Dataset Information

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.

ABSTRACT: Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.

SUBMITTER: Tomuro K

PROVIDER: S-EPMC11347596 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.

Tomuro Kotaro K Mito Mari M Toh Hirotaka H Kawamoto Naohiro N Miyake Takahito T Chow Siu Yu A SYA Doi Masao M Ikeuchi Yoshiho Y Shichino Yuichi Y Iwasaki Shintaro S

Nature communications 20240826 1

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ri ...[more]

PMID: 39187487

Similar Datasets

Project description:To understand translational capacity on a genome-wide scale across three developmental stages of immature soybean seed cotyledons, ribosome profiling was performed in combination with RNA sequencing and cluster analysis. Transcripts representing 216 unique genes demonstrated a higher level of translational activity in at least one stage by exhibiting higher translational efficiencies (TEs) in which there were relatively more ribosome footprint sequence reads mapping to the transcript than were present in the control total RNA sample. The majority of these transcripts were more translationally active at the early stage of seed development and included 12 unique serine or cysteine proteases and 16 2S albumin and low molecular weight cysteine-rich proteins that may serve as substrates for turnover and mobilization early in seed development. It would appear that the serine proteases and 2S albumins play a vital role in the early stages. In contrast, our investigation of profiles of 19 genes encoding high abundance seed storage proteins, such as glycinins, beta-conglycinins, lectin, and Kunitz trypsin inhibitors, showed that they all had similar patterns in which the TE values started at low levels and increased approximately 2 to 6-fold during development. The highest levels of these seed protein transcripts were found at the mid-developmental stage, whereas the highest ribosome footprint levels of only up to 1.6 TE were found at the late developmental stage. These experimental findings suggest that the major seed storage protein coding genes are primarily regulated at the transcriptional level during normal soybean cotyledon development. Finally, our analyses also identified a total of 370 unique gene models that showed very low TE values including over 48 genes encoding ribosomal family proteins and 95 gene models that are related to energy and photosynthetic functions, many of which have homology to the chloroplast genome. Additionally, we showed that genes of the chloroplast were relatively translationally inactive during seed development.

Project description:Transport of therapeutic agents across epithelial barriers is an important element in drug delivery. Transepithelial flux is widely used as a measure of transit across an epithelium, however it is most typically employed as a relative as opposed to absolute measure of molecular movement. Here, we have used the calcium switch approach to measure the maximum rate of paracellular flux through unencumbered intercellular junctions as a method to calibrate the flux rates for a series of tracers ranging in 0.6-900kDa in size across barriers composed of human colon epithelial (Caco-2) cells. We then examined the effects of nanostructured films (NSFs) on transepithelial transport. Two different NSF patterns were used, Defined Nanostructure (DN) 2 imprinted on polypropylene (PP) and DN3 imprinted on polyether ether ketone (PEEK). NSFs made direct contact with cells and decreased their barrier function, as measured by transepithelial resistance (TER), however cell viability was not affected. When NSF-induced transepithelial transport of Fab fragment (55kDa) and IgG (160kDa) was measured, it was unexpectedly found to be significantly greater than the maximum paracellular rate as predicted using cells cultured in low calcium. These data suggested that NSFs stimulate an active transport pathway, most likely transcytosis, in addition to increasing paracellular flux. Transport of IgG via transcytosis was confirmed by immunofluorescence confocal microscopy, since NSFs induced a significant level of IgG endocytosis by Caco-2 cells. Thus, NSF-induced IgG flux was attributable to both transcytosis and the paracellular route. These data provide the first demonstration that transcytosis can be stimulated by NSFs and that this was concurrent with increased paracellular permeability. Moreover, NSFs with distinct architecture paired with specific substrates have the potential to provide an effective means to regulate transepithelial transport in order to optimize drug delivery.

Dataset Information

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.

Publications

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets