PXD026828-1

PXD026828 is an original dataset announced via ProteomeXchange.

Title	Nascent Ribo-Seq measures ribosomal loading time and reveals kinetic impact on ribosome density
Description	mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.
HostingRepository	PRIDE
AnnounceDate	2021-06-22
AnnouncementXML	Submission_2021-06-22_04:01:15.408.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Tamar Geiger
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	biotinylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2021-06-21 03:03:45	ID requested
⏵ 1	2021-06-22 04:01:15	announced

Dataset with its publication pending

submitter keyword: translatome, ribo-seq, nascent peptides

Georg Stoecklin
contact affiliation	German Cancer Research Center
contact email	georg.stoecklin@medma.uni-heidelberg.de
lab head
Tamar Geiger
contact affiliation	Tel Aviv University
contact email	geiger@tauex.tau.ac.il
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD026828
     1. Label: PRIDE project
     2. Name: Nascent Ribo-Seq measures ribosomal loading time and reveals kinetic impact on ribosome density