Ribosomal Fraction Research Articles

Respiratory Syncytial Virus (RSV) optimizes the translational landscape during infection.

Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5'-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5'-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation.

Effect of biological sex and short-term high-fat diet on cellular proliferation, ribosomal biogenesis, and targeted protein abundance in murine articular cartilage

ObjectiveTo identify factors contributing to sex-differences in OA risk by evaluating the short-term effect of high-fat (HF) diet on sex-specific changes in cartilage cell proliferation, ribosomal biogenesis, and targeted extra-cellular and cellular protein abundance. Materials and methodsKnee cartilage was harvested to the subchondral bone from 20-week-old female and male C57BL/6J mice fed a low-fat or HF diet for 4 weeks and labeled with deuterium oxide for 1, 3, 5, 7, 15, or 21 days. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Protein concentration was measured using targeted high resolution accurate mass spectrometry. ResultsHF diet increased the maximal deuterium incorporation into DNA from approximately 40 to 50%, albeit at a slower rate. These findings, which were magnified in female versus male mice, indicate a greater number of proliferating cells with longer half-lives under HF diet conditions. HF diet caused distinct sex-dependent effects on deuterium incorporation into RNA, increasing the fraction of ribosomes undergoing biogenesis in male mice and doubling the rate of ribosome biogenesis in female mice. HF diet altered cartilage protein abundance similarly in both sexes, except for matrilin-3, which was more abundant in HF versus LF conditions in female mice only. Overall, HF diet treatment had a stronger effect than sex on cartilage protein abundance, with most changes involving extracellular matrix and matrix-associated proteins. ConclusionsShort-term HF diet broadly altered cartilage matrix protein abundance, while sex-dependent effects primarily involved differences in cell proliferation and ribosomal biogenesis.

How total mRNA influences cell growth

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically motivated regime where RNA polymerases compete for genes and ribosomes for transcripts and gives general expressions relating growth rate, mRNA concentrations, ribosome, and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast and Escherichia coli bacteria: i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.

Abstract PO1-24-01: ADAR1 drives ribosomal biogenesis by regulating NOP14 and stabilizing FBL-NOP58 complex in breast cancer

Abstract Background Breast cancer (BC) has been widely accepted as a highly heterogeneous disease. Transcriptional and translational dysregulation is commonly found in BC patients. Adenosine-to-inosine (A-to-I) editing on RNA is the most abundant RNA editing form in human, mediated mainly by ADARs. Though the role of ADAR1 in RNA editing and immune response has been deeply investigated, the change of the translational profile and the biology sequalae after base editing are still unclear. In this study, we uncovered a previously unknown role of ADAR1 in the regulation of ribosome biosynthesis and translational dysregulation in BC cells. Method RNA-seq was used for gene set enrichment analysis (GSEA) and A-to-I editing analysis. Transmission electron microscopy (TEM) was conducted to visualize the nucleolar architecture. Nuclear rRNA abundances of 18S and 28S rRNAs were monitored using Qsep100. Ribosomal fractions were isolated with a Beckman SW41Ti rotor and quantified on a 2100 Bioanalyzer. 3' Untranslated region (3′UTR) reporter construction and dual-luciferase reporter assay were used to validate the correlation. In-vitro transcription, RNA pull-down assay and sanger sequencing were used to directly confirm the direct interaction and A-to-I editing status. Cell proliferation, migration assay, apoptosis assay in vitro and xenograft mouse model in vivo were performed for phenotype study. Co-immunoprecipitation (Co-IP) was used to identify the interactive protein. Immunofluorescence (IF) assay was applied to indicate the co-localization. Immunohistochemistry (IHC) staining was applied to tissue microarrays and assess of expression. Survival analysis was made by Kaplan-Meier method. RNA polymerase I inhibitor CX-5461 was used for treatment. Result Through bioinformatic analysis with RNA-seq data from cell lines and clinical samples, we found the expression of ADAR1 was correlated with signatures of ribosomes. Consistently, BC cells with overexpression of ADAR1 showed larger nucleoli and contained more 18S and 28S rRNAs. To uncover the potential ribosomal genes involved in this process, we analyzed the transcriptional profile of 88 BC samples in combination with ADAR1 editing profiles identified by comparing ADAR1-null and control cells. Nucleolar Protein 14 (NOP14), a nucleolar ribosome biogenesis gene, was found to be hyper-edited. Phenotype experiments revealed that NOP14 promotes tumor growth and invasion in vitro and in vivo. The RNA and protein level of NOP14 is regulated by ADAR1 through A-to-I editase activity. Mechanically, ADAR1 binds directly with NOP14 transcripts in the 3’UTR region through Sanger sequencing and RNA-pull down. The binding region of NOP14 with ADAR1 was also a target of miR-939-3p, while the ADAR1 mediated-editing led to less binding of miR-939-3p with NOP14 and resulted in NOP14 up-regulation. Notably, NOP14 facilitates the assembly of the box C/D small nucleolar ribonucleoprotein (snoRNP) and stabilize the interaction of fibrillarin (FBL)-nucleolar Protein 58 (NOP58) with this complex. Loss of NOP14 impaired the nucleolar distribution of FBL and the abundance of ribosomes. CX-5461, an RNA polymerase I inhibitor, suppressed the overexpression of ribosomes in ADAR1 high cells and displayed a strong inhibition efficacy on tumor growth both in vitro and in vivo. Conclusion Taken together, these results shed light on the role of ADAR1 in ribosomal biogenesis in breast cancer. The promising results also provide potential targets in BCs with high ADAR1 by suppressing translational dysregulation. Citation Format: Liren Wangxu, Hengyu Ren, Zhibo Shao, Jiong Wu, Yayun Chi. ADAR1 drives ribosomal biogenesis by regulating NOP14 and stabilizing FBL-NOP58 complex in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-01.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h−1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

A coarse-grained resource allocation model of carbon and nitrogen metabolism in unicellular microbes.

Coarse-grained resource allocation models (C-GRAMs) are simple mathematical models of cell physiology, where large components of the macromolecular composition are abstracted into single entities. The dynamics and steady-state behaviour of such models provides insights on optimal allocation of cellular resources and have explained experimentally observed cellular growth laws, but current models do not account for the uptake of compound sources of carbon and nitrogen. Here, we formulate a C-GRAM with nitrogen and carbon pathways converging on biomass production, with parametrizations accounting for respirofermentative and purely respiratory growth. The model describes the effects of the uptake of sugars, ammonium and/or compound nutrients such as amino acids on the translational resource allocation towards proteome sectors that maximized the growth rate. It robustly recovers cellular growth laws including the Monod law and the ribosomal growth law. Furthermore, we show how the growth-maximizing balance between carbon uptake, recycling, and excretion depends on the nutrient environment. Lastly, we find a robust linear correlation between the ribosome fraction and the abundance of amino acid equivalents in the optimal cell, which supports the view that simple regulation of translational gene expression can enable cells to achieve an approximately optimal growth state.

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides

SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.

High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development.

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

Environment-specificity and universality of the microbial growth law

As the nutrient quality changes, the fractions of ribosomal proteins in the proteome are usually positively correlated with the growth rates due to the auto-catalytic nature of ribosomes. While this growth law is observed across multiple organisms, the relation between the ribosome fraction and growth rate is often more complex than linear, beyond models assuming a constant translation speed. Here, we propose a general framework of protein synthesis considering heterogeneous translation speeds and protein degradations. We demonstrate that the growth law curves are generally environment-specific, e.g., depending on the correlation between the translation speeds and ribosome allocations among proteins. Our predictions of ribosome fractions agree quantitatively with data of Saccharomyces cerevisiae. Interestingly, we find that the growth law curve of Escherichia coli nevertheless appears universal, which we prove must exhibit an upward bending in slow-growth conditions, in agreement with experiments. Our work provides insights on the connection between the heterogeneity among genes and the environment-specificity of cell behaviors.

Emergent Role of IFITM1/3 towards Splicing Factor (SRSF1) and Antigen-Presenting Molecule (HLA-B) in Cervical Cancer

The IFITM restriction factors play a role in cancer cell progression through undefined mechanisms. We investigate new protein–protein interactions for IFITM1/3 in the context of cancer that would shed some light on how IFITM1/3 attenuate the expression of targeted proteins such as HLA-B. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the SRSF1 splicing factor and transporter of mRNA to the ribosome. Using in situ proximity ligation assays, we confirmed a predominant cytosolic protein–protein association for SRSF1 and IFITM1/3. Accordingly, IFITM1/3 interacted with HLA-B mRNA in response to IFNγ stimulation using RNA–protein proximity ligation assays. In addition, RT-qPCR assays in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that HLA-B gene expression at the mRNA level does not account for lowered HLA-B protein synthesis in response to IFNγ. Complementary, shotgun RNA sequencing did not show major transcript differences between IFITM1/IFITM3 null cells and wt-SiHa cells. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to wild type. It was partially reverted by IFITM1/3 complementation. Our data link IFITM1/3 proteins to HLA-B mRNA and SRSF1 and, all together, our results begin to elucidate how IFITM1/3 catalyze the synthesis of target proteins. IFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. Our study has identified new proteins associated with IFITMs which support their role in mediating protein expression; a pivotal function that is highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 affect the expression of targeted proteins; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs in mediating protein abundance is relevant, as it has the potential for regulating the expression of viral and oncogenic proteins.

Altered tRNA dynamics during translocation on slippery mRNA as determinant of spontaneous ribosome frameshifting

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and –1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the –1-frame triplet, resulting in slippage followed by recruitment of the –1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.

Mitochondrial rRNA Methylation by Mettl15 Contributes to the Exercise and Learning Capability in Mice.

Mitochondrial translation is a unique relic of the symbiotic origin of the organelle. Alterations of its components cause a number of severe human diseases. Hereby we report a study of mice devoid of Mettl15 mitochondrial 12S rRNA methyltransferase, responsible for the formation of m4C839 residue (human numbering). Homozygous Mettl15−/− mice appeared to be viable in contrast to other mitochondrial rRNA methyltransferase knockouts reported earlier. The phenotype of Mettl15−/− mice is much milder than that of other mutants of mitochondrial translation apparatus. In agreement with the results obtained earlier for cell cultures with an inactivated Mettl15 gene, we observed accumulation of the RbfA factor, normally associated with the precursor of the 28S subunit, in the 55S mitochondrial ribosome fraction of knockout mice. A lack of Mettl15 leads to a lower blood glucose level after physical exercise relative to that of the wild-type mice. Mettl15−/− mice demonstrated suboptimal muscle performance and lower levels of Cox3 protein synthesized by mitoribosomes in the oxidative soleus muscles. Additionally, we detected decreased learning capabilities in the Mettl15−/− knockout mice in the tests with both positive and negative reinforcement. Such properties make Mettl15−/− knockout mice a suitable model for mild mitochondriopathies.

A Mutant Era GTPase Suppresses Phenotypes Caused by Loss of Highly Conserved YbeY Protein in Escherichia coli.

Ribosome assembly is a complex fundamental cellular process that involves assembling multiple ribosomal proteins and several ribosomal RNA species in a highly coordinated yet flexible and resilient manner. The highly conserved YbeY protein is a single-strand specific endoribonuclease, important for ribosome assembly, 16S rRNA processing, and ribosome quality control. In Escherichia coli, ybeY deletion results in pleiotropic phenotypes including slow growth, temperature sensitivity, accumulation of precursors of 16S rRNA, and impaired formation of fully assembled 70S subunits. Era, an essential highly conserved GTPase protein, interacts with many ribosomal proteins, and its depletion results in ribosome assembly defects. YbeY has been shown to interact with Era together with ribosomal protein S11. In this study, we have analyzed a suppressor mutation, era(T99I), that can partially suppress a subset of the multiple phenotypes of ybeY deletion. The era(T99I) allele was able to improve 16S rRNA processing and ribosome assembly at 37°C. However, it failed to suppress the temperature sensitivity and did not improve 16S rRNA stability. The era(T99I) allele was also unable to improve the 16S rRNA processing defects caused by the loss of ribosome maturation factors. We also show that era(T99I) increases the GroEL levels in the 30S ribosome fractions independent of YbeY. We propose that the mechanism of suppression is that the changes in Era’s structure caused by the era(T99I) mutation affect its GTP/GDP cycle in a way that increases the half-life of RNA binding to Era, thereby facilitating alternative processing of the 16S RNA precursor. Taken together, this study offers insights into the role of Era and YbeY in ribosome assembly and 16S rRNA processing events.

Protein degradation sets the fraction of active ribosomes at vanishing growth.

Growing cells adopt common basic strategies to achieve optimal resource allocation under limited resource availability. Our current understanding of such "growth laws" neglects degradation, assuming that it occurs slowly compared to the cell cycle duration. Here we argue that this assumption cannot hold at slow growth, leading to important consequences. We propose a simple framework showing that at slow growth protein degradation is balanced by a fraction of "maintenance" ribosomes. Consequently, active ribosomes do not drop to zero at vanishing growth, but as growth rate diminishes, an increasing fraction of active ribosomes performs maintenance. Through a detailed analysis of compiled data, we show that the predictions of this model agree with data from E. coli and S. cerevisiae. Intriguingly, we also find that protein degradation increases at slow growth, which we interpret as a consequence of active waste management and/or recycling. Our results highlight protein turnover as an underrated factor for our understanding of growth laws across kingdoms.

Alternative Polyadenylation Utilization Results in Ribosome Assembly and mRNA Translation Deficiencies in a Model for Muscle Aging.

Aging-associated muscle wasting is regulated by multiple molecular processes, whereby aberrant mRNA processing regulation induces muscle wasting. The poly(A)-binding protein nuclear 1 (PABPN1) regulates polyadenylation site (PAS) utilization, in the absence of PABPN1 the alternative polyadenylation (APA) is utilized. Reduced PABPN1 levels induce muscle wasting where the expression of cellular processes regulating protein homeostasis, the ubiquitin-proteasome system, and translation, are robustly dysregulated. Translation is affected by mRNA levels, but PABPN1 impact on translation is not fully understood. Here we show that a persistent reduction in PABPN1 levels led to a significant loss of translation efficiency. RNA-sequencing of rRNA-depleted libraries from polysome traces revealed reduced mRNA abundance across ribosomal fractions, as well as reduced levels of small RNAs. We show that the abundance of translated mRNAs in the polysomes correlated with PAS switches at the 3′-UTR. Those mRNAs are enriched in cellular processes that are essential for proper muscle function. This study suggests that the effect of PABPN1 on translation efficiency impacts protein homeostasis in aging-associated muscle atrophy.

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

IntroductionRibosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation.MaterialsSnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling.ResultsAs a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5–6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation.ConclusionThe expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

Global pleiotropic effects in adaptively evolved Escherichia coli lacking CRP reveal molecular mechanisms that define the growth physiology.

Evolution facilitates emergence of fitter phenotypes by efficient allocation of cellular resources in conjunction with beneficial mutations. However, system-wide pleiotropic effects that redress the perturbations to the apex node of the transcriptional regulatory networks remain unclear. Here, we elucidate that absence of global transcriptional regulator CRP in Escherichia coli results in alterations in key metabolic pathways under glucose respiratory conditions, favouring stress- or hedging-related functions over growth-enhancing functions. Further, we disentangle the growth-mediated effects from the CRP regulation-specific effects on these metabolic pathways. We quantitatively illustrate that the loss of CRP perturbs proteome efficiency, as evident from metabolic as well as ribosomal proteome fractions, that corroborated with intracellular metabolite profiles. To address how E. coli copes with such systemic defect, we evolved Δcrp mutant in the presence of glucose. Besides acquiring mutations in the promoter of glucose transporter ptsG, the evolved populations recovered the metabolic pathways to their pre-perturbed state coupled with metabolite re-adjustments, which altogether enabled increased growth. By contrast to Δcrp mutant, the evolved strains remodelled their proteome efficiency towards biomass synthesis, albeit at the expense of carbon efficiency. Overall, we comprehensively illustrate the genetic and metabolic basis of pleiotropic effects, fundamental for understanding the growth physiology.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and 'damage control' during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid &lt;em&gt;In Vivo&lt;/em&gt; Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and 'damage control' during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.