Biology subjects

Kramer, G.

Publications and source records attributed to Kramer, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Molecular physiological characterization of the dynamics of persister formation in Staphylococcus aureus.

Bacteria possess the ability to enter a growth arrested state known as persistence in order to survive antibiotic exposure. Clinically, persisters are regarded as the main causative agents for chronic and recurrent infectious diseases. To combat this antibiotic-tolerant population, a better understanding of the molecular physiology of persisters is required. In this study, we collected samples at different stages of the biphasic kill curve to reveal the dynamics of the cellular molecular changes that occur in the process of persister formation. After exposure to antibiotics with different modes of action, namely vancomycin and enrofloxacin, similar persister levels were obtained. Both shared and distinct stress responses were enriched for the respective persister populations. However, the dynamics of the presence of proteins linked to the persister phenotype throughout the biphasic kill curve and the molecular profiles in a stable persistent population did show large differences depending on the antibiotic used. This suggests that persisters at the molecular level are highly stress specific, emphasizing the importance of characterizing persisters generated under different stress conditions. Additionally, although generated persisters exhibited cross-tolerance toward tested antibiotics, combined therapies were demonstrated to be a promising approach to reduce persister levels. In conclusion, this investigation sheds light on the stress-specific nature of persisters, highlighting the necessity of tailored treatment approaches and the potential of combined therapy. ImportanceBy monitoring proteome and metabolites during Staphylococcus aureus persister formation under vancomycin and enrofloxacin exposure, we revealed the dynamic information of the molecular physiology of persister formation upon exposure to two different antibiotics with different modes of action. The data shows that cells that phenotypically are similarly classified as persisters, do have several molecular characteristics in common but, remarkably so, differ substantially in a significant number of other aspects of their molecular makeup. These contrasts provided valuable insights into persister eradication, which holds considerable clinical relevance.

microbiology↗

bioRxiv2023

Multi-omics analysis of Bacillus. subtilis spores formed at different environmental temperatures reveal differences at the morphological and molecular level.

Spore-forming bacteria play an essential role in the food industry and public health. Through sporulation, bacteria can withstand extreme environmental conditions that vegetative cells cannot survive. Although it is well established that the same environmental factors that affect the growth of vegetative cells also profoundly influence sporulation, the mechanisms of how growth conditions affect spore structure and function remain unknown. Prior research has shown that spores prepared at higher temperatures are more heat resistant than those prepared at lower temperatures. The present study examines, both at metabolomic and proteomic levels, the effect of different sporulation temperatures (25, 37 and 42{degrees}C) on the small molecule and protein composition of spores (strain PY79) of the model organism B. subtilis. Through differential harvesting times, spores of the same developmental stage were obtained for each temperature regime. The heat resistance, dipicolinic acid content, germination kinetics and spore morphology were assayed to compare spore properties. Metabolome and proteome analysis yielded unparalleled broad molecular detail of the spores formed at different environmental temperatures. Our findings indicate that peptidoglycan biosynthesis and 28 outer-layer proteins play a crucial role in the functional diversity of spores produced by B. subtilis under varying temperatures.

microbiology↗

bioRxiv2023

Integrative metabolomics and proteomics allow the global intracellular characterization of Bacillus subtilis cells and spores.

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular datasets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol), and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition and the quality of proteomics profiles. The analytical workflow comprises (1) parallel metabolite and protein extraction, (2) monophasic or biphasic sample extraction, (3) proteomics comparison and (4) multi-omics-based data visualization. The 60% EtOH protocol proved to be the easiest in sample processing and more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrated that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. Correlation analysis further demonstrated that the metabolome and proteome datasets were highly correlated in content for components annotated to be part of the pyrimidine metabolism pathway. The results obtained by the 60% EtOH protocol provide a comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/545067v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@18b0206org.highwire.dtl.DTLVardef@1321433org.highwire.dtl.DTLVardef@cc5e6eorg.highwire.dtl.DTLVardef@14bac12_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Whitefly effector G4 interacts with tomato proteins of which MIPDB141 affects whitefly performance

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate defense responses. Here, we present a set of proteins that was identified in artificial diet on which B. tabaci was salivating. We studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of the flg22-induced ROS response when transiently expressed in Nicotiana benthamiana. In addition, G4 was able to supress ROS in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). Fused to a fluorescence tag, G4 localized in the cytoplasm in N. benthamiana. A yeast two-hybrid screen combined with a luciferase bimolecular complementation and co-localization assays resulted in the identification of two target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato, using virus-induced gene silencing, reduced whitefly fecundity up to 40% demonstrating that the protein is involved in susceptibility to B. tabaci. Together our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with the ROS production and via a direct interaction with tomato susceptibility protein MIPDB141.

plant biology↗

bioRxiv2023

Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Candida krusei.

The pathogenic yeast Candida krusei is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in C. krusei. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in C. krusei is similar to Saccharomyces cerevisiae and C. albicans and is comprised of {beta}-1,3-glucan, {beta}-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, C. krusei cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of C. krusei yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 32 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in C. krusei including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of C. krusei. AUTHOR SUMMARYThe yeast Candida krusei is among the five most prevalent causal agents of candidiasis but its mechanisms underlying pathogenicity have been scarcely studied. This is also true for its cell wall structure, an essential organelle that governs primary host-pathogen interactions and host immune responses. Solid knowledge about cell wall synthesis and dynamics is crucial for the development of novel antifungal strategies against this pathogenic yeast. Here, through a combination of comparative genomics, protein structure modeling, and biochemical and proteomic analysis of purified walls, we present a detailed study of the cell wall composition in C. krusei and identify important architectural differences compared to C. albicans cell walls. Cell walls of C. krusei contain higher mannan and protein levels with altered mannan branching patterns, governed by expansions and reductions in gene families encoding mannosyltransferases. We also show that, in contrast to other Candida species, static cultures produce floating biofilms. Comparative wall proteomic studies of these biofilms show increased abundance of flocculins and hydrolytic enzymes, protein classes implicated in biofilm formation and primary host-pathogen interactions leading to tissue colonization. In conclusion, our study uncovers important keys towards a better molecular understanding of the virulence mechanisms of the important pathogen C. krusei.

cell biology↗

bioRxiv2022

Cross-link scrambling in peptide pairs

Identification of peptides and their linked amino acids from chemically cross-linked protein complexes with bifunctional N-hydroxysuccinimidyl (NHS) esters can reveal interacting proteins and their spatial arrangements. With NHS esters both amide- and ester cross-links can be formed. Retention time prediction for strong cation exchange chromatography (SCXC) of cross-linked peptides at pH 3 can distinguish between ester and amide cross-links based on their charge differences. By this approach we show that about 98 % of cross-links are formed by two amide bonds. However MS/MS analysis revealed the presence of an ester linkage in more than 5% of peptide pairs predicted by SCXC to be linked by amide bonds. This discrepancy can be explained by intra-peptide amide-ester rearrangement in the gas phase during MS/MS analysis. So, SCXC retention time prediction can be used to distinguish amide-amide, amide-ester and ester-ester linkages actually formed in the cross-linking reaction and to detect scrambling of cross-linked sites. This information is important for studies aimed to understand the spatial arrangement of protein complexes by cross-linking at the highest possible resolution.

biochemistry↗

bioRxiv2022

Translational Activity Controls Ribophagic Flux and Turnover of Distinct Ribosome Pools

Ribosomes are among the most abundant and complex machineries in the cell, however, the turnover of their subunits remains poorly understood. Here, we apply proteomic flux and cryo-electron microscopy analyses to interrogate the ribosome life cycle in human cells. We show that subpopulations of ribosomal subunits coexist, which vary in turnover kinetics and structure. Specifically, 80S ribosomes have a much longer half-life than free 40S and 60S ribosomal subunits, indicating that they represent distinct subunit pools that poorly intermix. Translation inhibition starkly increases the pool-size of 80S ribosomes in a translationally idle state and induces ribophagy of old ribosomes, ultimately rejuvenating the ribosome fleet. Our findings provide a comprehensive model for ribosome turnover and its regulation via translational activity.

biochemistry↗

bioRxiv2022

Genetic elements orchestrating Lactobacillus crispatus glycogen metabolism in the vagina

Glycogen in the female lower reproductive tract is a major carbon source for vaginal colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously we identified the pullulanase gene pulA in Lactobacillus crispatus, correlating with its ability to autonomously utilize glycogen for growth. Here we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacilllus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the pullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective pullulanase variant or in a pullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available human vaginal metagenome dataset indicates that 15 out of 272 samples, containing a Lactobacillus crispatus pulA gene, contain a defective variant of this gene. Another 23 out of 272 samples show large deletions or transposon insertions. Taken together, these results demonstrate that both environmental as well as genetic factors explain variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

microbiology↗

bioRxiv2022

Interferon regulates stem cell output via post-transcriptional repression of Sox2 independent of its antiviral function in the brain

Stem cells show intrinsic interferon signalling, which protects them from viral infections at all ages. In the ageing brain, interferon signalling in stem cells also reduces their ability to activate. Whether these functions are linked and at what time interferons start taking on a role in stem cell functioning is unknown. Additionally, the molecular link between interferons and activation in neural stem cells and how this relates to productivity is not well understood. Here we combine single-cell transcriptomics, RiboSeq and animal models of interferon to show that this pathway is important for proper stem cell function at all ages. Interferon orchestrates cell cycle and mTOR activity to post-transcriptionally repress Sox2 and drive the exit from stem cell activation. The interferon response then decreases in the subsequent maturation states. Mathematical simulations indicate that this regulation is beneficial for the young and harmful for the old brain. Our study establishes molecular mechanisms of interferon in stem cells and interferons as genuine regulators of stem cell homeostasis and a potential therapeutic target to repair the ageing brain.

neuroscience↗

bioRxiv2021

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone Hsp90 is well known to undergo important conformational changes, which depend on nucleotide, co-chaperones, substrate interactions and post-translational modifications. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally, yet central to its function. Here, using optical tweezers and luciferase and glucocorticoid receptor substrates, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable kinases and receptor clients, regulates their activity, and how Hsp90 cooperates with Hsp70.

biophysics↗

bioRxiv2021

High resolution analysis of proteome dynamics during Bacillus subtilis sporulation

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (blue, brown, green and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue, is negatively correlated with modules brown and green, and contained ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green. We speculate that they may also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation. We speculate on their possible role in guiding or helping assembly of other coat proteins, after which they can be disposed of, but such a hypothesis remains to be experimentally addressed.

microbiology↗

bioRxiv2020

A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at a proteome wide-scale and at the peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker that enables isolation of target peptides and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 for analysis of a previously obtained LCMSMS dataset from exponentially growing Bacillus subtilis treated in culture with the cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of < 5%. To also obtain a FDR < 5% for inter-protein cross-linked peptide pairs additional thresholds values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions to be assigned for both composite peptides. Threshold values were based on a set of decoy sequences from yeast and human sequence databases. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After this filtering, pLink 2 identified more than 80% of previously reported protein-protein interactions. In addition the use of pLink 2 revealed interesting new inter-protein cross-linked peptide pairs, among others showing interactions between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/097956v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@974dd1org.highwire.dtl.DTLVardef@1760d10org.highwire.dtl.DTLVardef@1539d5eorg.highwire.dtl.DTLVardef@808e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIImproved protocol for identification of PPIs at low FDR by in vivo cross-linking with BAMG C_LIO_LIThe use of all intra-protein cross-linked peptide pairs as true positives C_LIO_LIThe cytosolic aminopeptidase (AMPA_BACSU) interacts with the 50S ribosomal protein L17 C_LIO_LIThe transition state regulator AbrB interacts with elongation factor Tu C_LIO_LIThe essential protein YlaN of unknown function interacts with the iron uptake repressor Fur C_LI SignificanceImportant for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20-25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.

biochemistry↗

bioRxiv2020

The Ice Nucleating Protein InaZ is Activated by Low Temperature

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Utilizing specialized ice-nucleating proteins (INPros), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds and may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INPro-induced freezing have remained largely elusive. In the present study, we investigated the folding and the structural basis for interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae strain R10.79. Using vibrational sum-frequency generation and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a {beta}-helical structure in solution and at water surfaces. In this configuration, hydrogen bonding between INPros and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with spectral calculations and molecular-dynamics simulations shows that the INPro reorients at lower temperatures. We suggest that the reorientation can enhance order-inducing water interactions and, thereby, the effectiveness of ice nucleation by InaZ.

biophysics↗

bioRxiv2020

Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates

Variation in translation-elongation kinetics along a transcripts coding sequence plays an important role in the maintenance of cellular protein homeostasis by regulating co-translational protein folding, localization, and maturation. Translation-elongation speed is influenced by molecular factors within mRNA and protein sequences. For example, the presence of proline in the ribosomes P- or A-site slows down translation, but the effect of other pairs of amino acids, in the context of all 400 possible pairs, has not been characterized. Here, we study Saccharomyces cerevisiae using a combination of bioinformatics, mutational experiments, and evolutionary analyses, and show that many different pairs of amino acids and their associated tRNA molecules predictably and causally encode translation rate information when these pairs are present in the A- and P-sites of the ribosome independent of other factors known to influence translation speed including mRNA structure, wobble base pairing, tripeptide motifs, positively charged upstream nascent chain residues, and cognate tRNA concentration. The fast-translating pairs of amino acids that we identify are enriched four-fold relative to the slow-translating pairs across Saccharomyces cerevisiaes proteome, while the slow-translating pairs are enriched downstream of domain boundaries. Thus, the chemical identity of amino acid pairs contributes to variability in translation rates, elongation kinetics are causally encoded in the primary structure of proteins, and signatures of evolutionary selection indicate their potential role in co-translational processes.

molecular biology↗

bioRxiv2019

Thyroid and androgen receptor signaling are antagonized by CRYM in prostate cancer

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein -Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRa/TRb) in target tissues. We show in this study that low CRYM expression levels in PCa patient samples are associated with early BCR and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in PET/MRI imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3 and androgen-mediated signalling. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth. HighlightsO_LIThyroid and androgen hormone driven pathways in prostate cancer (PCa) are antagonized by - Crystallin (CRYM). C_LIO_LI[18F]fluoromethylcholine uptake and prognostic values in PCa correlate with CRYM protein levels. C_LIO_LIReduced CRYM expression predicts early biochemical recurrence (BCR) in PCa patients. C_LI

cancer biology↗

bioRxiv2019

Nα-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles in Saccharomyces cerevisiae

SO_SCPLOWUMMARYC_SCPLOWN-terminal (Nt)-acetylation is a highly prevalent co-translational protein modification in eukaryotes, catalyzed by at least five Nt-acetyltransferases (Nat) with differing specificities. Nt-acetylation has been implicated in protein quality control but its broad biological significance remains elusive. We investigated the roles of the two major Nats of S. cerevisiae, NatA and NatB, by performing transcriptome, translatome and proteome profiling of natA{Delta} and natB{Delta} mutants. Our results do not support a general role of Nt-acetylation in protein degradation but reveal an unexpected range of Nat-specific phenotypes. NatA is implicated in systemic adaptation control, as natA{Delta} mutants display altered expression of transposons, sub-telomeric genes, pheromone response genes and nuclear genes encoding mitochondrial ribosomal proteins. NatB predominantly affects protein folding, as natB{Delta} mutants accumulate protein aggregates, induce stress responses and display reduced fitness in absence of the ribosome-associated chaperone Ssb. These phenotypic differences indicate that controlling Nat activities may serve to elicit distinct cellular responses.

molecular biology↗

bioRxiv2019

Translational regulation of Pmt1 and Pmt2 by Bfr1 affects unfolded protein O-mannosylation

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding GFP as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance, but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

molecular biology↗

bioRxiv2019

Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

Translation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5 cap. Consequently, only one ribosome scans a 5UTR at a time, and 5UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of [~]12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.\n\nHIGHLIGHTSO_LISelective 40S FPing visualizes regulation of translation initiation on mRNAs in vivo\nC_LIO_LIScanning ribosomes are cap-tethered in human cells\nC_LIO_LIOnly one ribosome scans a 5UTR at a time in human cells\nC_LIO_LIRibosomes retain eIFs during early translation, allowing reinitiation after uORFs\nC_LI

molecular biology↗