We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae . mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across gradient quantitative microarray analysis, providing profile ribosome association with for thousands genes. For most genes, majority molecules associated ribosomes presumably engaged translation. This systematic approach enabled us to recognize genes unusual behavior. 43 not ribosomes, suggesting...

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction translocase outer membrane (TOM) complex with precursor while it is translated, thereby leading association polysomal mitochondria. To test this hypothesis, we isolated fractions from yeast cells lacking major import receptor, Tom20, and compared their mRNA repertoire that wild-type by DNA microarrays. Most...

Abstract It is well established that import of proteins into mitochondria can occur after their complete synthesis by cytosolic ribosomes. Recently, an additional model was revived, proposing some are imported co-translationally. This entails association ribosomes with the mitochondrial outer membrane, shown to be mediated through ribosome-associated chaperone nascent chain-associated complex (NAC). However, receptor this unknown. Here, we identify Saccharomyces cerevisiae membrane protein...

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates these steps determine both the number position ribosomes along mRNA, but traditional velocity sedimentation assays for translational status only bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage polysomal followed by separation on sucrose gradient northern analysis, to associated with specified portions...

Genome-wide studies of steady-state mRNA levels revealed common principles underlying transcriptional changes in response to external stimuli. To uncover that govern other stages the gene-expression response, we analyzed translational and its coordination with transcriptome following exposure severe stress. Yeast cells were grown for 1 h medium containing M NaCl, which elicits a maximal but transient translation inhibition, nonpolysomal or polysomal pools subjected DNA-microarray analyses....

Ribosome queuing is a fundamental phenomenon suggested to be related topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this hasn't been quantified yet at genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints) are usually calibrated capture only single protected footprints (mRPFs) thus limited in their ability detect queuing. On the other hand, most of models field assume analyze...

Mitochondria exert their many functions through a repertoire of hundreds proteins. The vast majority these proteins are encoded in the nuclear genome, translated cytosol and imported into mitochondria. Current models, derived mainly from work yeast, suggest that translation can occur close vicinity to mitochondria outer membrane by localized ribosomes. Here, we applied ribosome-proximity biotin labeling address this possibility. A clear biotinylation ribosomes mitochondrial Tom20-BirA fusion...

Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one the most common RNA modifications; however, only few modifiers have been identified to date, and no pseudouridine reader known. Here, we applied novel genome-wide approach identify regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found clear enrichment were previously described contain (Ψ). Follow-up in vitro vivo analyses on prime target...

Aminoacyl-tRNA synthetases (aaRSs) are well studied for their role in binding and charging tRNAs with cognate amino acids. Recent RNA interactome studies had suggested that these enzymes can also bind polyadenylated RNAs. Here, we explored the mRNA repertoire bound by several yeast aaRSs. immunoprecipitation (RIP) followed deep sequencing revealed unique sets of mRNAs each aaRS. Interestingly, every tested aaRSs, a preferential association its own was observed, suggesting an autoregulatory...

Nuclear-encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the mechanism of transport for those distal locations is not fully understood. Here, we describe axonal co-transport Cox7c with mitochondria. Fractionation analysis single-molecule fluorescence in situ hybridization (smFISH) assay revealed that endogenous mRNA encoding was preferentially associated mitochondria a mouse cell line primary motor neuron axons,...

Fragment-based screening is an efficient method for early-stage drug discovery. In this study, we aimed to create a fragment library optimized producing high hit rates against RNA targets. has historically been underexplored target, but recent research suggests potential optimizing small molecule libraries binding. We extended concept produce fluorinated library. then screened library, alongside two non-RNA libraries, three targets: the human cytoplasmic A-site and S. cerevisiae tRNAAsp...

A major challenge in cell and developmental biology is the automated identification quantitation of cells complex multilayered tissues. We developed CytoCensus: an easily deployed implementation supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection challenging datasets with ill-defined boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software accuracy speed detection. used count stem their...

Cotranslational synthesis of proteins into the endoplasmic reticulum is preceded by targeting translating mRNA once a signal peptide emerges from ribosome exit tunnel. Many mRNAs, however, are unlikely to be targeted this process because they encode that do not contain or too short recognized recognition particle. Herein we tested possible involvement 3′-UTR in localization an encodes very Saccharomyces cerevisiae protein (Pmp1). We found density mapping, sedimentation analysis, differential...

Abstract Contemporary research aims to understand biological processes not only by identifying participating proteins, but also characterizing the dynamics of their interactions. Because Förster's Resonance Energy Transfer (FRET) is invaluable for latter undertaking, its usage steadily increasing. However, FRET measurements are notoriously error-prone, especially when inherent efficiency low, a uncommon situation. Furthermore, many methods either difficult implement, appropriate observation...

Cracking the encryption: Parallel computing with molecular finite-state automata and fluorescently labeled DNA molecules has been used to decipher two different images encrypted onto a single chip (see picture). The were deciphered by mixture of input that processed biomolecular automata, strategy potentially offers huge diversity images.

Many nuclear‐transcribed mRNAs encoding mitochondrial proteins are localized near the outer membrane. A yet unresolved question is whether protein synthesis important for transport of these to their destination. Herein we present a connection between mRNA localization in yeast and chaperone Ssa1. Ssa1 depletion lowered association with mitochondria while its overexpression increased it. genome‐wide analysis revealed that Ssa preferentially affect hydrophobic proteins, which expected targets...

PUF proteins bind mRNAs and regulate their translation, stability, localization. Each protein binds a selective group of mRNAs, enabling coordinate control. We focus here on the specificity Puf2p Puf1p Saccharomyces cerevisiae , which copurify with overlapping groups mRNAs. applied an RNA-adapted version DRIM algorithm to identify putative binding sequences for both proteins. first identified novel motif in 3′ UTRs previously shown associate Puf2p. This consisted two UAAU tetranucleotides...

During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of transition between proliferation differentiation. Prospero level low in neural stem cells their immediate progeny, but upregulated larval neurons it unknown how this process controlled. Here, we use single molecule fluorescent situ hybridisation to show that selectively transcribe a long prospero mRNA isoform containing 15 kb 3’ untranslated region, which bound...

DNA microarrays have been used extensively in recent years to study mRNA expression profiles of different cell types under various growth conditions. These steady-state provide a wealth information about cellular functions and responses. However, they do not necessarily reflect the ultimate gene profile since step translation might lead discrepancies between actual functioning unit cell, protein (e.g., ,).