C, 10 min; 37 °C, 10 min; 42 °C, 45 min. Next, 0.25 μM of primers (X14–X17, Supplementary Table 2) containing a 7-nt index and three guanosine ribonucleotides at their 3′ end were added to the reaction mix and reactions were performed at 42 °C for 30 min. Four microlitres of the resultant cDNA were used as template for amplification with primers X18 and X19 (Supplementary Table 2) and Advantage 2 polymerase mix (Clontech) for 30 cycles, according to the manufacturer’s instructions. PCR products were precipitated and washed with 75% ethanol. DNA libraries were sequenced as 36-bp single-end reads on an Illumina Genome Analyzer. Reads were mapped to sacCer2 genome with Bowtie software with default settings.

Reads were excluded from further analysis if their location was outside of the boundaries of an mRNA or other transcribed regions of the genome as previously defined³⁶. Genes were only considered to be bound by Ded1p, if more than 10 FPKM mapped to the respective mRNA gene.

iCLIP

iCLIP experiments were performed independently with two different approaches, (i) using Ded1p–HTBH and (ii) Ded1p–His₆–FLAG₃, both on the endogenous DED1 allele. For iCLIP with Ded1p–HTBH, cell growth, cross-linking and tandem affinity purification on Ni²⁺-agarose and streptavidin beads was performed as described above for the XL-RAP–seq procedure. Streptavidin beads were washed twice in 1× PNK buffer and split into two samples (80%, L; 20%, H). 200 ng RNase I was added per gram dry pellet weight to the L-sample and 0.1 ng RNase I to the H-sample. The samples were incubated for 5 min at 37 °C on a rotator. Resins were subsequently washed with ~1.5 ml wash buffer 2 (2% (w/v) SDS), and then with 1.5 ml wash buffer 2 without SDS, and finally with 1.5 ml 1× PNK buffer. The supernatant was removed and beads were re-suspended in 67 μl RNase-free water, 3 μl alkaline phosphatase (NEB) and 2 μl RNasin (Roche), and incubated for 20 min at 37 °C. Beads were washed twice with 1.5 ml 1× PNK buffer.

3′ ligation was performed by re-suspending the resin in 32 μl RNase-free water with 8 μl of 20 μM RL3 RNA Linker (Supplementary Table 3). Reactions were performed overnight at 4 °C in 40 μl, containing 22 μl RNase-free water, 8 μl 10× T4 RNA ligase buffer, 8 μl BSA (0.2 μg μl⁻¹), 3 μl T4 RNA ligase (all NEB).

The resin was washed with 1.5 ml 1× TEV-salt buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT) and twice with 1.5 ml 1× PNK buffer. Beads were re-suspended in 64 μl RNase-free water, 8 μl 10× PNK Buffer, 4 μl ³²P-γ-ATP, 4 μl T4 PNK (all NEB). Reactions were performed for 50 min in a shaking thermoblock at 37 °C. Beads were washed twice with 1.5 ml TEV salt buffer and twice with 1.5 ml TEV-elution buffer. Beads were subsequently mixed with SDS loading dye and subjected to PAGE on a 10% NEXT gel (Amresco) according to the manufacturer’s conditions. Gels were subsequently blotted with nitrocellulose membranes (Amersham Protran, GE Healthcare) and exposed to X-ray film. RNA was then liberated by Proteinase K treatment as described³⁷. The purified RNAs were re-suspended in 89 μl RNase- free water, 11 μl 10× DNase I Buffer, 5 μl RNasin, 5 μl RQ1 DNase (all Promega) and incubated for 20 min at 37 °C. The reaction was stopped by standard phenol–chloroform extraction and RNA was ethanol-precipitated overnight.

The DNase-treated RNA was re-suspended in 1.4 μl RNase-free water, 0.2 μl 10 mM dNTPs, 1 μl 20 nM reverse transcription primer X97 (Supplementary Table 2), and incubated for 5 min at 65 °C. Next, 0.8 μl 5× first-strand buffer, 0.2 μl 1M DTT, 0.2 μl RNase Inhibitor, 0.2 μl Superscript III (all Invitrogen) were added and incubated for 30 min at 50 °C. RNA was degraded by alkaline hydrolysis (after addition of 0.5 μl of 1N NaOH and incubation at 98 °C for 15 min). After addition of loading buffer, cDNA was applied to 10% denaturing PAGE and staining with SYBR Gold. Fragments of 100–125 nt were cut from the gel. The gel slices were crushed and cDNA was recovered by incubation in 500 μl diffusion buffer (20 mM Tris-HCl pH 7.5, 250 mM NaOAc, 1 mM EDTA, 0.25% (w/v) SDS) overnight at 4 °C with subsequent ethanol precipitation.

The cDNA was suspended in 15 μl RNase-free water and circularized with CircLigase I (Epicentre) according to the manufacturer’s instructions. The circularized cDNA was used for amplification with Phusion polymerase (NEB) and primers X98 and X99 (Supplementary Information 2). PCR settings were: 30 s at 98 °C and then 24 PCR cycles (10 s at 98 °C, 30 s at 58 °C, 30 s at 72 °C). PCR products were applied to 10% non-denaturing PAGE and visualized by SYBR Gold. Products with 75–90bp were extracted from cut gel slices, and ethanol precipitated as described above. PCR products were amplified with Phusion polymerase and primers X100 and X101 for five cycles using the same PCR settings as above. PCR products were then separated on a 2% agarose gel, cut out and subjected to Illumina sequencing using primer X102.

For iCLIP with Ded1p–His₆–FLAG₃, cells were grown in SD–Trp medium to OD_600 nm = 0.5–0.6. The culture was subsequently transferred to a 245 mm × 245 mm × 25 mm square Petri dish. UV cross-linking was performed in a Stratalinker 2400 (150 mJ cm⁻², 254 nm) at room temperature. Cells were collected by centrifugation for 5 min at 2000g, washed twice in ice-cold PBS, frozen in liquid nitrogen, and stored at −80 °C.

Frozen cells were lysed in CLIP lysis buffer (50 mM Tris-HCl, pH 7.8, 300 mM NaCl, 1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail (Roche)) with glass beads six times for 1 min in a Disruptor Genie system (Scientific Industries). Lysates were centrifuged after removal of the glass beads at 10,000g, twice for 5 min. Cleared lysates (~26.5 A₂₆₀ units) were incubated with anti-FLAG M2 Magnetic Beads (20 μl slurry pre-equilibrated in CLIP Lysis Buffer; Sigma) in a total volume of 1 ml overnight at 4 °C. Beads were washed twice in 1 ml FLAG Wash Buffer (50 mM Tris-HCl pH 7.8, 1 M NaCl, 0.1% NP-40) and twice in 1 ml FLAG Elution Buffer (50 mM Tris-HCl pH7.8, 150 mM NaCl, 0.1% NP-40). Proteins were eluted twice in 95 μl FLAG Elution Buffer containing 150 ng μl 3× FLAG tag peptide (Sigma). Pooled eluates were incubated with 10 μl RNase I (Ambion), diluted 1:500,000 in FLAG elution buffer for 15 min at room temperature. Reactions were quenched with 960 μl 8 M guanidine–HCl, 90 μl Dilution Buffer (600 mM Tris-HCl pH 7.8, 3.93 M NaCl), 6.4 μl 2 M imidazole, 10.8 μl 10% NP-40and 12.8 μl 500 mM β-mercaptoethanol. RNase-treated eluates were incubated further with Ni-NTA magnetic agarose beads (50 μl slurry pre-equilibrated in Ni-NTA binding buffer (50mM Tris-HCl pH 7.8, 300 mM NaCl, 10 mM imidazole, 6 M guanidine-HCl, 0.1% NP-40, 5 mM β-mercaptoethanol); Qiagen) overnight at 4 °C.

Ni²⁺-beads were washed twice in 1 ml CLIP wash buffer I (50 mM Tris-HCl pH 7.8, 500 mM NaCl, 10 mM imidazole, 6 M guanidine-HCl, 0.1% NP-40, 5 mM β-mercaptoethanol) and three times in 1 ml 1× PNK Buffer (50 mM Tris-HCl pH 7.8, 10 mM MgCl₂, 0.5% NP-40, 10 mM β-mercaptoethanol). Beads were subsequently incubated with 30 μl dephosphorylation mix (50 mM Tris-HCl pH 7.8, 10 mM MgCl₂, 10 mM β-mercaptoethanol, 3 M BU TSAP (Promega), 30 U SUPERase-In (Ambion)) for 30 min at 37 °C in a thermomixer at 1000 rpm. Reactions were terminated by adding 1 ml CLIP wash buffer I, and beads were washed three times in 1 ml 1× PNK buffer, re-suspended in 30 μl ligation mix (50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 10 mM β-mercaptoethanol, 10% PEG8000 (NEB), 10% DMSO, 2 μM 3′ adenylated adaptor X103, 30 U T4 RNA Ligase 1 (NEB), 30 U SUPERase-In (Ambion)) and incubated for 3 h at 22 °C in a thermomixer at 1000 rpm. Reactions were terminated by adding 1 ml CLIP wash buffer I. Beads were washed three times in 1 ml 1× PNK Buffer, re-suspended in 30 μl Kinase Mix (50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 10 mM β-mercaptoethanol, 30 U PNK (NEB), 5 μCi γ- ³²P-ATP, 30 U SUPERase-In (Ambion)) and incubated for 30 min at 37 °C in a thermomixer (1000 rpm). Reactions were terminated by adding 1 ml CLIP Wash Buffer I. Beads were washed three times in 1 ml CLIP Wash Buffer I, and three times in 1 ml CLIP wash buffer II (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, 10 mM imidazole, 0.1% NP-40, 5 mM β-mercaptoethanol).

Proteins were eluted with 35 μl CLIP wash buffer II containing 200 mM imidazole three times for 5 min at 22 °C in a thermomixer (1000 rpm).

Pooled eluates were digested with Proteinase K (Invitrogen) at 50 °C for 1 h. The reaction was stopped by standard phenol–chloroform extraction. Released RNA was precipitated by ethanol precipitation overnight in the presence of 1 μl GlycoBlue (Ambion) and 1 pmol reverse- transcription DNA primer X104. RNA was converted to first-strand cDNA in a 10 μl standard reaction mix with 0.5 μl Superscript III (Invitrogen) and the co-precipitated DNA primer. The reaction conditions were as follows: 25 °C for 5 min, 42 °C for 20 min, 50 °C for 40 min. RNA was degraded with 1.67 μl of 1 M NaOH for 10 min at 90 °C. The cDNA was ethanol precipitated with GlycoBlue for >2 h. cDNA fragments of 120–200 nt were gel purified on a urea-denaturing 6% acrylamide gel and ethanol precipitated with GlycoBlue overnight. cDNA was circularized in a 10 μl reaction mix using 0.5 μl CircLigase I (Epicentre) in the presence of 1 M betaine at 60 °C for 1h. The reaction was then supplemented with additional 0.5 μl CircLigase I and incubated at 60 °C for 1 h. The enzyme was inactivated at 80 °C for 10 min. PCR and formamide-gel purification of PCR products were performed as described³⁸, using 20 cycles of PCR and isolating 120–200 nt ssDNA fragments.

Sequencing reads were processed as previously described^37,39. In brief, after the trimming of adaptor sequences, reads were mapped to sacCer3 with Bowtie2 or TopHat software with similar settings used for the ribosome profiling data, outlined above. Identical reads were subsequently collapsed and duplications removed. The cross-link position of Ded1p to RNA was defined as 1 nt 5′ of the 5′ mapped nucleotide of a sequencing read³⁷.

DMS-MaPseq

Yeast strains (wild-type DED1 and ded1-95) were grown in YPD at 30 °C. Overnight cultures were diluted to OD_600 nm of ~ 0.09 and grown to an OD_600 nm = 0.6. An equal volume of 44 °C YPD medium was added to achieve an immediate temperature shift to 37 °C, as outlined for the ribosome profiling experiments. Cultures were incubated in a 37 °C water bath for 3 min. At this time, DMS (Sigma) was added to a 5% (v/v) final concentration and incubation was continued with stirring for 3 min. DMS was quenched by adding 30 ml of ice-cold stop solution (30% β-mercaptoethanol, 50% (v/v) isoamyl alcohol). Cells were quickly transferred to ice, collected by centrifugation at 3,500g at 4 °C for 4 min, and washed with 10 ml 30% β- mercaptoethanol solution. Cells were re-suspended in 0.6 ml RNA lysis buffer (6 mM EDTA, 45 mM NaOAc, pH 5.5). Total RNA was purified with hot acid phenol (Ambion) and ethanol precipitation. Sequencing libraries were prepared as previously described¹⁷.

Raw fastq files were stripped of linker sequences and filtered for overall quality using the FASTX-Toolkit Clipper and Quality Filter functions (http://hannonlab.cshl.edu/fastx_toolkit/), respectively, requiring that 80% of sequenced bases have a quality score >25. Reads were aligned against the yeast genome (sacCer3) using Tophat v2.1.0 with Bowtie2 with the following settings for a 50 bp sequencing run: --no-novel-juncs -N 5 --read-gap-length 7 --read-edit-dist 7 --max-insertion-length 5 --max-deletion-length 5 -g 3. All non-uniquely aligned reads were then removed. Owing to empirically determined mutation enrichment from non-template addition, 2 nt was trimmed from the 5′ end of each read. Mismatches located within 3 nt of an indel were also discarded for future analysis.

Bioinformatic analyses

Yeast genomic sequence conservation scores were obtained from S. cerevisiae genome database (https://www.yeastgenome.org). Positional coordinates of mRNAs including transcription start sites and polyadenylation sites are based on sacCer3 and reported measurement⁴⁰. Genome-wide datasets were visualized by IGV software⁴¹. Structural models of the small and large ribosomal subunits including initiation factors were generated with the Chimera software^30,42. Analyses of Gene Ontology term enrichment were carried out with GOrilla software using a single ranked list of genes⁴³.

RNA structure prediction was carried out with sequences 0–99 nt 3′ of the first nucleotide of an alternative start codon using the RNAfold web server⁴⁴. Constraint settings were derived from DMS-MaPseq data as follows: a nucleotide was set as ‘unpaired’ if the DMS-MaPseq counts of a given nucleotide exceeded the value of 0.49 relative to the third highest count number in the range of 100 nt downstream of an ATIS.

Statistical significance of enrichment or depletion in certain regions (for example, Figs. 2c, 3d, e, Extended Data Fig. 10a) was determined by comparing weighted data vectors of the observed variable to the background value. To this end, we calculated t-values with the wtd.t.test function in R. The algorithm is based on the mean and 1/(standard errors)² as an estimate of the means accuracy. The given P values correspond to a two-tailed t-test.

Further bioinformatic analyses and multiple linear regressions were performed with R⁴⁵ with customized scripts using RStudio Software (https://www.rstudio.com/). Code is available upon request. Normalization of the datasets including ribosome protected fragments, monosome-protected fragments, Ded1p iCLIP–seq and DMS-MaPseq counts were performed relative to the total number of counts of the entire mRNA⁴².

To compute Ded1p binding density, DMS-MapSeq ratios, or sequence conservation values in the vicinity of ATIS, it was important to normalize for inherent positional trends within the exact region in the respective iCLIP, DMS-MapSeq and sequence conservation datasets. For example, values for DMS-MapSeq ratios (counts ded1-95/ counts wild type), and iCLIP reads show an upward trend with increasing distance from the 5′ cap in 5′ UTRs. To normalize for inherent positional trends, we calculated a background distribution for the vicinity of each ATIS. We randomly choose a position in the respective section of a given mRNA, and determined the signal distribution in the vicinity of this position (for example, position −5 relative to the 5′ nt of an ATIS). This process was repeated four times. The background value reflects the average of these five calculated values. Reported enrichment values represent the ratio of the measured signal over the background value at each indicated position. Values are given in all plots as log₂(measured signal/background signal). Statistical significance of enrichment or depletion was determined by calculating the t-value of the observed variable on the basis of the mean and s.d. of the background value.

Metagene profiles were calculated by averaging normalized Ded1p iCLIP counts and DMS- MaPseq counts after binning transcript coordinates from 5′ UTRs, ORFs and 3′ UTRs in bins reflecting 2% of each section of mRNA. Ded1p binding sites and the midpoint of RNA secondary structures were determined by Piranha peak calling software (http://smithlabresearch.org).

Calling parameters were optimized on the basis of visual inspection. To call peak sites of RNA secondary structures, a genome-wide dataset of log₂(counts of DMS-MaPseq wild type/ded1-95) was used as input file.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

The data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) repository with the accession code GSE93959. All other data are available from the corresponding author upon reasonable request.

References

Sharma, D. & Jankowsky, E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases. Crit. Rev. Biochem. Mol. Biol. 49, 343–360 (2014).
Article PubMed CAS Google Scholar
Bol, G. M., Xie, M. & Raman, V. DDX3, a potential target for cancer treatment. Mol. Cancer 14, 188 (2015).
Article PubMed PubMed Central CAS Google Scholar
Oh, S. et al. Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress. Oncotarget 7, 28169–28182 (2016).
PubMed PubMed Central Google Scholar
Pugh, T. J. et al. Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations. Nature 488, 106–110 (2012).
Article PubMed PubMed Central ADS CAS Google Scholar
Snijders Blok, L. et al. Mutations in DDX3X are a common cause of unexplained intellectual disability with gender-specific effects on Wnt signaling. Am. J. Hum. Genet. 97, 343–352 (2015).
Article PubMed PubMed Central CAS Google Scholar
Valiente-Echeverría, F., Hermoso, M. A. & Soto-Rifo, R. RNA helicase DDX3: at the crossroad of viral replication and antiviral immunity. Rev. Med. Virol. 25, 286–299 (2015).
Article PubMed CAS Google Scholar
Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protocols 7, 1534–1550 (2012).
Article PubMed CAS Google Scholar
Putnam, A. A. et al. Division of labor in an oligomer of the DEAD-box RNA helicase Ded1p. Mol. Cell 59, 541–552 (2015).
Article PubMed PubMed Central CAS Google Scholar
Burckin, T. et al. Exploring functional relationships between components of the gene expression machinery. Nat. Struct. Mol. Biol. 12, 175–182 (2005).
Article PubMed CAS Google Scholar
Chuang, R. Y., Weaver, P. L., Liu, Z. & Chang, T. H. Requirement of the DEAD-box protein Ded1p for messenger RNA translation. Science 275, 1468–1471 (1997).
Article PubMed CAS Google Scholar
Sen, N. D., Zhou, F., Ingolia, N. T. & Hinnebusch, A. G. Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A. Genome Res. 25, 1196–1205 (2015).
Article PubMed PubMed Central CAS Google Scholar
Heyer, E. E. & Moore, M. J. Redefining the translational status of 80S monosomes. Cell 164, 757–769 (2016).
Article PubMed CAS Google Scholar
Hinnebusch, A. G., Ivanov, I. P. & Sonenberg, N. Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science 352, 1413–1416 (2016).
Article PubMed ADS CAS Google Scholar
Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).
Article PubMed PubMed Central ADS CAS Google Scholar
Brar, G. A. et al. High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science 335, 552–557 (2012).
Article PubMed ADS CAS Google Scholar
Kolitz, S. E., Takacs, J. E. & Lorsch, J. R. Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation. RNA 15, 138–152 (2008).
Article PubMed CAS Google Scholar
Berthelot, K., Muldoon, M., Rajkowitsch, L., Hughes, J. & McCarthy, J. E. Dynamics and processivity of 40S ribosome scanning on mRNA in yeast. Mol. Microbiol. 51, 987–1001 (2004).
Article PubMed CAS Google Scholar
Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat. Methods 14, 75–82 (2017).
Article PubMed CAS Google Scholar
Huppertz, I. et al. iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274–287 (2014).
Article PubMed PubMed Central CAS Google Scholar
Hinnebusch, A. G. The scanning mechanism of eukaryotic translation initiation. Annu. Rev. Biochem. 83, 779–812 (2014).
Article PubMed CAS Google Scholar
Gavin, A. C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006).
Article PubMed ADS Google Scholar
Krogan, N. J. et al. High-definition macromolecular composition of yeast RNA-processing complexes. Mol. Cell 13, 225–239 (2004).
Article PubMed CAS Google Scholar
Kozak, M. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc. Natl Acad. Sci. USA 87, 8301–8305 (1990).
Article PubMed ADS CAS Google Scholar
Gao, Z. et al. Coupling between the DEAD-box RNA helicases Ded1p and eIF4A. eLife 5, e16408 (2016).
Article PubMed PubMed Central Google Scholar
Hilliker, A., Gao, Z., Jankowsky, E. & Parker, R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex. Mol. Cell 43, 962–972 (2011).
Article PubMed PubMed Central CAS Google Scholar
Putnam, A. A. & Jankowsky, E. AMP sensing by DEAD-box RNA helicases. J. Mol. Biol. 425, 3839–3845 (2013).
Article PubMed PubMed Central CAS Google Scholar
Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).
Article PubMed PubMed Central CAS Google Scholar
Andrews, S. J. & Rothnagel, J. A. Emerging evidence for functional peptides encoded by short open reading frames. Nat. Rev. Genet. 15, 193–204 (2014).
Article PubMed CAS Google Scholar
Tang, H. L. et al. Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J. Biol. Chem. 279, 49656–49663 (2004).
Article PubMed CAS Google Scholar
Aylett, C. H., Boehringer, D., Erzberger, J. P., Schaefer, T. & Ban, N. Structure of a yeast 40S–eIF1–eIF1A–eIF3–eIF3j initiation complex. Nat. Struct. Mol. Biol. 22, 269–271 (2015).
Article PubMed CAS Google Scholar
Hu, W., Sweet, T. J., Chamnongpol, S., Baker, K. E. & Coller, J. Co-translational mRNA decay in Saccharomyces cerevisiae. Nature 461, 225–229 (2009).
Article PubMed PubMed Central ADS CAS Google Scholar
Smith, J. E. et al. Translation of small open reading frames within unannotated RNA transcripts in Saccharomyces cerevisiae. Cell Reports 7, 1858–1866 (2014).
Article PubMed PubMed Central CAS Google Scholar
Goecks, J., Nekrutenko, A. & Taylor, J. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010).
Article PubMed PubMed Central Google Scholar
Andreev, D. E. et al. Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. eLife 4, e03971 (2015).
Article PubMed PubMed Central Google Scholar
Matz, M. et al. Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 27, 1558–1560 (1999).
Article PubMed PubMed Central CAS Google Scholar
Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).
Article PubMed PubMed Central ADS CAS Google Scholar
König, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).
Article PubMed PubMed Central CAS Google Scholar
Subtelny, A. O., Eichhorn, S. W., Chen, G. R., Sive, H. & Bartel, D. P. Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508, 66–71 (2014).
Article PubMed PubMed Central ADS CAS Google Scholar
Licatalosi, D. D. et al. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008).
Article PubMed PubMed Central ADS CAS Google Scholar
Park, D., Morris, A. R., Battenhouse, A. & Iyer, V. R. Simultaneous mapping of transcript ends at single-nucleotide resolution and identification of widespread promoter-associated non-coding RNA governed by TATA elements. Nucleic Acids Res. 42, 3736–3749 (2014).
Article PubMed PubMed Central CAS Google Scholar
Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Article PubMed PubMed Central CAS Google Scholar
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Article PubMed CAS Google Scholar
Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10, 48 (2009).
Article PubMed PubMed Central Google Scholar
Gruber, A. R., Bernhart, S. H. & Lorenz, R. The ViennaRNA web services. Methods Mol. Biol. 1269, 307–326 (2015).
Article PubMed CAS Google Scholar
The R Core Team. A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria., http://www.R-project.org (2013).
Google Scholar
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).
MathSciNet MATH Google Scholar
Miyasaka, H. The positive relationship between codon usage bias and translation initiation AUG context in Saccharomyces cerevisiae. Yeast 15, 633–637 (1999).
Article PubMed CAS Google Scholar
Nielsen, K. H. et al. Synergistic activation of eIF4A by eIF4B and eIF4G. Nucleic Acids Res. 39, 2678–2689 (2011).
Article PubMed CAS Google Scholar
Floor, S. N., Condon, K. J., Sharma, D., Jankowsky, E. & Doudna, J. A. Autoinhibitory interdomain interactions and subfamily-specific extensions redefine the catalytic core of the human DEAD-box protein DDX3. J. Biol. Chem. 291, 2412–2421 (2016).
Article PubMed CAS Google Scholar
Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005).
Article PubMed PubMed Central CAS Google Scholar

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Acknowledgements

This study was supported by the National Institutes of Health (NIH) (GM118088 to E.J., GM107331 to D.D.L.) and by a postdoctoral fellowship from the German Research Council (GU 1146/1-1 to U.-P.G.). D.P.B. and J.S.W. are investigators of the Howard Hughes Medical Institute. We thank M. Adams for help with the initial data analysis; N. Al-Huseini and J. Coller for discussion and materials; A. Tambe, M. Hannigan, and R. Backofen for advice on bioinformatic data analysis; B. Klaus for critical advice on statistical analysis and M. Hentze for discussion.

Reviewer information

Nature thanks P. Todd and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

These authors contributed equally: Ulf-Peter Guenther, David E. Weinberg, Meghan M. Zubradt.

Authors and Affiliations

Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA
Ulf-Peter Guenther, Frank A. Tedeschi, Brittany N. Stawicki, Leah L. Zagore, Donny D. Licatalosi & Eckhard Jankowsky
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA
David E. Weinberg, Meghan M. Zubradt & Jonathan S. Weissman
Howard Hughes Medical Institute Whitehead Institute for Biomedical Research, Cambridge, MA, USA
David E. Weinberg & David P. Bartel
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
David E. Weinberg & David P. Bartel
Howard Hughes Medical Institute California Institute for Quantitative Biomedical Research, San Francisco, CA, USA
Meghan M. Zubradt & Jonathan S. Weissman
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
Gloria A. Brar
Department of Physics, Case Western Reserve University, Cleveland, OH, USA
Eckhard Jankowsky

Authors

Ulf-Peter Guenther
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David E. Weinberg
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Meghan M. Zubradt
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Frank A. Tedeschi
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Brittany N. Stawicki
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Leah L. Zagore
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Gloria A. Brar
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Donny D. Licatalosi
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David P. Bartel
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Jonathan S. Weissman
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Eckhard Jankowsky
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Contributions

U.-P.G.: conceptualization, material generation, ribosome profiling, XL-RAP–seq, iCLIP, molecular biology techniques, bioinformatic analysis, statistical analysis and manuscript writing; D.E.W.: conceptualization and iCLIP; M.M.Z.: conceptualization, DMS-MaPseq and bioinformatic analysis; F.A.T.: iCLIP, bioinformatic analysis, western blots; B.N.S.: material generation and northern blots; L.L.Z. and D.D.L.: iCLIP; G.A.B.: material generation and meiosis strains; D.P.B.: conceptualization and study supervision; J.S.W.: conceptualization and study supervision.; E.J.: conceptualization, study supervision and manuscript writing. All authors commented on and edited the manuscript.

Corresponding author

Correspondence to Eckhard Jankowsky.

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Extended data figures and tables

Extended Data Fig. 1 mRNA expression, and translation profiles in wild-type DED1 and ded1-95.

a, Correlation of ribosome footprint counts between two biological replicates in wild-type DED1 at 30 °C (n = 5,523). b, Correlation of mRNA expression levels between two biological replicates in wild-type DED1 at 30 °C (n = 5,372). c, Correlation of ribosome footprint counts between two biological replicates in wild-type DED1, 5 min after temperature shift to 37 °C (n = 5,523). d, Correlation of mRNA expression levels between two biological replicates in wild-type DED1, 5 min after temperature shift to 37 °C (n = 5,372). e, Correlation of ribosome footprint counts between two biological replicates in ded1-95 at 30 °C (n = 5,523). f, Correlation of mRNA expression levels between two biological replicates in ded1-95 at 30 °C (n = 5,372). g, Correlation of ribosome footprint counts between two biological replicates in ded1-95, 5 min after temperature shift to 37 °C (n = 5,523). h, Correlation of mRNA expression levels between two biological replicates in ded1-95, 5 min after temperature shift to 37 °C (n = 5,372). i, Correlation of mRNA expression levels between wild-type DED1 and ded1-95 at 30 °C (n = 2,976). Each data point represents the average of at least two replicates. j, Correlation of translational efficiencies between wild-type DED1 and ded1-95 at 30 °C (n = 2,976). Each data point represents the average of at least two replicates. k, Representative polysome profiles of wild-type DED1 and ded1-95 strains at 30 °C and 5 min after shift to 37 °C. Similar results were obtained in three independent experiments. l, Changes in translational efficiencies (∆TE) for mRNAs in ded1-95, compared to wild-type DED1, 5 min after temperature shift (mean of two biological replicates). The dotted line indicates no change. m, Fraction of 18S rRNA in polysome fractions, compared to the entire sample, at 30 °C and 5 min after temperature shift to 37 °C. Each bar represents the average of three independent experiments. Empty circles represent each replicate. n, Cumulative distribution of translational efficiencies of wild-type DED1 and ded1-95, 5 min after temperature shift to 37 °C (n = 2,976). Each data point represents the average of at least two replicates.

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Extended Data Fig. 2 A subset of mRNAs is largely insensitive to Ded1p.

a, mRNA groups defined by Gene Ontology term with translation that is strongly affected (green) or largely unaffected by Ded1p (blue). Box plots (group median) of change in translational efficiencies. Box boundaries, upper and lower quartiles; error bars, 1.5 × interquartile range. The black box plot marks changes in translational efficiencies for all mRNAs (Fig. 1b). mRNAs for each Gene Ontology term were extracted from the Saccharomyces Genome Database (https://www.yeastgenome.org). The false discovery rate q value indicates the enrichment P value according to a hypergeometric model after correction for multiple testing using the Benjamini and Hochberg method⁴⁶. b, Box plots (as in a) of 5′ UTR lengths and median of the shift in the normalized centre of ribosome density (Fig. 1b) for Gene Ontology term defined mRNA groups, colour-coded as in a.

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Extended Data Fig. 3 Activation of ATIS in ded1-95 upon temperature shift.

a, Fraction of ribosome footprints on 5′ UTRs in wild-type DED1 and ded1-95, (5 min, 37 °C, n = 3,273). The red line indicates the mean. Statistical significance for the difference between ded1-95 and wild-type DED1: P = 1.2 × 10⁻¹¹⁹ (two tailed t-test). A similar result was obtained in an independent replicate (P = 5.4 × 10⁻⁴⁷). b, Changes in the fraction of ribosomes on 5′ UTRs for all mRNAs (n = 2,660) in wild-type DED1 compared to ded1-95, 5 min after temperature shift to 37 °C. The values on the x-axis represent the ratio (log₂) of the fraction of ribosomes on each 5′ UTR in the wild type, divided by the fraction of ribosomes on the same 5′ UTR in ded1-95. Each value represents the mean of two independent biological replicates. c, Representative northern blots of PSA1 after sucrose gradient centrifugation for wild-type DED1 and ded1-95, at 30 °C. A similar result was obtained in an independent biological replicate. d, Fraction of ribosome footprints on 5′ UTRs in wild-type DED1 and ded1-95, (5 min, 37 °C), measured only in 80S monosomes (n = 973, reads from two independent experiments combined). Statistical significance for the difference between ded1-95 and wild-type DED1: P = 1.2×10⁻⁵⁰ (two tailed t-test). e, Mean ribosome occupancy within 10 nt 3′ and 5′ of the high-confidence ATIS in 5′ UTRs (moving average ± 1 nt, 5 min, 37 °C), measured without cycloheximide. f, Mean ribosome occupancy within 10 nt 3′ and 5′ of the high-confidence ATIS in 5′ UTRs (n = 274) in 80S monosomes (moving average ± 1 nt, 5 min, 37 °C). g, Mean ribosome occupancy within 10 nt 3′ and 5′ of all near-cognate initiation codons (n = 61,614; excluding medium-confidence ATIS) in 5′ UTRs (moving average ± 1 nt, 5 min, 37 °C). h, Ribosome occupancy of 3′ ends in small upstream open reading frames (smORFs) initiating at high-confidence ATIS in ded1-95 before (t = 0) and after (t = 5 min) temperature shift. smORFs were included in this analysis if its length exceeds three codons and if the smORF terminates at least 11 nt upstream of the main AUG codon (n = 76). i, Ribosome occupancy of 3′ ends of smORFs defined in e in wild-type DED1 before (t = 0) and after (t = 5 min) temperature shift. j, Ribosome occupancy 4 nt 5′ and 20 nt 3′ of high-confidence ATIS on 5′ UTRs (n = 274) for ded1-95, 5 min after temperature shift. The dashed lines indicate the first nucleotide of the marked in-frame codons. k, Ribosome occupancy 4 nt 5′ and 20 nt 3′ of the main AUG (mAUG) of mRNAs containing high-confidence ATIS on 5′ UTRs for ded1-95, 5 min after temperature shift. For mRNAs with multiple high-confidence ATIS in their 5′ UTR, the main AUG was counted only once. The dashed lines indicate the first nucleotide of the marked in-frame codons. l, Ribosome occupancy 4 nt 5′ and 20 nt 3′ of high-confidence ATIS-matched random positions (averaged from five randomizations) on 5′ UTRs (n = 274) for ded1-95, 5 min after temperature shift. The dashed line indicates the first nucleotide.

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Extended Data Fig. 4 Characteristics of small open reading frames associated with activated ATISs.

a, Enrichment or depletion of each near-cognate codon in ATISs over the background distribution of the codon. P values determined using a two-tailed t-test. b, Mean translation initiation site score (positions −6 to +6, excluding +1 to +3), calculated according to previously published methods⁴⁷, for high-stringency ATIS (n = 274, red), and TIS of main ORFs (n = 4,972, grey). A TIS score exceeding 0.01 is considered a potential translational initiation site¹⁴. c, Changes in translational efficiencies (ΔTE) for mRNAs in ded1-95, compared to wild-type DED1, 5 min after temperature shift for all mRNAs (Fig. 1b) and ATIS-containing mRNAs. d, Length of the small open reading frames (smORFs) associated with ded1-95-activated ATIS. smORFs encoding N-terminal extensions were excluded from the analysis. e, Type of smORFs associated with ded1-95-activated ATIS. The bar graphs show the fraction of smORFs that falls into each category. The distribution of changes in translation efficiency (ΔTE) for RNAs with each type of smORF did not differ significantly.

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Extended Data Fig. 5 mRNA structure unwinding by Ded1p in cells using DMS MaPSeq.

a, Schematic for DMS-MaPseq approach to monitor RNA structure unwinding by Ded1p. All DMS-MapSeq experiments were performed 5 min after temperature shift. b, Representative DMS MaPSeq tracks in the PSA1 5′ UTR 5 min after temperature shift for wild-type DED1 (grey) and ded1-95 (red). Bars show normalized reverse transcription stops. A similar result was obtained in an independent replicate. The average Pearson’s correlation coefficient of DMS-MaPseq counts per 5′ UTR between two replicates (5 min after temperature shift to 37 °C) were R = 0.57 (n = 864) for the wild type and R = 0.63 (n = 692) for ded1-95. The ribosome occupancy track for ded1-95 is shown for reference. c, Unwinding of mRNA structure by Ded1p for different mRNA regions. Similar results were obtained in two independent experiments.

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Extended Data Fig. 6 XL-RAP–seq and iCLIP

a, Correlation of sequence reads (fragments per kilobase of exon per million fragments mapped, FPKM) per mRNA for two independent biological XL-RAP–seq replicates (n = 2,992) b, Fraction of mRNA (40%) and rRNA (44%) cross-linked to wild-type Ded1p as a fraction of all sequencing reads (mean of two independent experiments). n = 4,280 mRNAs exceed a minimal read count of FPKM ≥ 10. c, Correlation of the number of reverse transcription stops (FPKM) per mRNA for the two independent iCLIP approaches. Replicate 1, FLAG-tagged Ded1p; replicate 2, HTBH-tagged Ded1p; n = 4,007.

Extended Data Fig. 7 Ded1p binding sites on 18S RNA and mRNAs.

a, Ded1p binding sites on helix 720 (exit) and helix 16 (entry) (red) are in close proximity to the binding sites of eIF3c (purple) and eIF3b (green) on the 40S ribosomal subunit⁴² (rRNA, grey; ribosomal proteins, cyan). b, Localization of Ded1p (apricot) on helix 16 of the PIC. Schematic model of the yeast PIC with eIF3 (http://www.bangroup.ethz.ch/research/eukaryotic_translation_initiation.html and references therein); the positioning of eIF4G^572-853 and eIF4A is derived from previously published work⁴⁸. The position of the eIF4G C terminus is hypothetical. The helicase core of Ded1p was modelled in analogy to the DDX3 core structure⁴⁹, with the RNA binding site in contact with helix 16 at the main iCLIP cross-link sites. The position of the low-complexity N terminus of Ded1p is hypothetical. c, Sequence logo of Ded1p binding sites on mRNAs. Sets of 10⁴ binding sites were randomly sampled from all Ded1p cross-linking sites and used as input to create a sequence logo (http://weblogo.berkeley.edu). All subsets yielded essentially the same sequence logo as shown here. Position zero denotes the reverse transcription stop.

Extended Data Fig. 8 Representative northern blots of PSA1 (Δ2°) and ATP5 (ΔATIS, Δ2°) for wild-type DED1 and ded1-95.

a, Representative RNA blots (5 min, 37 °C) for the PSA1 mRNA with altered secondary structure, 3′ of the ATIS (Δ2°). Similar results were obtained in three independent biological replicates. b, Quantification of RNA blots for accumulation of the PSA1 Δ2° mRNA in monosomes in ded1-95, compared to wild-type DED1. The line indicates the average. The P value for the difference in monosome accumulation was determined using a one-tailed t-test. c, Representative ribosome profiling tracks for the 5′ UTR of ATP5 in wild-type DED1 and ded1-95 (at 30 °C and after temperature shift). The near-cognate initiation codon is highlighted by a star. For comparison, Ded1p cross-linking (yellow track) and differential DMS-MaPseq tracks (log₂(ded1-95/wild type), unwound mRNA regions are marked by red bars and have negative values) for the 5′ UTR of the ATP5 mRNA are also shown. Similar results were obtained in two independent experiments. d, DMS-MapSeq-constrained secondary-structure model of a fragment of the ATP5 mRNA 5′ UTR. The ATIS is marked by a line. Ded1p cross-linking (iCLIP) and unwinding (DMS-MapSeq) for each nucleotide are indicated. The ratio of normalized DMS-MapSeq counts of wild type/ded1-95 in two categories: yellow triangles, 0.6 – 1.0 (moderately unwound) and red triangles, >1.0 (strongly unwound). e, Representative RNA blots (5 min, 37 °C) for wild-type ATP5 mRNA and the same mRNA with mutations in the ATIS (ΔATIS) or with altered secondary structure 3′ of the ATIS (Δ2°) for wild-type DED1 and ded1-95. Similar results were obtained in three independent experiments. f, Quantification of RNA blots for accumulation of the ATP5 ΔATIS and Δ2° mRNA in monosomes in ded1-95, compared to wild-type DED1. Lines indicate averages and P values were determined using a one-tailed t-test.

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Extended Data Fig. 9 Ded1p binding and mRNA remodelling can occur without decreased translation efficiency if no near-cognate initiation codon is present.

Ded1p iCLIP track, differential DMS-MaPseq track (5 min, 37 °C) and ribosome occupancy tracks (5 min, 37 °C) of wild-type DED1 and ded1-95 for ADH3 mRNA, the translation of which is largely unaffected by Ded1p (ΔTE = −0.1). 5′ UTR and ORF are marked. iCLIP and DMS-MaPseq tracks show Ded1p binding and remodelling of the 5′ UTR, ribosome profiling tracks indicate no significant accumulation of ribosomes in the 5′ UTR. Similar results were obtained in two independent experiments.

Extended Data Fig. 10 ATIS conservation across fungi and Ded1p-mediated activation of upstream ORFs starting from near-cognate initiation codons.

a, Sequence conservation in fungi around high-confidence ATIS (moving average of ±1 nt). Positive values indicate higher sequence conservation than the average of five randomly chosen positions on the same 5′ UTR for each ATIS, negative values indicate less sequence conservation (conservation scores were obtained from the sacCer3 phastCons7way dataset, on the basis of sequence homology between the following species: S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus, Naumoovozyma castellii and Lachancea kluyveri)⁵⁰. b, Ribosome occupancy tracks (30 °C and 5 min, 37 °C) of wild-type DED1 and ded1-95 for ALA1 mRNA. 5′ UTR and ORF are indicated. The ACG initiation codon (−25, marked) has been previously shown to function as an ATIS for the mitochondrial isoform of Ala1p²⁹. Similar results were obtained in two independent biological replicates for each experiment. c, Ribosome occupancy tracks (5 min, 37 °C) of ded1-95 and wild-type DED1 (vegetative control and anaphase II) for PSA1 mRNA. ATISs are marked by dashed lines. Similar results were obtained in two (vegetative control) and four (anaphase II) independent experiments. d, Ribosome-protected fragments mapping to DED1 in vegetative cells and cells in anaphase II. Data are the mean of two (vegetative) and four (anaphase II) independent experiments, circles represent each replicate.

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Guenther, UP., Weinberg, D.E., Zubradt, M.M. et al. The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs. Nature 559, 130–134 (2018). https://doi.org/10.1038/s41586-018-0258-0

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Received: 30 January 2017
Accepted: 08 May 2018
Published: 27 June 2018
Issue Date: 05 July 2018
DOI: https://doi.org/10.1038/s41586-018-0258-0

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Yeast strains, plasmids and oligonucleotides

Generation of a yeast strain expressing Ded1p–HTBH

Generation of a yeast strain expressing Ded1p–His6–FLAG3

Generation of a yeast strain expressing wild-type, ∆ATIS and secondary structure mutants of PSA1 and ATP5 mRNAs

Polysome analysis

Northern blot analysis

Western blot analysis

Ribosome profiling

XL-RAP–seq

iCLIP

DMS-MaPseq

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Generation of a yeast strain expressing Ded1p–His₆–FLAG₃