Selective ribosome profiling to study interactions of translating ribosomes in yeast
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ABSTRACT:
PROVIDER: PRJNA507750 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8267410.fastq.gz | Fastqsanger.gz | |||
| SRR8267411.fastq.gz | Fastqsanger.gz | |||
| SRR8267404.fastq.gz | Fastqsanger.gz | |||
| SRR8267405.fastq.gz | Fastqsanger.gz | |||
| SRR8267406.fastq.gz | Fastqsanger.gz |
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Project description: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 5’UTR at a time, and 5’UTR 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.
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Project description:Selective ribosome profiling of the DHX29-bound ribosomes in HEK293T cells
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Project description:Translating ribosome affinity purification (TRAP) methods allow cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. In this study, we purified and analysed the polyribosome-associated RNA fraction of zebrafish embryo´s retinas at 22 hours post-fertilization. To this aim, we generated a transgenic strain in which the tagged ribosomes expression is restricted to the retina cells due to the activity of a specific promoter.
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Project description:<p>During translation, transfer RNAs (tRNAs) deliver specific amino acids to the ribosome in a coordinated manner with the sequence encoded by the mRNA. Despite their central role in protein synthesis, the precise contribution of tRNAs to the modulation of translation remains poorly understood, primarily due to lack of methods to characterise tRNA abundances and their modifications from actively translating ribosomes. Here we develop RiboNano-tRNAseq, a novel method to quantify native ribosome-associated tRNAs (ribo-tRNAs) from actively translating ribosomes, achieved by coupling tag-free pulldown of active ribosomes with native tRNA nanopore sequencing. Applying RiboNano-tRNAseq, we investigate how the ribosome-associated tRNAome varies under diverse stress conditions. Our work reveals highly tailored tRNA responses depending on the stress type. For example, under leucine starvation, we observe enrichment of a subset of tRNALeu isoacceptors in ribo-tRNAs. By contrast, methionine starvation leads to a decrease in methyl-based tRNA modifications, such as m1A or m2,2G, reflecting the decreased availability of the methyl donor S-adenosylmethionine (SAM). Strikingly, arsenite starvation does not affect tRNA modification profiles, but instead causes a drastic fragmentation of tRNAs, predominantly within ribosome-associated tRNAs. Altogether, our work findings demonstrate that RiboNano-tRNAseq enables comprehensive characterization of the tRNAome and its dynamics by simultaneously capturing tRNA abundance, modification and fragmentation patterns, both in total and actively translating tRNA pools.</p>
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