Computational and Structural Biotechnology Journal 16 (2018) 167–176

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/csbj

Mini Review

Following Ribosome Footprints to Understand Translation at a Genome

Wide Level
Guillermo Eastman a, Pablo Smircich a,b, José R. Sotelo-Silveira a,c,⁎
a
Department of Genomics, Instituto de Investigaciones Biológicas Clemente Estable, MEC, Av. Italia 3318, Montevideo, CP 11600, Uruguay
b
Laboratory of Molecular Interactions, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo, CP 11400, Uruguay
c
Department of Cell and Molecular Biology, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo, CP 11400, Uruguay

a r t i c l e i n f o a b s t r a c t

Article history: Protein translation is a key step in gene expression. The development of Ribosome Profiling has allowed the
Received 16 January 2018 global analysis of this process at sub-codon resolution. In the last years the method has been applied to several
Received in revised form 6 April 2018 models ranging from bacteria to mammalian cells yielding a surprising amount of insight on the mechanism
Accepted 10 April 2018
and the regulation of translation. In this review we describe the key aspects of the experimental protocol and
Available online 1 May 2018
comment on the main conclusions raised in different models.
Keywords:
© 2018 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural
Ribo-seq Biotechnology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Translation
Translatome
Transcriptome
Ribosome profiling

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
2. Ribosome Profiling Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2.1. Protocol Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2.2. Protocol Variants, User Decisions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3. Biological Models and Contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
3.1. Bacteria: Translational Pausing, Codon Use and Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
3.2. Yeast: Start Codons, uORFs and Translational Pauses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
3.3. Mammalian Cells: uORFs, Pauses, Initiation Sites and lncRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
3.3.1. uORFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
3.3.2. Translational Pauses and Elongation Speed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
3.3.3. Translation Initiation Sites (TIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
3.3.4. Long Non Coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
3.4. Others Biological Models: Zebrafish, Drosophila, C. elegans, Trypanosomatids and Virus . . . . . . . . . . . . . . . . . . . . . . . . . 173
4. Applications, Challenges and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

1. Introduction

The decreasing cost of obtaining Next Generation Sequencing (NGS)

⁎ Corresponding author at: Department of Genomics, Instituto de Investigaciones data [1–3] together with the huge information sets arising from these
Biológicas Clemente Estable, Av. Italia 3318, Montevideo, CP 11600, Uruguay.
technologies is revolutionizing several research fields of life sciences
E-mail addresses: geastman@iibce.edu.uy (G. Eastman), psmircich@iibce.edu.uy
(P. Smircich), jsotelosilveira@iibce.edu.uy (see an example in [4] or in disease biology [5,6]). Ingenuity is continu-
URL: http://www.iibce.edu.uy (J.R. Sotelo Silveira). ously leading to the development of new methods, a very interesting

https://doi.org/10.1016/j.csbj.2018.04.001
2001-0370/© 2018 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
168 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176

case is an application named Ribosome Profiling (RP), or Ribo-Seq, de- freezing becomes crucial in cases where using translation inhibitors
veloped by Ingolia & Weissman in 2009 [7] where the deep sequencing are to be avoided.
of mRNA fragments covered by ribosomes during translation yielded an The RNAse protection assay, also called nuclease footprinting, is an-
original view of translation at a genome wide scale. The footprints of other critical step in RP protocol. Several RNAses had been used, mainly
active ribosomes are obtained using an RNAse protection assay, where RNAse I and micrococcal nuclease (MNAse) in eukaryotic cell models
controlled digestion generates small mRNA fragments/footprints of ap- and bacterial cells, respectively. At this step, controlling factors like reac-
proximately 30 nucleotides [8]. Therefore, after data processing, transla- tion time and enzyme concentration are critical to ensure an appropri-
tion can be observed at an unprecedented resolution in a variety of ate mRNA digestion, for example it has been stablished that the ratio
biological settings. Before performing the digestion, ribosomes are between RNA and RNAse controls footprints size [13].
halted over the mRNAs using translation inhibitory drugs or by quick The third step is one of the most laborious in terms of protocol. Dif-
deep freezing the sample to avoid ribosome run-off. The resulting ferent strategies had been used to isolate ribosome protected fragments
fragments, i.e. the ribosome footprints, are purified and used to con- or ribosome footprints, but all of them imply a ribosome/poly-ribosome
struct sequencing libraries to feed short read sequencers. In this sce- purification step. Even though commercial columns are available to pu-
nario, a transcriptome wide picture of the translating ribosomes rify monosomes, the most used approach is the differential sedimenta-
location over mRNAs is obtained, together with an estimation of the tion of ribosomes through a sucrose cushion during ultracentrifugation.
mRNAs translation rates. These expression levels estimated by RP define The use of this technique of subcellular fractionation ensures the purifi-
what is called translatome, in analogy to the term transcriptome. cation of monosomes with bound ribosome footprints. Once mono-
Translatome estimations of gene expression levels correlate better somes are purified, a polyacrylamide gel electrophoresis in denaturing
with proteomic data than transcriptome-derived estimations (see conditions is run to separate the complex sample by length. Using ap-
below). This increased correlation evidences the existence of mecha- propriate size markers, the gel is cut at the corresponding length of
nisms operating in the control of translation that fine tune the synthesis 28-30 nt using a dark field transilluminator, even if footprints are not
of cellular proteins. visible as it is usually the case. After disrupting the gel slices, precipita-
In the context of the rich data obtained in a RP experiment, an inter- tion and re-purification of ribosome footprints, samples are ready to
esting outcome was the definition of two concepts: translational effi- proceed to library preparation.
ciency and periodicity. The first concept refers to how much an mRNA Library preparation implies a set of protocol steps common in many
is translated considering the level of its coding mRNA, so it is an impor- high-throughput sequencing experiments like end repair, 3′ adaptor
tant parameter yielding information on translation regulation. ligation, reverse transcription and PAGE cDNA purification, circulariza-
Translational efficiency is calculated as the ratio between translation tion of cDNA and PCR amplification. After checking length and concen-
(derived from counts of footprints per mRNA) over transcription tration of the ribosome footprints library, they can be submitted to
(derived from RNA-seq mRNA levels) of particular mRNA. The second, sequencing according to user-preferred sequencing technologies. Due
refers to the three bases mapping periodicity observed for the reads de- to footprints small size, neither long reads nor paired-end reads are
rived from footprints as a consequence of ribosome movement along needed. Nevertheless, due to ribosomal rRNA presence in the footprints
mRNA. Since the ribosome moves codon by codon, the 5′-end of the ri- fraction purified, depletion of rRNA, coupled with extra sequencing
bosome footprints tend to map at the same position of each codon depth are usually needed.
throughout the whole coding sequence. Finally, the bioinformatic analysis of data is the most user-
Several aspects concerning protocol have been discussed, revised dependent step. A typical analysis would include quality control of
and modified since the original protocol was established. Some aim to raw reads, mapping, count normalization and gene expression levels es-
adapt the protocol to different biological models, like eukaryotic or pro- timation. It could also include, for example, differential gene expression
karyotic cells, specific tissues, etc. Other aspects have been intensely analysis if two biological conditions are contrasted. Table 1 show a list of
discussed, for example what the appropriate method to stop translation some of the software available to perform classical analysis over RP data.
is or how to define the correct translation frame from ribosome foot- Nevertheless, how deeply the data is interrogated is on user's hands,
prints. Nevertheless, RP protocol is currently a widely used approach here we will discuss some of these downstream analyses later.
to study gene expression in different biological models from virus and
bacteria to complex mammalian tissues (examples in [9–11]). In this 2.2. Protocol Variants, User Decisions
mini-review we will discuss the main and critical steps in the RP proto-
col, its uses and main findings obtained in different biological models Up to this point we have reviewed the main steps in RP protocol con-
and the contributions to our knowledge of cellular and molecular sidering the classical approaches most used in literature. Henceforth we
biology. will mention some protocol variants and why they could be used if is
necessary (Fig. 1B). Considering the chronological order of the protocol,
we will start with one of the steps where more variants are described in
2. Ribosome Profiling Protocol the literature: how to stop translation at the moment the experimental
design requires to do so. Efficient stop of translation avoids ribosome
2.1. Protocol Description run off, sharpening the picture taken of the translatome at a given
time point. In the original protocol, a classical translation inhibitor like
Ribosome Profiling comprise mainly five steps: sample preparation, cycloheximide was used to specifically target translation elongation.
RNAse protection assay, isolation of ribosome footprints, high- However, as it does not interfere with pre-initiation complex scanning
throughput sequencing and bioinformatic analysis (Fig. 1A) [12]. and translation initiation, treatment with cycloheximide causes a signif-
Sample preparation refers to steps necessary to process the biological icant accumulation of ribosomes at initiation sites of mRNAs actively
sample and obtain a post mitochondrial supernatant where lysis condi- translated. This could represent a source of bias since a lot of ribosome
tions ensure to preserve in vivo ribosome positioning and RNA integrity. footprints will be generated by initiating ribosomes while elongation
Among others, alternative inputs could be tissue homogenates, isolated is stopped. This issue was highly covered in the literature, with some au-
tagged ribosomes or a bacterial cell lysate. Critical aspects concerning thors proposing that this accumulation is actually due to an enrichment
this step are: ensuring enough biological material to produce quantifi- of slow codons after the initiation and others are in line with the bias
able ribosome footprints and avoiding ribosome run-off. For the last, hypothesis that generates a skewed distribution. Alternatively, it is
either drugs inhibitors of translation or physical methods like flash- possible to stop translation using liquid nitrogen and dry ice [12]. In
freezing using liquid nitrogen and dry ice can be used. Indeed, fast this scenario, ribosomes are flash-frozen and stopped just by reducing
 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176 169

Fig. 1. Ribosome Profiling protocol description. A general description of RP protocol is shown in A, representing the main steps described in the text. The protocol variants discussed are
summarized in B, linked to the corresponding step where would be applied. Variants that correspond to prokaryotes are marked in italic.

kinetic energy to a minimum. This alternative seems to not affect enzyme selected. In this case, the amount of RNA that is digested and
ribosome density and expression measurements but it's not the most other reaction conditions are well established, but when a new RNAse
extended approach, maybe because of availability of liquid nitrogen in is being used, parameters like enzyme units and time of the digestion
the laboratories. When working with prokaryotes, besides flash- needs to be specifically determined to ensure a correct ribosome foot-
freezing, drugs like chloramphenicol and 5′-Guanylyl imidodiphosphate print production. It has been useful the use of enzymes, like Benzonase,
had been used [9]. Finally, it is worth mentioning that other drugs that or the above mentioned MNAse, that produce digestion products that
target translation had been used to reveal specific aspects of translation. allow a more straight forward ligation of the linkers required to prepare
One of the most extended example is the use of harringtonine or the com- NGS molecular libraries [17–20] simplifying the library preparation
bined use of cycloheximide and harringtonine. Since harringtonine it is an protocol.
inhibitor of translation initiation, the use of this drug alone could reveal Once cells are harvested, lysed and the RNAse protection assay is
translation initiation sites exclusively. Also, if harringtonine is first carried out, the next step is to collect ribosomes and specifically purify
applied, and cycloheximide is applied after at different time points, it is ribosome footprints. As we mentioned above, ribosome purification
possible to measure very specific translation properties like translation could be one of the most laborious step. Despite commercial columns
elongation speed [14,15]. are available to purify ribosomes, more classical strategies tend to be
The second step we mentioned it is the RNAse protection assay. In used, like monosome separation by ultracentrifugation in sucrose
this step enzyme selection is critical [16]. In first place the biological cushions or gradients. While sucrose gradients fractionation is challeng-
model (eukaryotic or prokaryotic) already limits the options. In the ing, sucrose cushions give similar results with less technical challenges.
literature, enzymes used for eukaryotic systems are mainly RNAse I, A, Other approaches to collect ribosomes are available, like genetic manip-
S7, T1 and MNAse, also used in prokaryotes. Since the method has ulation to add epitope tags to ribosomes, allowing affinity purification
been mainly applied to eukaryotic cells, RNAse I is the more common [21–23]. In any case, after ribosome isolation, footprints purification is
170 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176

Table 1
Software available to analyze, interpret and visualize RP-derived data.
A list of some of the software used to analyze RP data is briefly described, indicating its main features and the adequate environment to use it.

Name Functions/description Enviroment Ref.

riboSeqR Parsing data, align reads, plotting functions, frameshift detection and inferring alternative ORFs. R [101]
RiboProfiling Quality assessment, read start position recalibration, counting of reads on CDS, 3′UTR, and 5′UTR, plotting of count data: pairs, log R [102]
fold-change, codon frequency and coverage assessment, principal component analysis on codon coverage.
RiboGalaxy On-line tools for the analysis and visualization of ribo-seq data (some of them use riboSeqR) Galaxy webserver [103]
Plastid A handful of scripts for common high-throughput sequencing and ribosome profiling analyses, like: determining P-sites offsets Python Library [104]
Ribomap Generates isoform-level ribosome profiles from ribosome profiling data Unix [105]
RiboTraper Identifies translated regions Unix [106]
Rfoot Identifies RNA regions protected by non-ribosomal protein complex present in Ribo-Seq data Perl [107]
anota Analysis of differential translation and results visualization R [108]
RiboDiff An statistical tool to detect changes in protein translation efficiency Unix [109]
Xtail An analysis pipeline that identifies differentially translated genes in pairwise comparisons R [110]
RiboTools Detection of translational ambiguities, stop codon readthrough events and codon occupancy. Provides plots for the visualization Galaxy webserver [111]
of these events.
Proteoformer Genome-wide visualization of ribosome occupancy and a translation initiation site calling algorithm. A protein database can be Galaxy webserver [112]
incorporated to increase protein identification
ORFscore Small ORF identification In SPECTtre [106]; [75]
python
ORF-RATER Coding sequence annotation Python [113]
FLOSS A metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions In SPECTtre [106]; [61]
python
tRanslatome Analysis of transcriptome, translatome and proteome data: Differentially expressed genes detection, gene ontology enrichment R [114]
comparison and analysis of regulatory elements
TranslatomeDB Differential gene expression, translation ratio, elongation velocity index and translational efficiency. Also comparision with other Online [115]
RP experiments can be done
systemPipeR Filter/trim sequences, quality control, alignments, counting, peak detection, differentially expressed genes detection, enrichment, R [116]
classification, several reports and graphs

the immediate follow step. Since the RNAses used are endonucleases, contaminating your footprints and how many mRNAs you will need to
they digest “unprotected” mRNA while also cutting fragments of rRNA quantify.
exposed in ribosome's surface. This digestion produces a very complex Finally, data interpretation implies a complete in silico analysis (see
mix of RNA fragments of diverse length that is separated by a denatur- Table 1), although this is the step more flexible and open to user aims,
ing PAGE. Using appropriate size markers (26 and 34 nt), the band cor- it represents several challenges due to the particular features of RP.
responding to ribosome footprints is excised from the gel and the RNA is For example, reads are short in length, may have relatively high error
isolated. Interestingly, a new population of small footprints of 20 nt in rates and depending on library construction protocol could have high
length was recently described [24]. This small population would not bias. Also, some fragments tend to be enriched, because accumulated ri-
be recovered if we use the size markers mentioned above. In this bosomes at translation initiation sites or pausing sites, leading to high
context, depending on the experiment being performed and on the re- read counts. Beyond this, most of the available tool to process and ana-
search goals, size selection can be modified accordingly. lyze experiments of RNA-Seq are suitable to use analyzing data from RP,
Since the original sample contains a lot of ribosomes, a very impor- specifically the ones used to short length reads and/or single-end reads.
tant fraction of the generated fragments comes from rRNA. This contam- Nevertheless, some aspects need to be considered due to the peculiari-
ination, still present in ribosome footprints expected band, is an ties of the data set analyzed. For example, gene isoforms studies are dif-
important issue. One possible strategy is to continue with the protocol ficult since ribosome footprints are short reads and mapping over splice
ignoring this contamination and go deep in sequencing to obtain junctions tend to be unreliable. Briefly, bioinformatic analysis implies in
enough mRNA derived sequences to achieve RNA-seq like coverage. general: quality and adaptor trimming, mapping against a specific data
However, this contamination can represent up to 90% of the sample, base of rRNA or ncRNAs to remove contamination, unmapped reads are
so a subtracting strategy is usually necessary. Ribosomal RNA removal aligned to an mRNA data base, counting reads, normalize counts and
can be achieved through streptavidin affinity purification using specific proceed to check statistical differences between conditions. As said
biotinylated rRNA probes available for mouse and human. If the above, diverse analysis can be done with data, just to mention some:
biological model it is not mouse or human, synthesis of specific rRNA check footprints periodicity, upstream Open Reading Frame (uORF)
complementary oligos can be considered, provided by previous knowl- search, detection of different translation initiation sites, codon usage
edge of the region of the rRNA protected in the model used. The later and search for translation pauses, among others. Even though general-
can be obtained by sequencing at low depth to determine the most purpose RNA-seq tools may be suitable, some specific software has
abundant protected fragments derived from rRNA. Because different en- been developed to apply to RP data set that explicitly consider the influ-
zymes can produce different protected rRNA due to allosteric impedi- ence of transcript levels on translatome determinations (see examples
ments or cleavage site sequence specificity, determining the identity in Table 1).
of contaminating rRNAs could be necessary.
When footprints are collected, library construction and high- 3. Biological Models and Contributions
throughput sequencing are the next in line. Depending on the RNAse
used, end repair could be necessary prior to linker ligation. While con- Up to date, the RP protocol has been applied to a large variety of bi-
ventional protocols require PCR amplification and purification of the ological models from viruses and bacteria to yeast, mammalian cells and
amplified PCR product by PAGE, as mentioned above some enzymes tissues, and embryos. In this section we will present the main contribu-
simplify these steps. Finally, sequencing is performed. While several tions done in each model, and also what we have learned about the
platforms are available to perform high-throughput sequencing, long translation mechanism using this methodology. In addition, in Table 2
reads are not necessary as footprints are naturally short. Usually the several RP works were grouped by the main topic analyzed, indicating
depth of coverage to be achieved is dependent on how much rRNA is in each case the different organisms used.
 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176 171

Table 2 re-analyzing that public data sets generated. In the original article,
Brief summary of RP works in several models, grouped by the main analyzed topic. Ingolia et al. [7] explored translation response to starvation. In this sem-
Topic Organism Ref. inal paper the terms translation efficiency and periodicity were defined
Genomic/translation characterization Virus [11,86–88]
for first time in this context (see Introduction). While translation effi-
Mycobacterium abscessus [35] ciency is usually calculated in every experiment using RP, periodicity
Mammalian cells [14] is not assessed so often, because it depends on RNAse amount used
Translation initiation sites Caulobacter crescentus [26] and digestion time.
Mammalian cells [59]
For first time, integrating all data obtained, correlations between ex-
Translation elongation Saccharomyces cerevisiae [24]
Caenorhabditis elegan [79] pression levels estimated by RNA-Seq (transcriptional levels), RP
Translational pausing Escherichia coli [9,27,32] (translational levels) and proteomics (protein levels) could be obtained,
Bacillus subtilis [9] reflecting the contribution of translational regulation in the fine tuning
Saccharomyces cerevisiae [46,67] of final proteins levels (please see examples in Fig. 2). In this sense,
Codon usage Escherichia coli [37]
Saccharomyces cerevisiae [47,49,50]
other efforts have been made to correlate translation ratios and protein
Small ORF Saccharomyces cerevisiae [51] abundance. For example, Wang et al. [43] by incorporating mRNA
Zebra fish [75] length as a key factor, found a strong multivariate linear correlation be-
Drosophila melanogaster [77] tween protein levels and translation ratios estimated by ribosome-
Mammalian cells [65]
nascent chain complex sequencing (RNC-Seq). The correlation between
Translation dynamics on different stages Plasmodium falciparum [81,82]
Trypanosoma cruzi [17] translational and protein levels estimated by RP and proteomics, respec-
Trypanosoma brucei [83,84] tively, may be improved if elongation velocity index are incorporated in
Stress response Escherichia coli [41] the analysis, according to the authors [44] (please see Section 3.3.2).
Mycoplasma gallisepticum [34] Also, start codons were also precisely determined in this work, and
Arabidopsis thaliana [80]
initiation at non-AUG codons was observed as response to starvation.
Saccharomyces cerevisiae [7]
lncRNAs translation Mammalian cells [60–64] In the same way, detection of ribosome footprints at 5’-UTRs re-
veals translational activity in these regions mainly explained by the pres-
ence of uORFs. In this way, a new approach to uORF study and its
3.1. Bacteria: Translational Pausing, Codon Use and Antibiotics relationship with translation regulation was stablished, revealing a
completely new and complex field previously not covered in detail.
In bacteria, ribosome profiling was applied in first place to To highlight some of these contributions yeast models provided, we
Escherichia coli and Bacillus subtilis [9] to study the causes of transla- can mention that distinct population of ribosome footprints were dis-
tional pausing. The authors observed that the presence of Shine- covered and were assigned to distinct stages of translating ribosomes
Dalgarno-like features in coding sequences are the major determinants [24]. Furthermore, 80S ribosomes (monosomes) were detected as
of translation rates in these models. Instead of codon usage or the pres- translationally active, translating specific mRNAs encoding low abun-
ence of rare tRNAs, interactions between rRNA and these Shine- dance and regulatory proteins, among others [45]. In addition, codon
Dalgarno-like features in mRNA can impact on ribosomal movement usage, tRNA levels and how they influence translation was highly cov-
along mRNA, which in turn affect footprints location and abundance ered [46–50]. The hypothesis that arise more strongly in yeast is that
[25]. Later, Schrader et al. [26] also applied RP, in Caulobacter crescentus biochemical interactions between the nascent peptide and the ribo-
and arrived to the same conclusion: ribosomes tend to pause at internal somal exit tunnel (in particular the initial part of the tunnel) are
Shine-Dalgarno-like sequences in coding genes. Although the later hy- major determinants on ribosome stalling [46]. A stalling signal of pro-
pothesis regarding underling mechanisms of translation pausing in bac- line and arginine was detected, as others showed for bacteria [31,32].
teria is still controversial (see an example in [27]), with authors On the other hand, also the correlation between tRNA concentrations
supporting classical hypothesis of tRNA abundance as main modulator and codon decoding time was evaluated, finding a significant negative
of translation speed, this is still a new possible mechanism for regulating correlation, supporting the idea that translation efficiency is influenced
translation uncovered by the RP strategy. by tRNAs levels in the cells [48]. Also, RP was used to explore the
In another study Oh et al. [28], investigated a chaperone trigger factor genome-wide translation of small ORFs (b100 amino acids) and long
and how this protein regulates outer membrane proteins, using a RP pro- non coding RNAs (lncRNAs) [51], ribosome rescue in 3′-UTR [52], the
tocol modified later in [29]. Balakrishnan et al. [30] studied translation ini- yeast meiotic program with important contributions to the area [53],
tiation on E. coli using RP, while translation elongation was covered by and also how translation contributes to regulate gene-expression in
Elgamal et al. [31], where authors find translational pauses associated to yeast in an evolutionary view [54].
elongation factor P and amino acids motifs upstream to ribosome P-site
(also found in [32]). Other bacteria where RP was applied are Mycoplasma 3.3. Mammalian Cells: uORFs, Pauses, Initiation Sites and lncRNAs
gallisepticum [33,34], Mycobacterium abscessu [35] and Staphylococcus au-
reus [36]. RP as a powerful technique to measure translation rates at In mammalian cells, the first study carried out applied RP strategy to
subcodon resolution, has allowed scientist to focus on the relationship be- reveal aspects of microRNA's (miRNA) function in the cell [55]. The au-
tween translation efficiency and codon usage deriving in the optimization thors observed that miRNA predominantly affect mRNA levels, with
bacterial vectors for expression of heterologous recombinant proteins only a modest influence on translational efficiency. This study revealed
[37,38]. for first time that mRNA destabilization is the major consequence of
Also, RP has given new insights on the antibiotics mechanisms to in- miRNA regulation. So, from here to the end of this section we will
hibit translation [39]. Other studies have been using RP to investigate present some interesting research and their results in mammalian
mechanisms for biofilm formation in B. subtilis [40], ethanol effects on cells mainly, but also in other eukaryotic models.
translation [41] and mRNA cleavage by the endonuclease RelE [42]. A significant study in terms of results, conclusions and repercus-
sions, was done by the group who publish the RP protocol, but using
mouse embryonic stem cells (mESC) [14]. In this model, the authors
3.2. Yeast: Start Codons, uORFs and Translational Pauses identified thousands of pause sites and unannotated translation prod-
ucts like amino-terminal extension and uORFs with potential regulatory
Since RP was firstly described in the budding yeast Saccharomyces roles. In parallel, authors combine harringtonine and cycloheximide use
cerevisiae [7], a lot of research has been done using this model and by to monitoring kinetic of translation as we describe below, evidencing a
172 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176

Fig. 2. Correlations among RNA-Seq, RP and proteome-derived expression data sets. Genome-wide correlations of individual gene expression levels estimated by RNA-Seq, RP and
proteome techniques are shown. Each correlation value is referenced to its corresponding author, indicating also journal, year, organism involved and correlation test used, by the
same color code.

ribosome translation rate of 5.6 amino acids per second, consistent with (see also [46]). Nevertheless, the authors mention that they detect an
previous values [56], and that is independent of length, protein abun- unknown source of biases in the data that can interfere in ribosome pro-
dance, classes of mRNAs or codon use. files over mRNAs. Nevertheless, by experimentally assessing elongation
velocity, recently Lian & Guo et al. [44] found that these general conclu-
3.3.1. uORFs sions we have described may not be applicable to all individual cases. In
Ingolia et al. [14], using harringtonine in mESC, could identify trans- this work, information from RNA-Seq, RP and also RNC-mRNA was used
lation initiation sites, where AUG was present in almost 75% of canonical to define and calculate an elongation velocity index at individual genes
sites, but in b25% in upstream sites, where others near-AUG codons in human cells. This index was correlated with several mRNA features
were observed, like CUG and GUG (see also Section 3.3.3). Considering and also with biological conditions, where authors find an elongation
the initiation site defined, the reading frame associated was also inves- speed deceleration on malignant phenotype associated genes.
tigated and classified based on their relationship to the annotated ORF.
In this characterization, many uORF were detected, as well as alternate 3.3.3. Translation Initiation Sites (TIS)
protein products with amino-terminal extensions or truncations. The Combining more data sets, Michel et al. [59] designed a method to
authors also study the widespread translation of uORFs detected and estimate the probability of ribosomes initiating at individual start co-
their change during differentiation, highlighting the important regula- dons. This tool is able to discriminate between weak or strong initiation
tory role that these elements have affecting translation, particularly sites based on the accepted leaky scanning model of translation initia-
when the cell is under stress conditions [57]. A well-known example tion in eukaryotes. For example, analyzing the codon preference in TIS
is the uORF translation regulation that affects GCN4 expression in in human and mouse, a N 50% of AUG TIS and also almost 50% of AUG
yeast under starvation [7]. preference in downstream TIS was observed. Composition of upstream
TIS was more diverse: 25% are AUG codons, 30% CUG and 40% include
3.3.2. Translational Pauses and Elongation Speed other AUG-variants like UUG, GUG, AGG, ACG, among others [15].
Regarding translational stall sites, Ingolia et al. [14] observed in
mESC a consensus peptide motif of glutamate (preferentially GAA 3.3.4. Long Non Coding RNAs
codon) or aspartate in the A site of pauses, preceded by a proline or With no doubt, another striking finding of the work done by Ingolia
glycine, and then another proline (preferentially represented by et al. [14] in mESC was the detection of high levels of ribosome
CC[A/T] codons); while no evidence of rare codons enrichment was footprints in long intragenic noncoding RNAs (lincRNAs), with marked
seen in pausing sites. Also, Dana and Tuller [58], re-analyzed the data fo- initiation sites evidenced by harringtonine. They classify these RNAs as
cusing on elongation speed and ribosome profiles. Their analysis sug- sprcRNA: short, polycistronic ribosome-associated coding RNAs. If
gest that elongation speed is indeed determined by the tRNA pool, lincRNA encode or not a message to be translated by the ribosomes is
local mRNA folding and local charge of amino acids encoded; an idea a matter addressed specifically in two publications [60,61]. Guttman
that seems to be extended in different articles, as was mentioned before et al. [60] defined a Ribosome Release Score, that discriminate between
 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176 173

coding and noncoding transcripts. Using this score, authors claim that draws attention, is the low regulatory spectrum found in terms of
the ribosome occupancy observed on lincRNAs per se is not an indicator number of messengers: mTOR-regulated mRNAs were 253 and 144, re-
of active translation and describe possible reasons why noncoding RNAs spectively for each publication, a low number of targets considering the
show ribosome footprints. One of these possibilities is that these foot- central role of mTOR pathway in cellular metabolism and previous re-
prints actually come from ribonucleo protein particles or others RNA- sults of translation control resolution using RP. It is still an open ques-
protein complexes. Alternatively, footprints could be generated by real tion whether this number changes in different cell types or conditions,
engagement of ribosomes over ncRNAs that will not be functional at since there are still several factors downstream of mTOR that influences
the end. This interesting controversy was going to take an unexpected what is being translated.
turn when just over a year later, again Ingolia and Weissman described
a different metric to analyze footprints, that now classify lncRNAs as 3.4. Others Biological Models: Zebrafish, Drosophila, C. elegans,
coding [61]. This new metric called FLOSS (fragment length organiza- Trypanosomatids and Virus
tion similarity score) measure the magnitude of disagreement between
length distribution of a set of transcripts of interest and annotated Besides bacteria, yeast and mammalian cell lines, the RP method was
protein-coding transcripts. Based on FLOSS and other lines of evidence, used to study translation regulation in others biological models as
the authors proposed that lncRNAs has ribosome footprints that show zebrafish [63,75], the fruit fly Drosophila [76–78], C. elegans [79],
features of translation. In addition alternative hypothesis were Arabidopsis [80] and also parasites like Plasmodium falciparum [81,82],
discussed: i) translating ribosome could act as a potent helicase to re- Trypanosoma brucei [83,84] and T. cruzi [17]. Trypanosomatids undergo
model RNA structures and remove RNA-binding proteins; ii) translated a complex life cycle with several distinct developmental forms, each
sequences may also act as cis-acting elements over lncRNAs that origi- having particular morphologic and metabolic profiles. However, these
nate them and iii) the authors discuss about a possible contribution of organisms accomplish the associated gene expression changes without
the proteins synthetized by noncanonical translation to serve as possi- transcriptional control [85]. Indeed, translation regulation proved to be
ble antigens presented to the cellular immune system, expanding the a key mechanism controlling protein levels as revealed by drastic
universe of epitopes either in a viral infection or in a tumoral context. changes in translational efficiency for many developmentally regulated
In any case, the fact that some ncRNAs are associated with ribosomes, genes. For instance, the transition from a dividing to a non-diving
translationally active or not, generates both challenging and interesting parasite form was accompanied by a decrease in the translational effi-
questions that wait to be answered (see examples in [62–64]). ciency of ribosomal proteins which in turn may explain the observed
Using the data produced by RP on mESC, a lot of downstream analy- global decrease in protein synthesis. However, proteins required in
sis has been conducted. For example, an approach to search and predict the non-dividing stage scape this general trend and are actively trans-
putatively functional small ORF was developed to identify new classes lated as shown for the trans-sialidase family of virulence factors in T.
of bioactive peptides [65]. Another example is the work done by cruzi [17]. Besides, the data allowed the curation of the available ge-
Zupanic et al. [66], where the authors developed a method to study nomes in these non-model organisms [84].
mRNA translation regulation analyzing individual ribosome profiles. In- Also, RP was applied to study translation in virus like human
corporating RNA-Seq data to correct bias and artifacts, they look for cytomegalovirus and Kaposi's sarcoma-associated herpesvirus, both
changes in ribosome density along mRNAs to detect mechanisms of reg- herpesvirus, and also in Cricket paralysis virus and Influenza A virus
ulation, like premature termination or new transcript isoforms. (see [11,86–88], respectively).
Regarding bias, several articles have studied this important issue on
RP data. Some improvements have been done in terms of understand 4. Applications, Challenges and Perspectives
the bias source, and be able to correct it accurately [58,67,68].
The movement of the ribosome over the mRNA has been studied an- Besides classical applications we have been discussing above, like
alyzing in deep mapping periodicity leading to undercover mechanisms determine translation gene expression levels, pause associated motifs,
underlying translational frameshifts [59]. Also regions in the human ge- codon translational rates, uORF and frameshift events detection,
nome that are dually decoded were identified (~1% of human genome among others, here we will mention specific protocols that had evolved
approx.), either from different mRNAs as from the same, expanding from initial RP experiments, like how to determine TIS by Qian lab
our vision about translation regulation and even about central dogma [15,89]. In first place, they describe an approach named global transla-
[4,59]. tion initiation sequencing (or GTI-Seq) that combine the use of
In HeLa cells, RP was applied to explore the translational landscape lactimidomycin and cycloheximide to detect both initiation and elonga-
of cell cycle, and a widespread translation regulation was seen over tion ribosomes along transcripts, in human and mouse. The other, but
cell cycle progression [69,70]. Surprisingly, evidence of functional similar approach, named QTI-Seq (Quantitative Translation Initiation
bicistronic mRNAs with antiviral functions in the innate immune sys- Sequencing) evaluating not only TIS qualitatively, but also quantita-
tem was also revealed by RP in a human cell line [71]. Furthermore RP tively, so statistical comparisons can be made between two conditions.
was used in humans to investigate genetic variants in lymphoblastoid In bacteria also exist an approach to identify TIS genome-wide named
cells derived from a diverse group of 30 individuals and how some ge- tetracycline-inhibited RP [90].
netic differences may modulate ribosome occupancy [72]. Research on mitochondrial and chloroplast translation is also
The mTOR pathway is a very important target of different drugs and possible using RP [91–93]. Recently, an specific application of RP
has been implicated in several diseases, including cancer. Since this named mitochondrial ribosome (mitoribosome) profiling was devel-
complex regulates cell growth and proliferation by regulating mRNA oped [94]. In this case, the approach developed in yeast consist in the
translation, it is interesting to use RP protocol to elucidate translation immunoprecipitation of mitoribosomes from cell lysates to perform
control executed by mTOR. This was done by Sabatini's [73] and RNAse digestion. A similar approach but targeting reticulum-bound
Ruggero's [74] labs, and what they found was a surprising simple ribosomes was also used, in mammalian cells, to study translation
model of the mRNA features that mediates mTORC1-dependent transla- related to intracellular traffic of membranes [19].
tion: an established 5′ terminal oligopyrimidine (TOP) motifs. 5′-UTR Throughout this minireview we have shown how the RP method has
length or complexity was not associated with mTORC1 translation reg- provided the scientific community with a powerful system to study the
ulation. The later also identify another motif called PRTE (pyrimidine- translation mechanisms and regulation, and more generally a more
rich translational element) in 5′-UTR of mTOR targets mRNAs, which complete picture of regulation of gene expression in several models.
in conjunction with TOP motif were founded in almost 90% of mTOR- However even when the seminal paper will turn 10 years old next
sensitive genes. A common result of both works, which undoubtedly year many aspects of the technique are not completely resolved, as
174 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176

can be shown in the continuous development of new experimental [13] McGlincy NJ, Ingolia NT. Transcriptome-wide measurement of translation by ribo-
some profiling. Methods 2017;126:112–29. https://doi.org/10.1016/j.ymeth.2017.
protocols and analysis tools. Variations of the method are emerging to 05.028.
address particular cases, such as the development of protocols to assess [14] Ingolia NT, Lareau LF, Weissman JS. Ribosome profiling of mouse embryonic stem
localized translation. For instance, Williams et al. [95] reported cells reveals the complexity and dynamics of mammalian proteomes. Cell 2011;
147:789–802. https://doi.org/10.1016/j.cell.2011.10.002.
proximity-specific ribosome profiling to target translation of nuclear [15] Lee S, Liu B, Lee S, Huang S-X, Shen B, Qian S-B. Global mapping of translation ini-
encoded mitochondrial genes by tagging ribosomes in close contact tiation sites in mammalian cells at single-nucleotide resolution. Proc Natl Acad Sci
with the outer mitochondrial membrane. In this context, localized U S A 2012;109:E2424-2. https://doi.org/10.1073/pnas.1207846109.
[16] Gerashchenko MV, Gladyshev VN. Ribonuclease selection for ribosome profiling.
translation can be investigated in more difficult scenarios like protein Nucleic Acids Res 2017;45:e6. https://doi.org/10.1093/nar/gkw822.
synthesis in neuronal projections [96–98], specifically Holt lab per- [17] Smircich P, Eastman G, Bispo S, Duhagon MA, Guerra-Slompo EP, Garat B, et al.
formed ribosome tagging and analyzed mRNA associated to tagged Ribosome profiling reveals translation control as a key mechanism generating dif-
ferential gene expression in Trypanosoma cruzi. BMC Genomics 2015;16:197.
polysomes in the pre-synaptic area of a minute portion of the brain
https://doi.org/10.1186/s12864-015-1563-8.
[99] but the low input in mRNA would impair ribosome profiling. So, [18] Marcon BH, Holetz FB, Eastman G, Origa-Alves AC, Amorós M, de Aguiar AM, et al.
it is still necessary to develop methods that will allow the study of Downregulation of the protein synthesis machinery is a major regulatory event
systems where input material is a limiting factor. Some work is starting during early adipogenic differentiation of human adipose-derived stromal cells.
Stem Cell Res 2017. https://doi.org/10.1016/j.scr.2017.10.027.
to appear in this field [100]. [19] Reid DW, Nicchitta CV. Primary role for endoplasmic reticulum-bound ribosomes
Some intriguing questions have not been yet pursued, particularly in cellular translation identified by ribosome profiling. J Biol Chem 2012;287:
there are just a few reports where RP has been applied to disturbed 5518–27. https://doi.org/10.1074/jbc.M111.312280.
[20] Reid DW, Shenolikar S, Nicchitta CV. Simple and inexpensive ribosome profiling
systems, for example drug treated cells or pathological cells, as analysis of mRNA translation. Methods 2015;91:69–74. https://doi.org/10.1016/j.
neurodegenerative diseases tissue or cancer cells where translation ymeth.2015.07.003.
me be playing a key role in the etiology of the abnormal molecular [21] Sanz E, Yang L, Su T, Morris DR, McKnight GS, Amieux PS. Cell-type-specific
isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci
processes. U S A 2009;106:13939–44. https://doi.org/10.1073/pnas.0907143106.
[22] Heiman M, Schaefer A, Gong S, Peterson JD, Day M, Ramsey KE, et al. A translational
profiling approach for the molecular characterization of CNS cell types. Cell 2008;
Acknowledgements 135:738–48. https://doi.org/10.1016/j.cell.2008.10.028.
[23] Shigeoka T, Jung J, Holt CE, Jung H. Axon-TRAP-RiboTag: affinity purification of
We would like to acknowledge Dr. David Munroe for his inspiration, translated mRNAs from neuronal axons in mouse in vivo. Methods Mol Biol
1649;2018:85–94. https://doi.org/10.1007/978-1-4939-7213-5_5.
advices and support for the development of RP protocol and encourage-
[24] Lareau LF, Hite DH, Hogan GJ, Brown PO. Distinct stages of the translation elonga-
ment to study translation regulation. tion cycle revealed by sequencing ribosome-protected mRNA fragments. Elife
This work received financial support from: Master and PhD fellow- 2014;3:e01257. https://doi.org/10.7554/eLife.01257.
[25] O'Connor PBF, Li G-W, Weissman JS, Atkins JF, Baranov PV. rRNA:mRNA pairing al-
ships from Agencia Nacional de Investigación e Innovación (ANII) and
ters the length and the symmetry of mRNA-protected fragments in ribosome pro-
Comisión Académica de Posgrado (CAP) to GE (POS_NAC_2012_1_8584 filing experiments. Bioinformatics 2013;29:1488–91. https://doi.org/10.1093/
and POS_NAC_2016_1_129959), Comisión Sectorial de Investigación bioinformatics/btt184.
Científica (CSIC) for JRSS (CSIC grupos ID=108725 #144) and for GE [26] Schrader JM, Zhou B, Li G-W, Lasker K, Childers WS, Williams B, et al. The coding
and noncoding architecture of the Caulobacter crescentus genome. PLoS Genet
(CSIC Iniciación #370), ANII to JRSS (FCE_1_2017_1_136082) and to PS 2014;10:e1004463. https://doi.org/10.1371/journal.pgen.1004463.
(FCE_3_2016_1_126317), and from Programa de Desarrollo de las [27] Mohammad F, Woolstenhulme CJ, Green R, Buskirk AR. Clarifying the translational
Ciencias Básicas (PEDECIBA). pausing landscape in bacteria by ribosome profiling. Cell Rep 2016;14:686–94.
https://doi.org/10.1016/j.celrep.2015.12.073.
[28] Oh E, Becker AH, Sandikci A, Huber D, Chaba R, Gloge F, et al. Selective ribosome
References profiling reveals the cotranslational chaperone action of trigger factor in vivo.
Cell 2011;147:1295–308. https://doi.org/10.1016/j.cell.2011.10.044.
[1] Sboner A, Mu XJ, Greenbaum D, Auerbach RK, Gerstein MB. The real cost of se- [29] Latif H, Szubin R, Tan J, Brunk E, Lechner A, Zengler K, et al. A streamlined ribosome
quencing: higher than you think! Genome Biol 2011;12:125. https://doi.org/10. profiling protocol for the characterization of microorganisms. Biotechniques 2015;
1186/gb-2011-12-8-125. 58:329–32. https://doi.org/10.2144/000114302.
[2] Buermans HPJ, Dunnen den JT. Next generation sequencing technology: advances [30] Balakrishnan R, Oman K, Shoji S, Bundschuh R, Fredrick K. The conserved GTPase
and applications. Biochim Biophys Acta 2014;1842:1932–41. https://doi.org/10. LepA contributes mainly to translation initiation in Escherichia coli. Nucleic Acids
1016/j.bbadis.2014.06.015. Res 2014;42:13370–83. https://doi.org/10.1093/nar/gku1098.
[3] Muir P, Li S, Lou S, Wang D, Spakowicz DJ, Salichos L, et al. Erratum to: the real cost [31] Elgamal S, Katz A, Hersch SJ, Newsom D, White P, Navarre WW, et al. EF-P depen-
of sequencing: scaling computation to keep pace with data generation. Genome dent pauses integrate proximal and distal signals during translation. PLoS Genet
Biol 2016;17:78. https://doi.org/10.1186/s13059-016-0961-9. 2014;10:e1004553. https://doi.org/10.1371/journal.pgen.1004553.
[4] Mouilleron H, Delcourt V, Roucou X. Death of a dogma: eukaryotic mRNAs can code [32] Woolstenhulme CJ, Guydosh NR, Green R, Buskirk AR. High-precision analysis of
for more than one protein. Nucleic Acids Res 2016;44:14–23. https://doi.org/10. translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep
1093/nar/gkv1218. 2015;11:13–21. https://doi.org/10.1016/j.celrep.2015.03.014.
[5] Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW. Cancer [33] Fisunov GY, Evsyutina DV, Arzamasov AA, Butenko IO, Govorun VM. Profiling of
genome landscapes. Science 2013;339:1546–58. https://doi.org/10.1126/science. mycoplasma gallisepticum ribosomes. Acta Nat 2015;7:107–12.
1235122. [34] Fisunov GY, Evsyutina DV, Garanina IA, Arzamasov AA, Butenko IO, Altukhov IA, et al.
[6] Campbell BB, Light N, Fabrizio D, Zatzman M, Fuligni F, de Borja R, et al. Compre- Ribosome profiling reveals an adaptation strategy of reduced bacterium to acute
hensive analysis of hypermutation in human cancer. Cell 2017;171:1042–1056. stress. Biochimie 2017;132:66–74. https://doi.org/10.1016/j.biochi.2016.10.015.
e10. https://doi.org/10.1016/j.cell.2017.09.048. [35] Miranda-CasoLuengo AA, Staunton PM, Dinan AM, Lohan AJ, Loftus BJ. Functional char-
[7] Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS. Genome-wide analysis acterization of the Mycobacterium abscessus genome coupled with condition specific
in vivo of translation with nucleotide resolution using ribosome profiling. Science transcriptomics reveals conserved molecular strategies for host adaptation and persis-
2009;324:218–23. https://doi.org/10.1126/science.1168978. tence. BMC Genomics 2016;17:553. https://doi.org/10.1186/s12864-016-2868-y.
[8] Steitz JA. Polypeptide chain initiation: nucleotide sequences of the three ribosomal [36] Basu A, M-NF Yap. Ribosome hibernation factor promotes staphylococcal survival
binding sites in bacteriophage R17 RNA. Nature 1969;224:957–64. and differentially represses translation. Nucleic Acids Res 2016;44:4881–93.
[9] Li G-W, Oh E, Weissman JS. The anti-Shine-Dalgarno sequence drives translational https://doi.org/10.1093/nar/gkw180.
pausing and codon choice in bacteria. Nature 2012;484:538–41. https://doi.org/10. [37] Nakahigashi K, Takai Y, Shiwa Y, Wada M, Honma M, Yoshikawa H, et al. Effect of
1038/nature10965. codon adaptation on codon-level and gene-level translation efficiency in vivo.
[10] Castelo-Szekely V, Arpat AB, Janich P, Gatfield D. Translational contributions to tis- BMC Genomics 2014;15:1115. https://doi.org/10.1186/1471-2164-15-1115.
sue specificity in rhythmic and constitutive gene expression. Genome Biol 2017; [38] Li G-W. How do bacteria tune translation efficiency? Curr Opin Microbiol 2015;24:
18:116. https://doi.org/10.1186/s13059-017-1222-2. 66–71. https://doi.org/10.1016/j.mib.2015.01.001.
[11] Stern-Ginossar N, Weisburd B, Michalski A, Le VTK, Hein MY, Huang S-X, et al. [39] Marks J, Kannan K, Roncase EJ, Klepacki D, Kefi A, Orelle C, et al. Context-specific
Decoding human cytomegalovirus. Science 2012;338:1088–93. https://doi.org/10. inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase
1126/science.1227919. center. Proc Natl Acad Sci U S A 2016;113:12150–5. https://doi.org/10.1073/pnas.
[12] Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling 1613055113.
strategy for monitoring translation in vivo by deep sequencing of ribosome- [40] Subramaniam AR, Deloughery A, Bradshaw N, Chen Y, O'Shea E, Losick R, et al. A
protected mRNA fragments. Nat Protoc 2012;7:1534–50. https://doi.org/10.1038/ serine sensor for multicellularity in a bacterium. Elife 2013;2:e01501. https://doi.
nprot.2012.086. org/10.7554/eLife.01501.
 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176 175

[41] Haft RJF, Keating DH, Schwaegler T, Schwalbach MS, Vinokur J, Tremaine M, et al. [69] Stumpf CR, Moreno MV, Olshen AB, Taylor BS, Ruggero D. The translational land-
Correcting direct effects of ethanol on translation and transcription machinery con- scape of the mammalian cell cycle. Mol Cell 2013;52:574–82. https://doi.org/10.
fers ethanol tolerance in bacteria. Proc Natl Acad Sci U S A 2014;111:E2576-5. 1016/j.molcel.2013.09.018.
https://doi.org/10.1073/pnas.1401853111. [70] Aramayo R, Polymenis M. Ribosome profiling the cell cycle: lessons and challenges.
[42] Hwang J-Y, Buskirk AR. A ribosome profiling study of mRNA cleavage by the endonu- Curr Genet 2017;63:959–64. https://doi.org/10.1007/s00294-017-0698-3.
clease RelE. Nucleic Acids Res 2017;45:327–36. https://doi.org/10.1093/nar/gkw944. [71] Brubaker SW, Gauthier AE, Mills EW, Ingolia NT, Kagan JC. A bicistronic MAVS tran-
[43] Wang T, Cui Y, Jin J, Guo J, Wang G, Yin X, et al. Translating mRNAs strongly corre- script highlights a class of truncated variants in antiviral immunity. Cell 2014;156:
late to proteins in a multivariate manner and their translation ratios are phenotype 800–11. https://doi.org/10.1016/j.cell.2014.01.021.
specific. Nucleic Acids Res 2013;41:4743–54. https://doi.org/10.1093/nar/gkt178. [72] Cenik C, Cenik ES, Byeon GW, Grubert F, Candille SI, Spacek D, et al. Integrative
[44] Lian X, Guo J, Gu W, Cui Y, Zhong J, Jin J, et al. Genome-wide and experimental res- analysis of RNA, translation, and protein levels reveals distinct regulatory variation
olution of relative translation elongation speed at individual gene level in human across humans. Genome Res 2015;25:1610–21. https://doi.org/10.1101/gr.193342.
cells. PLoS Genet 2016;12:e1005901. https://doi.org/10.1371/journal.pgen. 115.
1005901. [73] Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, Sabatini DM. A unifying
[45] Heyer EE, Moore MJ. Redefining the translational status of 80S monosomes. Cell model for mTORC1-mediated regulation of mRNA translation. Nature 2012;485:
2016;164:757–69. https://doi.org/10.1016/j.cell.2016.01.003. 109–13. https://doi.org/10.1038/nature11083.
[46] Sabi R, Tuller T. A comparative genomics study on the effect of individual amino [74] Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, et al. The translational
acids on ribosome stalling. BMC Genomics 2015;16(Suppl. 10):S5. https://doi. landscape of mTOR signalling steers cancer initiation and metastasis. Nature
org/10.1186/1471-2164-16-S10-S5. 2012;485:55–61. https://doi.org/10.1038/nature10912.
[47] Pop C, Rouskin S, Ingolia NT, Han L, Phizicky EM, Weissman JS, et al. Causal signals [75] Bazzini AA, Johnstone TG, Christiano R, Mackowiak SD, Obermayer B, Fleming ES,
between codon bias, mRNA structure, and the efficiency of translation and elonga- et al. Identification of small ORFs in vertebrates using ribosome footprinting and
tion. Mol Syst Biol 2014;10:770. https://doi.org/10.15252/msb.20145524. evolutionary conservation. EMBO J 2014;33:981–93. https://doi.org/10.1002/
[48] Dana A, Tuller T. The effect of tRNA levels on decoding times of mRNA codons. embj.201488411.
Nucleic Acids Res 2014;42:9171–81. https://doi.org/10.1093/nar/gku646. [76] Dunn JG, Foo CK, Belletier NG, Gavis ER, Weissman JS. Ribosome profiling reveals
[49] Gardin J, Yeasmin R, Yurovsky A, Cai Y, Skiena S, Futcher B. Measurement of aver- pervasive and regulated stop codon readthrough in Drosophila melanogaster.
age decoding rates of the 61 sense codons in vivo. Elife 2014;3:198. https://doi.org/ Elife 2013;2:e01179. https://doi.org/10.7554/eLife.01179.
10.7554/eLife.03735. [77] Aspden JL, Eyre-Walker YC, Phillips RJ, Amin U, Mumtaz MAS, Brocard M, et al. Ex-
[50] Gorochowski TE, Ignatova Z, Bovenberg RAL, Roubos JA. Trade-offs between tRNA tensive translation of small open reading frames revealed by poly-Ribo-Seq. Elife
abundance and mRNA secondary structure support smoothing of translation elon- 2014;3:e03528. https://doi.org/10.7554/eLife.03528.
gation rate. Nucleic Acids Res 2015;43:3022–32. https://doi.org/10.1093/nar/ [78] Miettinen TP, Björklund M. Modified ribosome profiling reveals high abundance of
gkv199. ribosome protected mRNA fragments derived from 3′ untranslated regions. Nucleic
[51] Smith JE, Alvarez-Dominguez JR, Kline N, Huynh NJ, Geisler S, Hu W, et al. Transla- Acids Res 2015;43:1019–34. https://doi.org/10.1093/nar/gku1310.
tion of small open reading frames within unannotated RNA transcripts in Saccharo- [79] Stadler M, Fire A. Wobble base-pairing slows in vivo translation elongation in
myces cerevisiae. Cell Rep 2014;7:1858–66. https://doi.org/10.1016/j.celrep.2014. metazoans. RNA 2011;17:2063–73. https://doi.org/10.1261/rna.02890211.
05.023. [80] Juntawong P, Girke T, Bazin J, Bailey-Serres J. Translational dynamics revealed by
[52] Guydosh NR, Green R. Dom34 rescues ribosomes in 3′ untranslated regions. Cell genome-wide profiling of ribosome footprints in Arabidopsis. Proc Natl Acad Sci
2014;156:950–62. https://doi.org/10.1016/j.cell.2014.02.006. U S A 2014;111:E203-2. https://doi.org/10.1073/pnas.1317811111.
[53] Brar GA, Yassour M, Friedman N, Regev A, Ingolia NT, Weissman JS. High-resolution [81] Caro F, Ahyong V, Betegon M, DeRisi JL. Genome-wide regulatory dynamics of
view of the yeast meiotic program revealed by ribosome profiling. Science 2012; translation in the plasmodium falciparum asexual blood stages. Elife 2014;3:568.
335:552–7. https://doi.org/10.1126/science.1215110. https://doi.org/10.7554/eLife.04106.
[54] McManus CJ, May GE, Spealman P, Shteyman A. Ribosome profiling reveals post- [82] Bunnik EM, Chung D-WD, Hamilton M, Ponts N, Saraf A, Prudhomme J, et al. Poly-
transcriptional buffering of divergent gene expression in yeast. Genome Res some profiling reveals translational control of gene expression in the human ma-
2014;24:422–30. https://doi.org/10.1101/gr.164996.113. laria parasite plasmodium falciparum. Genome Biol 2013;14:R128. https://doi.
[55] Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly org/10.1186/gb-2013-14-11-r128.
act to decrease target mRNA levels. Nature 2010;466:835–40. https://doi.org/10. [83] Jensen BC, Ramasamy G, Vasconcelos EJR, Ingolia NT, Myler PJ, Parsons M. Exten-
1038/nature09267. sive stage-regulation of translation revealed by ribosome profiling of Trypanosoma
[56] Boström K, Wettesten M, Borén J, Bondjers G, Wiklund O, Olofsson SO. Pulse-chase brucei. BMC Genomics 2014;15:911. https://doi.org/10.1186/1471-2164-15-911.
studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep [84] Vasquez J-J, Hon C-C, Vanselow JT, Schlosser A, Siegel TN. Comparative ribosome
G2 cells. J Biol Chem 1986;261:13800–6. profiling reveals extensive translational complexity in different Trypanosoma
[57] Andreev DE, O'Connor PBF, Fahey C, Kenny EM, Terenin IM, Dmitriev SE, et al. brucei life cycle stages. Nucleic Acids Res 2014;42:3623–37. https://doi.org/10.
Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. Elife 1093/nar/gkt1386.
2015;4:e03971. https://doi.org/10.7554/eLife.03971. [85] Clayton CE. Life without transcriptional control? From fly to man and back again.
[58] Dana A, Tuller T. Determinants of translation elongation speed and ribosomal pro- EMBO J 2002;21:1881–8. https://doi.org/10.1093/emboj/21.8.1881.
filing biases in mouse embryonic stem cells. PLoS Comput Biol 2012;8:e1002755. [86] Stern-Ginossar N, Ingolia NT. Ribosome profiling as a tool to decipher viral com-
https://doi.org/10.1371/journal.pcbi.1002755. plexity. Annu Rev Virol 2015;2:335–49. https://doi.org/10.1146/annurev-virol-
[59] Michel AM, Andreev DE, Baranov PV. Computational approach for calculating the ogy-100114-054854.
probability of eukaryotic translation initiation from ribo-seq data that takes into ac- [87] Khong A, Bonderoff JM, Spriggs RV, Tammpere E, Kerr CH, Jackson TJ, et al. Tempo-
count leaky scanning. BMC Bioinf 2014;15:380. https://doi.org/10.1186/s12859- ral regulation of distinct internal ribosome entry sites of the dicistroviridae cricket
014-0380-4. paralysis virus. Virus 2016;8:25. https://doi.org/10.3390/v8010025.
[60] Guttman M, Russell P, Ingolia NT, Weissman JS, Lander ES. Ribosome profiling pro- [88] Bercovich-Kinori A, Tai J, Gelbart IA, Shitrit A, Ben-Moshe S, Drori Y, et al. A system-
vides evidence that large noncoding RNAs do not encode proteins. Cell 2013;154: atic view on influenza induced host shutoff. Elife 2016;5:600. https://doi.org/10.
240–51. https://doi.org/10.1016/j.cell.2013.06.009. 7554/eLife.18311.
[61] Ingolia NT, Brar GA, Stern-Ginossar N, Harris MS, Talhouarne GJS, Jackson SE, et al. [89] Gao X, Wan J, Liu B, Ma M, Shen B, Qian S-B. Quantitative profiling of initiating ri-
Ribosome profiling reveals pervasive translation outside of annotated protein- bosomes in vivo. Nat Methods 2015;12:147–53. https://doi.org/10.1038/nmeth.
coding genes. Cell Rep 2014;8:1365–79. https://doi.org/10.1016/j.celrep.2014.07. 3208.
045. [90] Nakahigashi K, Takai Y, Kimura M, Abe N, Nakayashiki T, Shiwa Y, et al. Compre-
[62] Cohen SM. Everything old is new again: (linc)RNAs make proteins! EMBO J 2014; hensive identification of translation start sites by tetracycline-inhibited ribosome
33:937–8. https://doi.org/10.1002/embj.201488303. profiling. DNA Res 2016;23:193–201. https://doi.org/10.1093/dnares/dsw008.
[63] Chew G-L, Pauli A, Rinn JL, Regev A, Schier AF, Valen E. Ribosome profiling reveals [91] Rooijers K, Loayza-Puch F, Nijtmans LG, Agami R. Ribosome profiling reveals fea-
resemblance between long non-coding RNAs and 5′ leaders of coding RNAs. Devel- tures of normal and disease-associated mitochondrial translation. Nat Commun
opment 2013;140:2828–34. https://doi.org/10.1242/dev.098343. 2013;4:2886. https://doi.org/10.1038/ncomms3886.
[64] Li L-J, Leng R-X, Fan Y-G, Pan H-F, Ye D-Q. Translation of noncoding RNAs: focus on [92] Zoschke R, Watkins KP, Barkan A. A rapid ribosome profiling method elucidates
lncRNAs, pri-miRNAs, and circRNAs. Exp Cell Res 2017;361:1–8. https://doi.org/10. chloroplast ribosome behavior in vivo. Plant Cell 2013;25:2265–75. https://doi.
1016/j.yexcr.2017.10.010. org/10.1105/tpc.113.111567.
[65] Crappé J, Van Criekinge W, Trooskens G, Hayakawa E, Luyten W, Baggerman G, [93] Chotewutmontri P, Barkan A. Dynamics of chloroplast translation during chloro-
et al. Combining in silico prediction and ribosome profiling in a genome-wide plast differentiation in maize. PLoS Genet 2016;12:e1006106. https://doi.org/10.
search for novel putatively coding sORFs. BMC Genomics 2013;14:648. https:// 1371/journal.pgen.1006106.
doi.org/10.1186/1471-2164-14-648. [94] Couvillion MT, Churchman LS. Mitochondrial ribosome (Mitoribosome) profiling
[66] Zupanic A, Meplan C, Grellscheid SN, Mathers JC, Kirkwood TBL, Hesketh JE, et al. for monitoring mitochondrial translation in vivo. Curr Protoc Mol Biol 2017;119:
Detecting translational regulation by change point analysis of ribosome profiling 4.28.1–4.28.25. https://doi.org/10.1002/cpmb.41.
data sets. RNA 2014;20:1507–18. https://doi.org/10.1261/rna.045286.114. [95] Williams CC, Jan CH, Weissman JS. Targeting and plasticity of mitochondrial pro-
[67] Artieri CG, Fraser HB. Accounting for biases in riboprofiling data indicates a major teins revealed by proximity-specific ribosome profiling. Science 2014;346:
role for proline in stalling translation. Genome Res 2014;24:2011–21. https://doi. 748–51. https://doi.org/10.1126/science.1257522.
org/10.1101/gr.175893.114. [96] Sotelo-Silveira JR, Holt CE. Introduction to the special issue on local protein synthe-
[68] Bartholomäus A, Del Campo C, Ignatova Z. Mapping the non-standardized biases of sis in axons. Dev Neurobiol 2014;74:207–9. https://doi.org/10.1002/dneu.22163.
ribosome profiling. Biol Chem 2016;397:23–35. https://doi.org/10.1515/hsz-2015- [97] Sotelo-Silveira JR, Calliari A, Kun A, Koenig E, Sotelo JR. RNA trafficking in axons.
0197. Traffic 2006;7:508–15. https://doi.org/10.1111/j.1600-0854.2006.00405.x.
176 G. Eastman et al. / Computational and Structural Biotechnology Journal 16 (2018) 167–176

[98] Calliari A, Farías J, Puppo A, Canclini L, Mercer JA, Munroe D, et al. Myosin Va asso- [107] Ji Z, Song R, Huang H, Regev A, Struhl K. Transcriptome-scale RNase-footprinting of
ciates with mRNA in ribonucleoprotein particles present in myelinated peripheral RNA-protein complexes. Nat Biotechnol 2016;34:410–3. https://doi.org/10.1038/
axons and in the central nervous system. Dev Neurobiol 2014;74:382–96. nbt.3441.
https://doi.org/10.1002/dneu.22155. [108] Larsson O, Sonenberg N, Nadon R. anota: analysis of differential translation in
[99] Shigeoka T, Jung H, Jung J, Turner-Bridger B, Ohk J, Lin JQ, et al. Dynamic axonal genome-wide studies. Bioinformatics 2011;27:1440–1. https://doi.org/10.1093/
translation in developing and mature visual circuits. Cell 2016;166:181–92. bioinformatics/btr146.
https://doi.org/10.1016/j.cell.2016.05.029. [109] Zhong Y, Karaletsos T, Drewe P, Sreedharan VT, Kuo D, Singh K, et al. RiboDiff:
[100] Hornstein N, Torres D, Sharma Das S, Tang G, Canoll P, Sims PA. Ligation-free detecting changes of mRNA translation efficiency from ribosome footprints.
ribosome profiling of cell type-specific translation in the brain. Genome Biol Bioinformatics 2017;33:139–41. https://doi.org/10.1093/bioinformatics/btw585.
2016;17:149. https://doi.org/10.1186/s13059-016-1005-1. [110] Xiao Z, Zou Q, Liu Y, Yang X. Genome-wide assessment of differential translations
[101] Chung BY, Hardcastle TJ, Jones JD, Irigoyen N, Firth AE, Baulcombe DC, et al. The use with ribosome profiling data. Nat Commun 2016;7:11194. https://doi.org/10.
of duplex-specific nuclease in ribosome profiling and a user-friendly software 1038/ncomms11194.
package for Ribo-seq data analysis. RNA 2015;21:1731–45. https://doi.org/10. [111] Legendre R, Baudin-Baillieu A, Hatin I, Namy O. RiboTools: a galaxy toolbox for
1261/rna.052548.115. qualitative ribosome profiling analysis. Bioinformatics 2015;31:2586–8. https://
[102] Popa A, Lebrigand K, Paquet A, Nottet N, Robbe-Sermesant K, Waldmann R, et al. doi.org/10.1093/bioinformatics/btv174.
RiboProfiling: a bioconductor package for standard Ribo-seq pipeline processing. [112] Crappé J, Ndah E, Koch A, Steyaert S, Gawron D, De Keulenaer S, et al.
F1000Res 2016;5:1309. https://doi.org/10.12688/f1000research.8964.1. PROTEOFORMER: deep proteome coverage through ribosome profiling and MS in-
[103] Michel AM, Mullan JPA, Velayudhan V, O'Connor PBF, Donohue CA, Baranov PV. tegration. Nucleic Acids Res 2015;43:e29-. https://doi.org/10.1093/nar/gku1283.
RiboGalaxy: a browser based platform for the alignment, analysis and visualization [113] Fields AP, Rodriguez EH, Jovanovic M, Stern-Ginossar N, Haas BJ, Mertins P, et al. A
of ribosome profiling data. RNA Biol 2016;13:316–9. https://doi.org/10.1080/ regression-based analysis of ribosome-profiling data reveals a conserved complex-
15476286.2016.1141862. ity to mammalian translation. Mol Cell 2015;60:816–27. https://doi.org/10.1016/j.
[104] Dunn JG, Weissman JS. Plastid: nucleotide-resolution analysis of next-generation molcel.2015.11.013.
sequencing and genomics data. BMC Genomics 2016;17:958. https://doi.org/10. [114] Tebaldi T, Dassi E, Kostoska G, Viero G, Quattrone A. tRanslatome: an R/
1186/s12864-016-3278-x. Bioconductor package to portray translational control. Bioinformatics 2014;30:
[105] Wang H, McManus J, Kingsford C. Isoform-level ribosome occupancy estimation 289–91. https://doi.org/10.1093/bioinformatics/btt634.
guided by transcript abundance with Ribomap. Bioinformatics 2016;32:1880–2. [115] Liu W, Xiang L, Zheng T, Jin J, Zhang G. TranslatomeDB: a comprehensive database
https://doi.org/10.1093/bioinformatics/btw085. and cloud-based analysis platform for translatome sequencing data. Nucleic Acids
[106] Calviello L, Mukherjee N, Wyler E, Zauber H, Hirsekorn A, Selbach M, et al. Detect- Res 2018;46:D206–12. https://doi.org/10.1093/nar/gkx1034.
ing actively translated open reading frames in ribosome profiling data. Nat [116] H Backman TW, Girke T. systemPipeR: NGS workflow and report generation
Methods 2016;13:165–70. https://doi.org/10.1038/nmeth.3688. environment. BMC Bioinf 2016;17:388. https://doi.org/10.1186/s12859-016-1241-0.