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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
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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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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
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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.
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