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A subcellular map of translational machinery composition and regulation at the single-molecule level [ALIBi_ER_vs_cyto]

ABSTRACT: A subcellular map of translational machinery composition and regulation at the single-molecule level [ALIBi_ER_vs_cyto]

PROVIDER: PRJNA1195970 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31650160_1.fastq.gz	Fastqsanger.gz
	SRR31650160_2.fastq.gz	Fastqsanger.gz
	SRR31650161_1.fastq.gz	Fastqsanger.gz
	SRR31650161_2.fastq.gz	Fastqsanger.gz
	SRR31650162_1.fastq.gz	Fastqsanger.gz

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A subcellular map of translational machinery composition and regulation at the single-molecule level [ALIBi_Nmd3_Gnl3_KD]

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A subcellular map of translational machinery composition and regulation at the single-molecule level [ALIBi_Lsg1_KD]

Project description:Millions of ribosomes are packed within mammalian cells, yet we lack tools to visualize them in toto and characterize their subcellular composition. Here, we present ribosome expansion microscopy (RiboExM) to visualize individual ribosomes and an optogenetic proximity-labeling technique (ALIBi) to probe their composition. We generated a super-resolution ribosomal map, revealing unexpected subcellular translational hotspots and enrichment of 60S subunits near polysomes at the endoplasmic reticulum (ER). We found that Lsg1 tethers 60S to the ER and regulates translation of select proteins. Additionally, we discovered ribosome heterogeneity at mitochondria guiding translation of metabolism-related transcripts. Finally, we visualized ribosomes in neurons, revealing a dynamic switch between monosomes and polysomes in neuronal translation. Together, these approaches enable exploration of ribosomal localization and composition at unprecedented resolution.

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A subcellular map of translational machinery composition and regulation at the single-molecule level [ALIBi_ER_vs_cyto]

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ROSALIND protects the mitochondrial translational machinery from oxidative damage

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Long read subcellular fractionation and sequencing reveals the translational fate of full length mRNA isoforms during neuronal differentiation

Project description:Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESC. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESC and NPC respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type-specific and subcellular fraction-associated RNA-binding protein signatures. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using a novel long-read sequencing-based method to study translational control.

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