Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA525707 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10953100_1.fastq.gz | Fastqsanger.gz | |||
| SRR10953100_2.fastq.gz | Fastqsanger.gz | |||
| SRR10953079_1.fastq.gz | Fastqsanger.gz | |||
| SRR10953080_1.fastq.gz | Fastqsanger.gz | |||
| SRR10953081_1.fastq.gz | Fastqsanger.gz |
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Project description:Eukaryotic genes often generate a variety of RNA isoforms that can lead to functionally distinct protein variants. The synthesis and stability of RNA isoforms is however poorly characterized. The reason for this is that current methods to quantify RNA metabolism use short-read sequencing that cannot detect RNA isoforms. Here we present nanopore sequencing-based Isoform Dynamics (nano-ID), a method that detects newly synthesized RNA isoforms and monitors isoform metabolism. nano-ID combines metabolic RNA labeling, long-read nanopore sequencing of native RNA molecules and machine learning. nano-ID derived RNA stability estimates enable a distinctive evaluation of stability determining factors such as sequence, poly(A)-tail length, RNA secondary structure, translation efficiency and RNA binding proteins. Application of nano-ID to the heat shock response in human cells reveals that many RNA isoforms change their stability. nano-ID also shows that the metabolism of individual RNA isoforms differs strongly from that estimated for the combined RNA signal at a specific gene locus. nano-ID enables studies of RNA metabolism on the level of single RNA molecules and isoforms in different cell states and conditions.
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Project description:Proteins regulate biological functions through formation of distinct protein complexes. Identification and characterization of these protein-protein interactions are critical to decipher their mechanism of action. Different antibody- or crosslinking-based methods have been developed to identify the protein-protein interactions. However, these methods require genetic engineering and/or other means to disrupt the native environments. To circumvent this limitation, we introduce here SMAP3-ID (Small Molecule Assisted identification of Protein-Protein interactions through Proximity) method to identify protein-protein interactions in native cells. This method entails a selective ligand for binding to a protein of interest for photocrosslinking, a live-cell compatible bioorthogonal click reaction with a trifunctional chemical probe and a final photocrosslinking reaction to covalently capture the interacting proteins. Using SMAP3-ID method and nuclear lamin as an example, we identified lamin-interactors in native cells. Significantly, we identified a number of mitochondrial enzymes as novel lamin A (LA)-interactors. The interactions between mitochondrial enzymes and LA were further validated, which provides mechanistic insights underlying the metabolic alterations caused by mutations in LA. We conclude that SMAP3-ID is a powerful and generalizable method to identify protein-protein interactions in the native cellular environment.
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