Translation Elongation Rate Research Articles

Co-translational folding of nascent polypeptides: Multi-layered mechanisms for the efficient biogenesis of functional proteins.

The coupling of protein synthesis and folding is a crucial yet poorly understood aspect of cellular protein folding. Over the past few years, it has become possible to experimentally follow and define protein folding on the ribosome, revealing principles that shape co-translational folding and distinguish it from refolding in solution. Here, we highlight some of these recent findings from biochemical and biophysical studies and their potential significance for cellular protein biogenesis. In particular, we focus on nascent chain interactions with the ribosome, interactions within the nascent protein, modulation of translation elongation rates, and the role of mechanical force that accompanies nascent protein folding. The ability to obtain mechanistic insight in molecular detail has set the stage for exploring the intricate process of nascent protein folding. We believe that the aspects discussed here will be generally important for understanding how protein synthesis and folding are coupled and regulated.

Small molecule Y-320 stimulates ribosome biogenesis, protein synthesis, and aminoglycoside-induced premature termination codon readthrough.

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

Full-length ribosome density prediction by a multi-input and multi-output model.

Translation elongation is regulated by a series of complicated mechanisms in both prokaryotes and eukaryotes. Although recent advance in ribosome profiling techniques has enabled one to capture the genome-wide ribosome footprints along transcripts at codon resolution, the regulatory codes of elongation dynamics are still not fully understood. Most of the existing computational approaches for modeling translation elongation from ribosome profiling data mainly focus on local contextual patterns, while ignoring the continuity of the elongation process and relations between ribosome densities of remote codons. Modeling the translation elongation process in full-length coding sequence (CDS) level has not been studied to the best of our knowledge. In this paper, we developed a deep learning based approach with a multi-input and multi-output framework, named RiboMIMO, for modeling the ribosome density distributions of full-length mRNA CDS regions. Through considering the underlying correlations in translation efficiency among neighboring and remote codons and extracting hidden features from the input full-length coding sequence, RiboMIMO can greatly outperform the state-of-the-art baseline approaches and accurately predict the ribosome density distributions along the whole mRNA CDS regions. In addition, RiboMIMO explores the contributions of individual input codons to the predictions of output ribosome densities, which thus can help reveal important biological factors influencing the translation elongation process. The analyses, based on our interpretable metric named codon impact score, not only identified several patterns consistent with the previously-published literatures, but also for the first time (to the best of our knowledge) revealed that the codons located at a long distance from the ribosomal A site may also have an association on the translation elongation rate. This finding of long-range impact on translation elongation velocity may shed new light on the regulatory mechanisms of protein synthesis. Overall, these results indicated that RiboMIMO can provide a useful tool for studying the regulation of translation elongation in the range of full-length CDS.

Functional Differences between β- and γ-Actin: From Translation Dynamics to Amino Acid Changes

The two mammalian cytoplasmic actins - β- and γ- actin - differ only by 4 conservative amino acids at the N-terminus, however their nucleotide coding sequences differ by ∼13% due to silent substitutions. These two actins show major functional differences: gene deletion of β-actin, unlike γ- actin, is embryonic lethal. The exact source of these differences is not well understood. We previously showed introducing 5 point mutations into mouse Actb gene (β-actin) to encode γ-actin protein (Actbc-g) does not affect mouse viability, despite the lack of β-actin protein. This result demonstrates β-actin's requirement for viability depends on its nucleotide, rather than amino acid sequence. Here we investigated the specific role of silent substitutions on β- and γ- actin function. Live cell translation measurements show β-actin translation elongation rate is 1.5-fold faster than γ-actin. Codon-switched constructs show this difference in translation elongation rates, leads to profound effects on cell migration and cell-substrate adhesion. Computational simulations confirmed lower actin synthesis rate near the focal adhesion results in shorter actin bundles. However, at the amino acid level, β- and γ- actin are conserved among all mammals, indicating selection pressure on the 4 conservative substitutions. We show that Actbc-g mice, which lack β-actin protein, show dramatic structural defects in cytoplasmic actin-rich structures, like microvilli in the small intestine and retina. In the retina these defects cause reduced light sensitivity in Actbc-g mice. Phalloidin proteomics of F-actin from Actbc-g tissues reveal altered affinity of specific actin binding proteins (ABPs) for actin in Actbc-g tissues. Together these data suggest actin isoforms’ N-termini co-evolved with ABPs to perform their specific functions in cells. Our results shed light on specific roles of nucleotide and amino acid sequence in actin isoform regulation.

How Elongator Acetylates tRNA Bases.

Elp3, the catalytic subunit of the eukaryotic Elongator complex, is a lysine acetyltransferase that acetylates the C5 position of wobble-base uridines (U34) in transfer RNAs (tRNAs). This Elongator-dependent RNA acetylation of anticodon bases affects the ribosomal translation elongation rates and directly links acetyl-CoA metabolism to both protein synthesis rates and the proteome integrity. Of note, several human diseases, including various cancers and neurodegenerative disorders, correlate with the dysregulation of Elongator’s tRNA modification activity. In this review, we focus on recent findings regarding the structure of Elp3 and the role of acetyl-CoA during its unique modification reaction.

Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

Light-induced psbA translation in plants is triggered by photosystem II damage via an assembly-linked autoregulatory circuit

The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair, and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts.

Cis-regulatory mechanisms and biological effects of translation elongation

Proteins are biological macromolecules essential for cells to maintain their metabolic activities. Proteins are synthesized during translation elongation, a synergistic process in which ribosomes decode the genetic information transmitted in mRNA, using tRNA. Numerous human diseases, such as neurodegenerative diseases and cancers, are known to be related to abnormal translation elongation. Translation elongation, as one of the two critical steps for the central dogma, used to be the focus of research in molecular biology. However, limitations in methodology had hindered further investigations on the dynamic process of translation elongation and its regulation. Recently, breakthroughs in methodology have revived this research field. Studies in the past decade or so have revealed that, beyond simple decoding of genetic information in mRNA, translation elongation entails sophisticated regulatory mechanisms and multifaceted biological consequences; such insights have provided a novel theoretical framework for understanding the maintenance of protein homeostasis and the development of diseases. In this review, we summarize the most updated methods that can be used to investigate the processes of translation elongation and highlight the mechanisms by which mRNA and protein sequences modulate the local rate of translation elongation. We further enumerate the consequences of dysregulation in translation elongation, from various aspects such as mRNA stability, protein synthesis and degradation, protein subcellular localization, and co-translational protein folding. We anticipate that this review will serve to draw the attention of scholars in various research fields to participate in the study of translation elongation.

Robust landscapes of ribosome dwell times and aminoacyl-tRNAs in response to nutrient stress in liver

Translation depends on messenger RNA (mRNA)-specific initiation, elongation, and termination rates. While translation elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here we combined ribosome and transfer RNA (tRNA) profiling to investigate the relations between translation elongation rates, (aminoacyl-) tRNA levels, and codon usage in mammals. We modeled codon-specific ribosome dwell times from ribosome profiling, considering codon pair interactions between ribosome sites. In mouse liver, the model revealed site- and codon-specific dwell times that differed from those in yeast, as well as pairs of adjacent codons in the P and A site that markedly slow down or speed up elongation. While translation efficiencies vary across diurnal time and feeding regimen, codon dwell times were highly stable and conserved in human. Measured tRNA levels correlated with codon usage and several tRNAs showed reduced aminoacylation, which was conserved in fasted mice. Finally, we uncovered that the longest codon dwell times could be explained by aminoacylation levels or high codon usage relative to tRNA abundance.

AMPK can stimulate eEF2 phosphorylation without regulating its cognate kinase eEF2K

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/Calmodulin (CaM)‐dependent protein kinase and the only known kinase that phosphorylates T56 of eukaryotic elongation factor 2 (eEF2). Phosphorylation of eEF2 at T56 inhibits its ribosomal affinity, thereby blocking tRNA translocation, and ultimately reducing translation elongation rates. The dysregulation of eEF2K is clinically relevant. Its overexpression positively correlates with poor prognosis in various cancers, such as those overexpressing Myc‐family transcription factors. eEF2K overexpression can promote cancer cell survival during severe nutrient deprivation through an AMPK‐dependent mechanism. AMPK, the ubiquitous cellular energy sensor, has been reported to activate eEF2K by directly phosphorylating it on S398. However, it remains unclear whether eEF2K is regulated directly by AMPK. Here using purified proteins, we show that phosphorylation by AMPK does not affect the in vitro enzymatic properties of eEF2K. We also show that the treatment of MCF10A cells with the allosteric activator of AMPK, A769662, results in the stimulation of AMPK activity. This activation leads to an increase in eEF2(T56) phosphorylation, but no rise in the level of activated eEF2K (as judged by T348 autophosphorylation). Thus, an unknown AMPK‐dependent mechanism that appears to have no impact on the activity status of eEF2K can regulate the level of eEF2 phosphorylation in cells. One possibility is that AMPK regulates a phospho‐eEF2 phosphatase.

Codon and amino acid content are associated with mRNA stability in mammalian cells.

Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in the cell and is a major control point for modulating gene expression. In yeast and other model organisms, codon identity is a powerful determinant of transcript stability, contributing broadly to impact half-lives. General principles governing mRNA stability are poorly understood in mammalian systems. Importantly, however, the degradation machinery is highly conserved, thus it seems logical that mammalian transcript half-lives would also be strongly influenced by coding determinants. Herein we characterize the contribution of coding sequence towards mRNA decay in human and Chinese Hamster Ovary cells. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Surprisingly, however, we also observe that the amino acid content of a gene is an additional determinant correlating with transcript stability. The impact of codon and amino acid identity on mRNA decay appears to be associated with underlying tRNA and intracellular amino acid concentrations. Accordingly, genes of similar physiological function appear to coordinate their mRNA stabilities in part through codon and amino acid content. Together, these results raise the possibility that intracellular tRNA and amino acid levels interplay to mediate coupling between translational elongation and mRNA degradation rate in mammals.

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase.

Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF-2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM-regulated kinases. We had previously identified a minimal construct of eEF-2K (TR) that is activated similarly to the wild-type enzyme by CaM in vitro and retains its ability to phosphorylate eEF-2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1-methyls of TR and Ile δ1- and Met ε-methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+ , CaM exclusively utilizes its C-terminal lobe (CaMC ) to engage the N-terminal CaM-binding domain (CBD) of TR in a high-affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+ -loaded N-terminal lobe of CaM (CaMN ) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+ -free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM-bound species without significantly modifying the conformation of TR within the final, active complex.

Slowing ribosome velocity restores folding and function of mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Bacterial stress defense: the crucial role of ribosome speed.

In nature, bacteria are constantly adapting to various stressful conditions. Timely activation of stress response programs is crucial for bacteria to smoothly survive under stressful conditions. Stress response, demanding the de novo synthesis of many defense proteins, is generally activated at the transcriptional level by specific regulators. However, the effect of the global protein translational status on stress response has been largely overlooked. The translational capacity is limited by the number of translating ribosomes and the translational elongation rate. Recent work has shown that certain environmental stressors (e.g. oxidative stress) could severely compromise the stress response progress of bacteria by causing either slow-down or even complete stalling of the translational elongation process. The maintenance of ribosome elongation rate, being crucial for timely synthesis of stress defense proteins, becomes the physiological bottleneck that limits the survival of bacteria in some stressful conditions. Here, we briefly summarize some recent progress on the translational status of bacteria under two distinct stress conditions, nutrient deprivation and oxidative stress. We further discuss several important open questions on the translational regulation of bacteria during stress. The ribosome translation should be investigated in parallel with traditional transcriptional regulation in order to gain a better understanding on bacterial stress defense.

ERF1 mediates codon usage effects on mRNA translation efficiency through premature termination at rare codons

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities.

Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.

To cope with harsh circumstances, bacterial cells must initiate cellular stress response programs, which demands the de novo synthesis of many stress defense proteins. Reactive oxygen species (ROS) is a universal environmental stressor for both prokaryotic cells and eukaryotic cells. However, the physiological burden that limits the survival of bacterial cells during oxidative stress remains elusive. Here we quantitatively characterize the cell growth and translational elongation rate of Escherichia coli cells treated with different doses of hydrogen peroxide. Cell growth is immediately arrested by low to moderate levels of hydrogen peroxide, but completely recovers after a certain lag time. The lag time depends positively on the dose of hydrogen peroxide. During the lag time, translational elongation rate drops by as much as ∼90% at initial stage and recovers to its normal state later, a phenomenon resulting from the dramatic alteration in cellular tRNA pools during oxidative stress. However, translational elongation is completely stalled at a certain threshold-level of hydrogen peroxide, at which cells ultimately fail to resume growth. Although the mRNA transcription of oxidative defense genes in oxyR regulon is dramatically induced upon hydrogen peroxide treatment, the extreme slow-down of translational elongation during high levels of hydrogen peroxide has severely compromised the timely synthesis of those oxidative defense proteins. Our study demonstrates that the tRNA-limited translational elongation is a key physiological bottleneck that the bacteria must overcome to counteract ROS, and the maintenance of translational elongation rate for timely synthesis of stress defense proteins is crucial for cells to smoothly get over the oxidative stress.

A chemical kinetic basis for measuring translation initiation and elongation rates from ribosome profiling data.

Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions, hence bioinformatics methods consistent with the laws of chemistry and physics are more likely to produce accurate results. Here, we derive simple equations based on chemical kinetic principles to measure the translation-initiation rate, transcriptome-wide elongation rate, and individual codon translation rates from ribosome profiling experiments. Our methods reproduce the known rates from ribosome profiles generated from detailed simulations of translation. By applying our methods to data from S. cerevisiae and mouse embryonic stem cells, we find that the extracted rates reproduce expected correlations with various molecular properties, and we also find that mouse embryonic stem cells have a global translation speed of 5.2 AA/s, in agreement with previous reports that used other approaches. Our analysis further reveals that a codon can exhibit up to 26-fold variability in its translation rate depending upon its context within a transcript. This broad distribution means that the average translation rate of a codon is not representative of the rate at which most instances of that codon are translated, and it suggests that translational regulation might be used by cells to a greater degree than previously thought.

A New Look at Codon Usage and Protein Expression.

%MinMax, a model of intra-gene translational elongation rate, relies on codon usage frequencies. Historically, %MinMax has used tables that measure codon usage bias for all genes in an organism, such as those found at HIVE-CUT. In this paper, we provide evidence that codon usage bias based on all genes is insufficient to accurately measure absolute translation rate. We show that alternative "High-ϕ" codon usage tables, generated by another model (ROC-SEMPPR), are a promising alternative. By creating a hybrid model, future codon usage analyses and their applications (e.g., codon harmonization) are likely to more accurately measure the "tempo" of translation elongation. We also suggest a High-ϕ alternative to the Codon Adaptation Index (CAI), a classic metric of codon usage bias based on highly expressed genes. Significantly, our new alternative is equally well correlated with empirical data as traditional CAI without using experimentally determined expression counts as input.