Active ribosome profiling with RiboLaceActive ribosome profiling with RiboLace . Massimiliano...

Active ribosome profiling with RiboLace

Massimiliano Clamer1,2*, Toma Tebaldi1, Fabio Lauria3, Paola Bernabò3, Rodolfo F.

Gómez-Biagi4, Elena Perenthaler3, Daniele Gubert3,5, Laura Pasquardini4, Graziano

Guella6, Ewout J.N. Groen7, Thomas H. Gillingwater7, Alessandro Quattrone1,

Gabriella Viero3*

1 Centre for Integrative Biology, University of Trento, Via Sommarive, 9 Povo (Italy). 2 IMMAGINA

Biotechnology srl, Via alla cascata 56/c, Povo (Italy). 3 Institute of Biophysics, CNR Unit at Trento, Via

Sommarive, 18 Povo (Italy). 4 Fondazione Bruno Kessler-LaBSSAH, Via Sommarive 18, Povo,

Trento, Italy. 5 Department of Information Engineering and Computer Science, University of Trento,

Povo (Italy). 6 Department of Physics, University of Trento, Povo (Italy). 7 Edinburgh Medical School:

Biomedical Sciences, University of Edinburgh, Edinburgh, UK *corresponding authors

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments

protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide

positional information concerning ribosomes flowing along transcripts, this method can be

used to shed light on mechanistic aspects of translation. However, current Ribo-Seq

approaches lack the ability to distinguish between fragments protected by ribosomes in

active translation or by inactive ribosomes. To overcome these significant limitation, we

developed RiboLace: a novel method based on an original puromycin-containing molecule

capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down

approach. RiboLace is fast, works reliably with low amounts of input material, and can be

easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of

active ribosome footprints at single nucleotide resolution.

The tightly regulated process of protein synthesis is a core regulator of numerous

critical physiological pathways, ranging from cell growth1 and development2,3 through

to immune responses4. Local protein synthesis in neurons5 also plays fundamental

roles in memory formation6–8 and synaptic plasticity9. Hence, dysregulation of

translation is a major driver of important pathologies, such as cancer10,11 and

neurodegenerative diseases12.

Over the last few years, newly developed methodological approaches such as

ribosome profiling (Ribo-Seq)13, have contributed to considerable new insights into

the translation process. Ribo-Seq has been largely employed to identify novel

translated RNAs (coding and non-coding), map novel upstream Open Reading

Frames (ORFs), and estimate translation levels in different biological conditions.

Indeed, Ribo-Seq has the potential to estimate translation efficiencies and the

“protein synthesis levels”14,15 in a variety of organisms, from prokaryotes15, to

yeast13, C. elegans16, zebrafish17,18, plants19, the mouse20, and human cell lines21–23.

Despite its undoubtful discrimination power and wide applicability, Ribo-Seq still

faces a number of challenges and presents with numerous limitations. For example,

translationally inactive mRNAs can be sequestered into ribonucleoprotein particles

(mRNP) or monosomes (80S), whose translational status remains a controversial

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 22, 2017. ; https://doi.org/10.1101/179671doi: bioRxiv preprint

https://doi.org/10.1101/179671

issue24–26. Importantly, mRNAs can be trapped within stalled or paused polysomes,

as has been shown especially in neurons12,27–33. As such, Ribo-Seq does not

necessarily discriminate “true” protected footprints of translating polysomes from

RNA fragments protected by the 80S ribosome or stalled ribosomes in polysomes,

leading to possible misinterpretations of translation occupancy profiles. Therefore,

Ribo-Seq still requires further optimization and refinements, from the bench to the

data analysis34, in order to generate maximal insights into the translation process.

Here, we present RiboLace, a novel methodological approach based on a newly

developed reagent, a puromycin analog molecule. RiboLace greatly improves the

study of translation both in vitro and in vivo, by selectively portraying mRNA

fragments protected by bona fide active ribosomes at single nucleotide resolution

and with unprecedented simplicity, requiring 30 times less biological material than

current protocols.

RESULTS

Design and synthesis of a new analog of puromycin

Puromycin is an aminonucleoside antibiotic able to bind the catalytic center of

the ribosome and the nascent peptide chain, causing ribosome disassembly and

disruption of protein synthesis35–38. Over the years, it has been used extensively to

quantify global of protein synthesis, taking advantage of radioactive39 and

biotinylated molecules40 or anti-puromycin antibodies41. Leveraging its ability to keep

contact with the ribosome42–45, puromycin has also been employed to covalently link

an mRNA to the corresponding protein during its synthesis46,47. In addition,

puromycin can be modified to create cell-permeable analogues suitable for direct

and in situ imaging of newly synthesized proteins48,49. All these methods require the

irreversible reaction of the α-amino group of puromycin with the carbon on its

carbonyl group, acylating the 3ʼ hydroxyl group of the peptidyl-tRNA buried in the P-

site of the ribosome.

Motivated by evidence that molecules containing α-amino group modified puromycin

can bind the large subunit of the active ribosome45, we covalently coupled puromycin

to a biotin moiety through two 2,2ʼ-ethylenedioxy-bis-ethylamine units, to obtain a

new compound still able to bind ribosomes by mimicking the tRNA entrance in the

acceptor site (A-site). We synthesized the new molecule at purity higher than 90%,

characterized it by NMR (Fig. 1a and Supplementary Fig. 1-6) and called it 3P.

Then, we verified the activity of the biotin moiety, taking advantage of its absorbance

spectrum and tested the binding on polystyrene or agarose beads. We observed that

the biotin group allows the binding of the 3P to commercially available streptavidin-

beads (Fig. 1b).

To demonstrate that 3P molecule maintains an inhibitory effect on translation,

we compared its effects to that exerted by puromycin using an eukaryotic in vitro

cell-free transcription-translation system (IVTT, rabbit reticulocyte lysate) and the

firefly luciferase as a reporter gene. We monitored total protein production by SDS-


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PAGE (Fig.1c, Supplementary Fig.7) and luminescence assay (Supplementary

Fig.7) in the presence of puromycin and 3P at different concentrations. As

expected, puromycin induced conspicuous decay of protein production at nanomolar

concentrations. In the case of 3P, we observed a decreased level of translation,

which reached ̴70% of inhibition at concentrations higher than 1 µM (Fig. 1c). We

concluded that, even if with slightly lower efficiency than puromycin, 3P can inhibit

eukaryotic translation in vitro, interfering with ribosome function, and can be used to

produce functionalized beads.

3P-functionalized magnetic beads captures mRNAs in active translation

in vitro.

Our finding that 3P is able to interact with the translation process, likely

through its puromycin moiety, prompted us to investigate whether 3P can capture

mRNAs under active translation.

First, we monitored the ability of 3P-functionalized magnetic beads (3P-beads) to

purify transcripts of reporter genes with different levels of protein expression in in

vitro translation systems. To purify mRNAs with 3P-beads, we developed the

following protocol (Fig. 2a): (i) 3P-beads and control beads functionalized with a

biotin-glycol conjugate (mP-beads, see Fig. 2a legend and methods for details) were

added to the in vitro translation system and the suspension was incubated for one

hour at 4°C in an appropriate buffer containing cycloheximide, to antagonize the

dissociation of ribosomal subunits exerted by the puromycin component of the

molecule; (ii) beads were pulled down using a magnet and washed two times; (iii)

protein and/or RNA were extracted for downstream analyses. In parallel, the

production of protein was followed by immunoblotting of whole protein extracts.

As expected, the EGFP reporter gene showed differential efficiency in protein

production depending on the expression vector used (pBluescript II KS+50, low

performance protein production and IPR- IBA251, high performance protein

production) (Fig. 2b, upper panel and Supplementary Fig. 8). Complete inhibition

of protein production was observed after addition of the translation inhibitor

harringtonine, as a control. RNA was purified and the relative abundance of the

EGFP mRNA, in both low and high performant conditions, was monitored by qRT-

PCR. We observed on 3P functionalized beads a 7 to 10-fold enrichment of the

reporter transcript in conditions of active translation, with respect to samples treated

with harringtonine (Fig. 2b lower panel, left), and in the absence of transcriptional

changes (Fig. 2b lower panel, right). The decreased enrichment in highly

performant conditions and at 120 min of incubation can be related to the fact that the

in vitro system is a closed one, causing the reaction to stop in the absence of

continuous supply of reactants during later stages of incubation.

Then, to demonstrate that this result was not dependent on the reporter used,

we applied RiboLace to a luciferase reporter system. In this case we observed a >

1.6-fold enrichment of luciferase transcript with respect to negative controls (mP-

beads) and an enrichment with respect to samples where translation had been


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inhibited (Fig. 2c), as previously observed for EGFP (Supplementary Fig. 9).

Finally, to understand if the observed enrichments were dependent on the puromycin

moiety of 3P, we pre-saturated the system with puromycin and induced ribosome

drop-off. Under these conditions, we found no evidence for mRNA enrichment.

Overall, these findings support the claim that 3P-beads can be used to capture

transcripts undergoing translation in eukaryotic in-vitro systems. We named the

method RiboLace.

RiboLace captures active ribosomes and associated mRNAs from whole

cellular lysates

Next, we wanted to establish whether RiboLace was capable of isolating

ribosomes and mRNAs under active translation from more complex samples than in

vitro mixtures. We used RiboLace on whole cellular lysates and under different

translational states, and monitored the resulting proteins and mRNAs associated

with the beads (Fig. 3a). We took advantage of established cellular stimuli that can

induce cells into translationally active or inactive states. To shut-down translation we

used cell starvation, oxidative, proteotoxic and heat stresses, known to globally

suppress protein synthesis52 (Supplementary Fig. S10a). To specifically activate

protein synthesis, we rescued cells from starvation by Epithelial Growth Factor

(EGF) or by Fetal Bovine Serum (FBS) stimulation53.

First, we monitored the enrichment on RiboLace of functional and structural

markers of ribosomes in lysates of immortalized human cells (HEK-293T).

Importantly, we used 2 x 105 cells, representing ̴1/30th of what classical polysomal

profiling approaches require. We monitored the ability of RiboLace to purify proteins

known to be associated with the translation machinery (eEF1α, calnexin, RPL26 and

RPS6). The elongation factor eEF1α is responsible for the delivery of aminoacyl-

tRNAs to the translation machinery and is associated to ribosomes in active

translation54,55. Calnexin is a chaperone protein in the endoplasmic reticulum that

associate with ribosomes, helping protein folding during translation56. Finally, RPL26

and RPS6 are ribosomal proteins belonging to the large and small subunits of the

ribosome, respectively. After immunoblot on the RiboLace eluted proteins, in lysates

from untreated cells (nt, Fig 3b and Fig. 3b), we observed an enrichment of all four

proteins (Fig. 3c and Fig.3d) with respect to serum starvation (st). When cells were

stimulated with EGF after starvation (st. + EGF 1 µg/mL, 4 hours, after starvation),

we observed a slight increase in the signal of the translational markers (Fig. 3c).

Since the background signal in controls (mP-beads) was the same in all conditions,

our results suggest that RiboLace can pull-down active ribosomes and therefore

monitoring the translational state of cells.

To further confirm this result, we compared the relative abundances on

RiboLace of eEF1α and eEF2 between no-stress and stress conditions. It is known

that eEF2-mediated translocation, as well as the switch of ribosome conformation

from the non-rotated to the rotated state57, is inhibited by cycloheximide58. We

observed that the two proteins differentially co-sedimented along the polysomal


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profile, with eEF1α mainly detected in the heavy fractions (Supplementary Fig. 10b)

indicating a preferential association of eEF1α to ribosomes in cycloheximide treated

cells. Then, in agreement with the hypothesis that RiboLace captures active

ribosomes, we found enrichment of eEF1α, but not of eEF2 (Fig. 3e and

Supplementary Fig. 10c and Fig 11), suggesting the capture of the pre-

translocation complex in the non-rotated conformation.

Next, we tested RiboLace in a different cell line, the widely used human tumor

cell line (MCF7), in control (nt) or starved (st) and control conditions , again

establishing levels of translational markers associated to RiboLace. In addition to

ribosomal proteins, we detected additional proteins known to be associated to

polysomes (PABP, eIF4B), or a marker of active translation (me(K9)H3)59 (Fig 3f

and Supplementary Fig. S10d). In agreement with results obtained for HEK-293T

cells, the decrease in translational markers associated to the beads in starved cells

suggests that RiboLace captures less ribosomes when global translation is

downregulated in different cell lines. To further validate this finding, we applied other

stresses known to elicit repression of global protein synthesis (i.e. proteotoxic stress,

heat shock and sodium arsenite). In all cases, translation markers were reduced

(Supplementary Fig. 10e). We then tested RiboLace on a mouse motor-neuron like

cell line, NSC-34, in normal growth conditions. Also in this case we observed an ̴8-

fold enrichment of RPL26 and ̴ 4-fold enrichment of RPS6 with respect to control

beads (Supplementary Fig. 10f, right), demonstrating that RiboLace can efficiently

capture ribosomes from both human and mouse cell lines. Lastly, we wanted to

establish whether RiboLace could capture components of the eukaryotic surveillance

mechanisms that target stalled elongation complexes. Among them, Pelota

(mammalian orthologue of the yeast Dom34)60,61 is a protein known to promote the

dissociation of stalled ribosomes. Strikingly, in control, starved or arsenite treated

lysates of HEK-293T and MCF7 cells, Pelota was not enriched on RiboLace beads

(Fig. 3g).

These findings prompted us to investigate whether RiboLace can provide an

improved estimation of translation efficiency, with respect to the use of total RNA or

polysomal RNA in profiling experiments (Fig. 3h and Supplementary Fig. S12). We

coupled RiboLace to Next Generation Sequencing (NGS) and identified sets of

differentially expressed mRNAs associated to RiboLace before and after EGF

stimulation (Supplementary Fig. 13 and Supplementary Fig. 14). We compared

these data with transcripts identified by classical transcriptome (RNA-Seq) or

translatome (POL-Seq, i.e. polysomal profiling62) analyses. Eight found differentially

expressed genes were selected for validation by RT-qPCR (PALLD, PLK3, IL27RA,

NCS1, VEGFA, DUSP5, PDCD4, PAPSS2), showing a good agreement with NGS

(Pearsonʼs r = 0.89) (Supplementary Fig.15). Then, we compared the protein levels

of four genes (PALLD, PLK3, hb-EGF and CYP27A1) to the relative RNA

abundances. PALLD and PLK3 proteins, evaluated by immunoblotting and

normalised for three different housekeeping proteins (ACTB, GAPDH and RPL26),

did not change upon EGF treatment (Fig 3i). Importantly, only RiboLace (RL) did not

show significant variation on both RT-qPCR and RNA-Seq measurements. The


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same concordance between protein level and RiboLace was obtained with hb-EGF

and CYP27A1, whose protein levels increased coherently with RiboLace

(Supplementary Fig. S16). Overall, these results establish the important proof-of-

concept that RiboLace can capture ribosomes under active translation and

determine protein variations more precisely than total or polysomal profiling, at least

in our case study.

In vivo active-ribosome profiling using RiboLace

Given the relative simplicity of the RiboLace protocol, and its ability to be used

with low amounts of input material, we next wanted to combine our purification

strategy with Ribo-Seq. This would establish whether RiboLace could be used to

capture active ribosome dynamics along transcripts, improving in vivo ribosome

profiling experiments. To facilitate this, we modified our original protocol by including

an endonuclease digestion step and applied it to lysates from mouse tissues (Fig.

4a).

To demonstrate that RiboLace can capture isolated ribosomes after

endonuclease digestion, we first measured the enrichment of eEF1α, calnexin,

RPL26 and RPS6 on RiboLace applied to control (nt), harringtonine treated (harr),

serum starved (st) or heat-shocked (h.s.) HEK-293T and HeLa cells (Fig. 4b). Our

results confirmed that RiboLace can selectively capture isolated ribosomes under

conditions of active translation. Then, we confirmed that RiboLace was able to enrich

ribosome protected fragments, by urea-gel electrophoresis and by the use of the

Bioanalyzer (Supplementary Fig. S17). After that, we probed RiboLace on 15 µL ( ̴

1/50thof the total lysate) samples of whole brain lysates from wild-type (WT) and

mice affected by the “wasted” mutation, consisting in a deletion of the elongation

factor eEF1α2 (Fig.4c), resulting in defective translation63. Our results, in agreement

with those previously obtained from cells, showed that RiboLace could selectively

enrich RPL26 only in the WT mouse tissue, extending its functionality from cell lines

to tissues.

Having established that RiboLace can capture isolated ribosomes from very

low amounts of starting material, we next isolated ribosome protected fragments 25-

35 nt long (RPFs) from ribosomes pulled down by RiboLace and, in parallel, from

polysomes isolated from whole mouse brain, following nuclease digestion (polysomal

Ribo-Seq, Fig. 4d and Supplementary Fig S18). After sequencing, we analyzed

both RiboLace and polysomal Ribo-Seq RPFs using the dedicated pipeline

RiboWaltz64 to obtain sub-codon information and identification of the trinucleotide

periodicity. The distribution of the reads showed a main population of length at ̴ 28

nt (Supplementary Fig. S19), in agreement with what has previously been observed

for ribosomes trapped on the mRNA by cycloheximide65,66. As expected for ribosome

footprints, we observed an enrichment of signal along the coding sequence in both

RiboLace and polysomal Ribo-Seq data (Fig. 4e). Occupancy meta-profiles showed

the typical trinucleotide periodicity of the ribosome P-site along coding sequences,

which is suggestive of signal derived from translating ribosomes (Fig.4f and g). The


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comparison between meta-profiles obtained with RiboLace and polysomal Ribo-Seq

highlights an accumulation of ribosomes at the start codon and at the 5th codon (Fig.

4h). Interestingly, the latter feature is associated with ribosome pauses necessary for

a productive elongation phase of translation67.

Taken together, these results confirm that RiboLace is capable of providing

positional data with nucleotide resolution and of enriching samples with active

ribosomes, thereby facilitating reliable descriptions of bona fide translational events

in vitro and in vivo.

DISCUSSION

During their lifetime in the cellular cytoplasm, mRNAs are regularly stored, degraded,

and transported, with only a fraction being actively translated to produce

proteins68,69. All these stages of the mRNA lifecycle are governed by cis- and trans-

factors that tightly regulate the uploading of mRNAs on polysomes, and subsequent

production of proteins. In order to generate a better understanding of these

sophisticated and dynamic processes, different methodological approaches have

been developed to determine, at a genome-wide scale changes in RNA steady state

levels (e.g. RNA-seq), the change in engagement with the translational machinery

(e.g. Ribo-Seq, polysomal profiling)20,62, and the change in protein production (e.g.,

SILAC, PUNCH-P)40,7071. Although Ribo-Seq remains a complex technology that

requires relatively large tissue samples, it has been shown to be extremely powerful

for identifying ORFs and translation initiation sites (i) from cell lysates or ribosome

pellets; (ii) from purified polysomal fractions (polysomal Ribo-Seq)19 or, more

recently, (iii) from tagged ribosomes72,73. Unfortunately, however, the use of cell

lysates and ribosome pellets often introduces unwanted background signals,

presumably mostly due to the presence of stalled ribosomes and fragments

protected by RNPs, or by the 80S monosome.

Here, we sought to significantly enhance Ribo-Seq approaches by developing a new

molecule (3P) that facilitates the selective capture of ribosomes under active

translation. We focused our attention on puromycin, the well-known structural

analogue of the 3′ end of aminoacyl-tRNA, and we suppressed its irreversible activity

by tethering the α-amino group to a biotinylated linker. Despite this modification on

the primary amino group of puromycin, we observed that 3P could still interfere with

eukaryotic translation in vitro. We used 3P- functionalized magnetic beads (a method

we called RiboLace) to capture and enrich transcripts undergoing translation in

eukaryotic in vitro and in vivo systems. We observed that the elongation factor

eEF1α, a key protein involved in delivering tRNAs to the ribosome, was the most

enriched protein on RiboLace in all our experiments. This may be explained with the

binding of 3P to the A-site of the ribosome in the not-rotated state, when the

acceptor site accommodates the aminoacyl-tRNA engaged by eEF1α. We then

demonstrated that RiboLace is capable of providing positional data with nucleotide


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resolution of translational events when used for ribosome profiling. Remarkably, we

observed > 95% of RPFs on the coding region with the characteristic trinucleotide

periodicity, suggestive of active ribosomes flowing along the transcripts. In our

biological model, RiboLace Ribo-Seq showed almost no signal on the 5′- and 3′-

untranslated regions of the mRNAs, and a peculiar pause of ribosomes at the 5th

codon. Overall, our data suggest that RiboLace possess significant advantages, in

terms of both sample yield and accuracy in RPFs detection, over classical Ribo-Seq

and polysomal Ribo-Seq approaches.

RiboLace protocols can be further adjusted to (i) isolate ribosomes from other

organisms, (ii) isolate ribosomes from specific eukaryotic cellular compartments such

as the Endoplasmic Reticulum or organelles like mitochondria, or (iii) to

characterized active specialized-ribosomes2,72,74 induced by different cell conditions.

We specifically designed RiboLace for ribosome profiling experiments, to facilitate

improved understanding of ribosome dynamics along transcripts and to allow better

estimates of translation levels based on ribosome footprints.

In summary, we report a major advancement in the technology available to

undertake ribosome profiling, providing the unique benefit of capturing ribosome in

active translation. Given its ability to enrich actively translating ribosome protected

fragments, RiboLace can be used in difficult samples with low input material, and

empowers accurate conclusions to be drawn concerning the actual translational

state of a biological system, paving the way for more detailed and accurate studies

of translation in the future.

METHODS

Methods and Supplementary Figures are available in the file Supplementary

Information.

ACKNOWLEDGMENTS

We thank Tocris Bioscience (a Biotechne brand) for the support in scaling up the 3P

synthesis. We thank Divya Kandala and Luca Minati for the helpful discussions. We

also thank Veronica Desanctis and Roberto Bertorelli of the CIBIO NGS for technical

support and Daniele Arosio for the kindly gift of the IPR- IBA2 plasmid.

AUTHOR CONTRIBUTION

MC and GV conceived the experiments. MC performed all the biological experiments

and contributed to the 3P synthesis. TT, DG and FL analyzed the RNA-seq and Ribo-

Seq data. PB and EP performed the Poly Ribo-Seq and helped with the RiboLace

Ribo-Seq. LP performed control experiments with mP-beads. RG performed a


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preliminary synthesis of 3P. GG designed the 3P synthesis, performed MS analysis,

COSY and NMR analysis of the 3P. EJNG and THG generated and provided the

mouse tissues. MC and GV drafted the manuscript. TT, FL and DG wrote the

methods related to RNA-seq and Ribo-Seq data analysis. GG wrote the methods

related to the chemical synthesis and characterization. GV, MC, TT, FL, GG, THG

and AQ reviewed the manuscript. All authors read and approved the final manuscript.

FUNDINGS

This work was supported by IMMAGINA BioTechnology s.r.l. and by the Provincia

Autonoma di Trento, Italy (AxonomiX research project)with additional grant funding

from the Wellcome Trust (to EJNG & THG) and UK SMA Research Consortium

(SMA Trust to THG).

COMPETING FINANCIAL INTEREST

MC is founder, director and a share-holder of IMMAGINA BioTechnology s.r.l., a

company engaged in the development of new technologies for gene expression

analysis at the ribosomal level. AQ and GG are shareholders and scientific

advisors of IMMAGINA BioTechnology s.r.l. GV is scientific advisor of

IMMAGINA BioTechnology s.r.l. All other authors declare no competing financial

interests. RiboLace is an IMMAGINA s.r.l. patented technology (WO2017013547

A1 - PCT/IB2016/054210).

Data availability. Raw and analyzed data for Ribosome profiling have been

deposited under GEO: GSE102354 for RiboLace Ribo-Seq, and GEO: GSE102318

for Poly Ribo-Seq


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REFERENCES

1. Goyer, C. et al. TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function. Mol. Cell. Biol. 13, 4860–4874 (1993).

2. Kondrashov, N. et al. Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning. Cell 145, 383–397 (2011).

3. Xue, S. et al. RNA regulons in Hox 5′ UTRs confer ribosome specificity to gene regulation. Nature 517, 33–38 (2014).

4. Piccirillo, C. A., Bjur, E., Topisirovic, I., Sonenberg, N. & Larsson, O. Translational control of immune responses: from transcripts to translatomes. Nat. Immunol. 15, 503–511 (2014).

5. Jung, H., Gkogkas, C. G., Sonenberg, N. & Holt, C. E. Remote control of gene function by local translation. Cell 157, 26–40 (2014).

6. Martin, K. C. et al. MAP Kinase Translocates into the Nucleus of the Presynaptic Cell and Is Required for Long-Term Facilitation in Aplysia. Neuron 18, 899–912 (1997).

7. Kandel, E. R., Dudai, Y. & Mayford, M. R. The Molecular and Systems Biology of Memory. Cell 157, 163–186 (2014).

8. Fioriti, L. et al. The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3. Neuron 86, 1433–1448 (2015).

9. McCamphill, P. K., Farah, C. A., Anadolu, M. N., Hoque, S. & Sossin, W. S. Bidirectional Regulation of eEF2 Phosphorylation Controls Synaptic Plasticity by Decoding Neuronal Activity Patterns. J. Neurosci. 35, 4403–4417 (2015).

10. Topisirovic, I. & Sonenberg, N. Translation and cancer. Biochim. Biophys. Acta - Gene Regul. Mech. 1849, 751–752 (2015).

11. Bhat, M. et al. Targeting the translation machinery in cancer. Nat. Rev. Drug Discov. 14, 261–278 (2015).

12. Darnell, J. C. et al. FMRP Stalls Ribosomal Translocation on mRNAs Linked to Synaptic Function and Autism. Cell 146, 247–261 (2011).

13. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. S. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–23 (2009).

14. Ingolia, N. T. et al. Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes. Cell Rep. 8, 1365–1379 (2014).

15. Li, G.-W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources. Cell 157, 624–635 (2014).

16. Stadler, M., Artiles, K., Pak, J. & Fire, A. Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets. Genome Res. 22, 2418–2426 (2012).

17. Chew, G.-L. et al. Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs. Development 140, 2828–2834 (2013).

18. Bazzini, A. A. et al. Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J. 33, 981–993 (2014).


https://doi.org/10.1101/179671

19. Juntawong, P., Girke, T., Bazin, J. & Bailey-Serres, J. Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 111, E203-12 (2014).

20. Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

21. Liu, B., Han, Y. & Qian, S.-B. Cotranslational Response to Proteotoxic Stress by Elongation Pausing of Ribosomes. Mol. Cell 49, 453–463 (2013).

22. Lee, S. et al. Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proc. Natl. Acad. Sci. 109, E2424–E2432 (2012).

23. Fritsch, C. et al. Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting. Genome Res. 22, 2208–2218 (2012).

24. WARNER, J. R., KNOPF, P. M. & RICH, A. A multiple ribosomal structure in protein synthesis. Proc. Natl. Acad. Sci. {U.S.A.} 49, 122–129 (1963).

25. Munro, A. J., Jackson, R. J. & Korner, A. Studies on the nature of polysomes. Biochem. J. 92, 289–99 (1964).

26. Heyer, E. E. & Moore, M. J. Redefining the Translational Status of 80S Monosomes. Cell 164, 757–769 (2016).

27. Chapman, R. E. & Walter, P. Translational attenuation mediated by an mRNA intron. Curr. Biol. 7, 850–9 (1997).

28. Verrier, S. B. & Jean-Jean, O. Complementarity between the mRNA 5’ untranslated region and 18S ribosomal RNA can inhibit translation. RNA 6, 584–97 (2000).

29. Sivan, G., Kedersha, N. & Elroy-Stein, O. Ribosomal slowdown mediates translational arrest during cellular division. Mol. Cell. Biol. 27, 6639–46 (2007).

30. Doma, M. K. & Parker, R. Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440, 561–564 (2006).

31. Higashi, S. et al. TDP-43 associates with stalled ribosomes and contributes to cell survival during cellular stress. J. Neurochem. 126, 288–300 (2013).

32. Ortiz, J. O. et al. Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ. J. Cell Biol. 190, 613–621 (2010).

33. Graber, T. E. et al. Reactivation of stalled polyribosomes in synaptic plasticity. Proc. Natl. Acad. Sci. U. S. A. 110, 16205–10 (2013).

34. Aeschimann, F., Xiong, J., Arnold, A., Dieterich, C. & Großhans, H. Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling. Methods 85, 75–89 (2015).

35. Yarmolinsky, M. B. & Haba, G. L. Inhibition By Puromycin of Amino Acid Incorporation Into Protein. Proc. Natl. Acad. Sci. U. S. A. 45, 1721–1729 (1959).

36. Welch, M., Chastang, J. & Yarus, M. An Inhibitor of Ribosomal Peptidyl Transferase Using the Transition-State Analogy. Biochemistry 34, 385–390 (1995).

37. Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).

38. Wilson, D. N. Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat. Rev. Microbiol. 12, 35–48 (2014).

39. Gambetti, P., Hirt, L., Stieber, A. & Shafer, B. Distribution of puromycin


https://doi.org/10.1101/179671

peptides in mouse entorhinal cortex. Exp. Neurol. 34, 223–228 (1972). 40. Aviner, R., Geiger, T. & Elroy-Stein, O. Genome-wide identification and

quantification of protein synthesis in cultured cells and whole tissues by puromycin-associated nascent chain proteomics (PUNCH-P). Nat. Protoc. 9, 751–60 (2014).

41. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275–277 (2009).

42. Pestka, S., Rosenfeld, H., Harris, R. & Hintikka, H. Studies on transfer ribonucleic acid-ribosome complexes. XXI. Effect of antibiotics on peptidyl-puromycin synthesis by mammalian polyribosomes. J. Biol. Chem. 247, 6895–6900 (1972).

43. Odom, O. W., Picking, W. D. & Hardesty, B. Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes. Biochemistry 29, 10734–10744 (1990).

44. Kukhanova, M. et al. The donor site of the peptidyltransferase center of ribosomes. FEBS Lett. 102, 198–203 (1979).

45. Schmeing, T. M. et al. A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits. Nat Struct Biol 9, 225–230 (2002).

46. Roberts, R. W. & Szostak, J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. U. S. A. 94, 12297–12302 (1997).

47. Biyani, M., Husimi, Y. & Nemoto, N. Solid-phase translation and RNA-protein fusion: A novel approach for folding quality control and direct immobilization of proteins using anchored mRNA. Nucleic Acids Res. 34, (2006).

48. Ge, J. et al. Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons. Angew. Chemie Int. Ed. n/a-n/a (2016). doi:10.1002/anie.201511030

49. Starck, S. R., Green, H. M., Alberola-Ila, J. & Roberts, R. W. A general approach to detect protein expression in vivo using fluorescent puromycin conjugates. Chem. Biol. 11, 999–1008 (2004).

50. Higashi, K. et al. Enhancement of +1 frameshift by polyamines during translation of polypeptide release factor 2 in Escherichia coli. J. Biol. Chem. 281, 9527–9537 (2006).

51. Arosio, D. et al. Spectroscopic and Structural Study of Proton and Halide Ion Cooperative Binding to GFP. Biophys. J. 93, 232–244 (2007).

52. Liu, B. & Qian, S.-B. Characterizing inactive ribosomes in translational profiling. Translation 4, e1138018 (2016).

53. Thomas, G. The effect of serum, EGF, PGF2α and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis. Cell 30, 235–242 (1982).

54. Andersen, G. R., Valente, L., Pedersen, L., Kinzy, T. G. & Nyborg, J. Crystal Structures of Nucleotide Exchange Intermediates in the eEF1A-eEF1Balpha Complex. Nat. Struct. Biol. 8, 531–534 (2001).

55. Lamberti, a et al. The translation elongation factor 1A in tumorigenesis, signal transduction and apoptosis: review article. Amino Acids 26, 443–448 (2004).

56. Lakkaraju, A. K. et al. Palmitoylated calnexin is a key component of the ribosome-translocon complex. EMBO J. 31, 1823–1835 (2012).

57. Lareau, L. F., Hite, D. H., Hogan, G. J. & Brown, P. O. Distinct stages of the


https://doi.org/10.1101/179671

translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments. Elife 2014, 1–16 (2014).

58. Schneider-Poetsch, T. et al. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat. Chem. Biol. 6, 209–217 (2010).

59. Rivera, C. et al. Methylation of histone H3 lysine 9 occurs during translation. Nucleic Acids Res. 43, 9097–9106 (2015).

60. Pisareva, V. P., Skabkin, M. A., Hellen, C. U., Pestova, T. V & Pisarev, A. V. Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 3093, 1804–1817 (2011).

61. Guydosh, N. R. & Green, R. Dom34 rescues ribosomes in 3′ untranslated regions. Cell 156, 950–962 (2014).

62. Tebaldi, T. et al. Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells. BMC Genomics 13, 220 (2012).

63. Chambers, D. M., Peters, J. & Abbott, C. M. The lethal mutation of the mouse wasted (wst) is a deletion that abolishes expression of a tissue-specific isoform of translation elongation factor 1alpha, encoded by the Eef1a2 gene. Proc. Natl. Acad. Sci. U. S. A. 95, 4463–8 (1998).

64. Lauria, F. et al. riboWaltz: optimization of ribosome P-site positioning in ribosome profiling data. BioRXiv (2017). doi:10.1101/169862

65. Archer, S. K., Shirokikh, N. E., Beilharz, T. H. & Preiss, T. Dynamics of ribosome scanning and recycling revealed by translation complex profiling. Nature 535, 570–4 (2016).

66. Lareau, L. F., Hite, D. H., Hogan, G. J. & Brown, P. O. Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments. Elife 2014, 1–16 (2014).

67. Han, Y. et al. Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation. Cell Res. 24, 842–851 (2014).

68. Wu, B., Eliscovich, C., Yoon, Y. J. & Singer, R. H. Translation dynamics of single mRNAs in live cells and neurons. Science (80-. ). 352, 1430–1435 (2016).

69. Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science (80-. ). 899, aaf0899 (2016).

70. Ong, S.-E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–60 (2006).

71. Liu, T.-Y. et al. Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics. Cell Syst. 4, 636–644.e9 (2017).

72. Simsek, D. et al. The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell 169, 1051–1065.e18 (2017).

73. Jan, C. H., Williams, C. C. & Weissman, J. S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling. Science (80-. ). 346, 1257521–1257521 (2014).

74. Shi, Z. et al. Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide. Mol. Cell 67, 71–83.e7 (2017).

75. Hansen, J. L., Moore, P. B. & Steitz, T. a. Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit. J. Mol. Biol. 330, 1061–1075 (2003).

76. Anger, A. M. et al. Structures of the human and Drosophila 80S ribosome.


https://doi.org/10.1101/179671

Nature 497, 80–5 (2013). 77. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. a. The complete

atomic structure of the large ribosomal subunit at 2. 4 A Resolut. Sci. 289, 905±920 (2000).

78. Afshar-Kharghan, V., Li, C. Q., Khoshnevis-Asl, M. & Lopez, J. A. Kozak sequence polymorphism of the glycoprotein (GP) Ibalpha gene is a major determinant of the plasma membrane levels of the platelet GP Ib-IX-V complex. Blood 94, 186–191 (1999).


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FIGURES

Figure 1. A new analog of puromycin inhibits translation and can be used for

functionalization of agarose and polystyrene beads. (a) Chemical formula of the

new puromycin analog: 3P. (b) 3P binding to streptavidin coated agarose and

polystyrene beads. Absorbance of the supernatant at 275 nm is measured after

addition of streptavidin coated magnetic beads to 100 pmol of 3P. Data represent the

mean of triplicate experiments (n = 3). The gray line identified the quantity of beads

(polystyrene, µg or agarose, µL of 10% slurry suspension) used in all experiments for

each sample. (c) Expression of the firefly luciferase in the presence of puromycin

(left panel) and 3P (right panel). ε-Labeled biotinylated lysine-tRNAs is used to

monitor the protein production by SDS–PAGE (top). The histograms represent the

relative quantification of the representative bands reported in the immunoblot with

respect to the control. The histogram on the right shows inhibition of luciferase

expression in the presence of different concentrations of 3P (0 µM, 0.01 µM, 0.1 µM,

1 µM and 10 µM (right panel). Error bars represent s.d. calculated from triplicate

experiments (n =3); (**) = t-test p-val < 0.05.


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Figure 2. 3P-beads can capture mRNAs in active translation in vitro. (a)

Experimental design: 3P-nbeads are used to pull-down transcripts in a cell-free in

vitro transcription-translation system. Briefly, from step 1 to 5: (1), Plasmids are

added to the in vitro transcription-translation reaction; (2), Beads are functionalized

with 3P; (3), The IVTT mix is added to 3P-beads and incubated for 1 hour in orbital

rotation at 2 rpm at 4°C; (4), Beads are washed without detaching them from the

magnet to remove unspecific binding; (4/a), The RNA is extracted, precipitated with

isopropanol, and digested with DNase I (4/b) to avoid possible DNA contaminations.

Finally, the cDNA is synthetized (4/c). (5), Samples are analyzed by RT-qPCR to

detect the reporter gene. (b) Top panel, immunoblotting of total EGFP protein at

different incubation time, without (-) or with harringtonine (+) at the reported

concentration. Middle panel, immunoblotting showing the comparison between the

total EGFP expression from the pPR-IBA2 plasmid and the EGFP expressed from

the pBluescript II KS+ plasmid. Bottom panel, EGFP RNA enrichment on RiboLace

(-, no harrigtonine) respect to the treated sample (harr, harringtonine treatment, 2

µg/mL for 3 min). On the right, the the total RNA content (gray histograms) without (-)

or with harringtonine (harr) for the two vectors as measured by RT-qPCR. (**) = t-test

pval = 0.01 (25 min); pval = 0.03 (120 min); n = 3. (c) Top panel, Sketch of

experimental protocol for in vitro transcription and translation of the firefly

luciferase(luc). Harringtonine and puromycin (puro, 5 µg/mL) are added to the

mixture. Then, (i) ribosomes in active translation are isolated with 3P-beads, by

using mP- beads as control; (ii) RNA is extracted, treated with DNAse I and retro-

transcribed to single stranded cDNA with random hexamers. (c) Bottom, Fold

change values relative to the total amount of transcript captured by 3P-beads

compared to the control beads (mP), henceforth referred as the ʼenrichmentʼ. (**) = t-


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test pval = 0.03 (luv vs luc+harr), pval = 0.02 (luc vs luc+puro); n = 5. Error bars

represent s.d.


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Figure 3. RiboLace is able to capture ribosomes and associated mRNAs under

active translation in cell cultures and tissues. (a) RiboLace protocol: magnetic

beads coated with streptavidin are functionalized with the 3P molecule (step 1).

RiboLace beads are then added to the cell lysate (step 2) (usually 5 - 20 µL,

corresponding to ~ 1.2 - 5 x 105 cells) and washed (step 3). Finally, both proteins

and RNA are recovered for further analysis (step 4). (b) Ponceau staining of a

nitrocellulose membrane containing (from left to right) the total protein extract after

applying the RiboLace protocol on HEK-293 and corresponding inputs in three


https://doi.org/10.1101/179671

different conditions (nt, not treated; st, starvation (0.5% FBS); st + EGF, starvation +

EGF stimulation). The lane intensity is reported as a percentage of the sum of the

three lysates. (c) Immunoblotting of eEF1ɑ, calnexin, RPL26 and RPS6 isolated after

applying the RiboLace protocol on HEK-293 (RiboLace, mP-beads, and in input). (d)

Quantification of proteins isolated with RiboLace in different stress conditions applied

to the cells. Immunoblots were scanned with a Chemidoc (Biorad) and quantified

with ImageJ v1.45s, n = 3, * indicates pval < 0.05. (e) Comparison between the

relative enrichment (no starvation vs starvation) of eEF2 and eEF1ɑ on RiboLace.

(*) t-test p-val = 0.02, n = 4 (f) Immunoblotting of eIf4B, PABP, eEF1ɑ, RPL14,

RPl26, me(K9)H3 and H3, detected on RiboLace in normal growing conditions (nt) or

under serum starvation (st) with relative inputs in MCF7 cytoplasmic lysates. (g)

Top, Immunoblotting of Pelota and eEF1α detected on RiboLace in HEK-293 not

treated, under starvation or after EGF stimulation, with relative inputs. Bottom,

Immunoblotting of Pelota and eEF1α detected on RiboLace in MCF7 treated (Ar) or

not treated (nt) with Arsenite. (#) indicates the number of each biological replicate

(h) Experimental design for identifying the global RNA repertoire of RNAs

associated to RiboLace by RNA-seq. MCF7 cells treated with EGF or serum-starved

in comparison to classical polysomal profiling (POL-Seq) and total RNA

transcriptomics analysis. After proteinase K digestion, RNA is extracted from

RiboLace and mP beads, and from both polyribosomal and total RNA from the same

profile. RiboLace, RL; Polyribosomal, P; Total, T.. Libraries are prepared using the

Illumina TruSeq library preparation kit and the sequencing performed with Illumina

HiSeq 2000. (i) Top, histograms representing RNA-seq (white bars) and RT-qPCR

(black bars) fold changes (FC) of four genes (NCS1, IL27RA, DUSP5, VEGFA) t-test

p-val < 0.05 (*). Bottom, Comparison between protein fold change and RNA fold

changes obtained with different methods (RL, P, T). The semi-quantitative analysis

of the protein band intensity (n = 3) for PALLD and PLK3 is reported in the

histogram. Black bars, RT-qPCR fold change; white bars, RNA-seq fold change; light

gray bars, protein fold change. Housekeeping: GAPDH, beta actin; ribosomal protein

L26. t-test (*) = p-val < 0.05.


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Figure 4. RiboLace Ribo-Seq. (a) Schematic RiboLace protocol for the separation

of single active ribosomes. Cell lysates (1) is treated with RNase I for 45 min (2) and

the quenched with an RNase inhibitor (3). Then, RiboLace beads are incubated with

the digested cell lysate (1h, 4°C) (4), washed (5) and then proteins or RNA extracted

(6). See methods for details. (b) Immunoblotting of eEF1α, calnexin, RPL26 and

RPS6 after applying the RiboLace protocol on HEK-293 and HeLa. Nt, not treated;

st, starvation (0.5% FBS), st + EGF, starvation + EGF stimulation; harr,

harringtonine; h.s., heat shock (c) Top, Schematic RiboLace protocol for the

extraction of single active ribosomes from mouse brain. Bottom, immunoblotting on

eEF1α, and RPL26 for RiboLace, mP-beads and relative inputs. (d) Scheme of the

protocol for comparative active Ribo-Seq and poly Ribo-seq. (e) Left, percentage of

P-sites mapping to the 5’ UTR, CDS and 3’ UTR of mRNAs from RiboLace and Poly

Ribo-Seq data. Right, percentage of region lengths in mRNAs sequences. Both

techniques show a clear enrichment in signal mapping to the CDS, consistent with


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ribosome protected fragments. (f) Percentage of P-sites corresponding to the three

possible reading frames along the 5’ UTR, CDS and 3’ UTR, stratified for read

length, comparing RiboLace (top panel) and Poly Ribo-Seq (bottom panel). For each

length and each region, the sum of the signal is normalized to 100%. The enrichment

in frame 0 is CDS specific in both cases. (g) Meta-gene profiles showing the density

P-sites around the translation initiation site (TIS) and translation termination site

(TTS) for RiboLace (top panel) and Poly Ribo-Seq (bottom panel). The peak

corresponding to the fifth codon is highlighted with an asterisk.

https://doi.org/10.1101/179671

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