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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Ribosome Profiling

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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RNA Structure01:23

RNA Structure

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Overview
The basic structure of RNA consists of a five-carbon sugar and one of four nitrogenous bases. Although most RNA is single-stranded, it can form complex secondary and tertiary structures. Such structures play essential roles in the regulation of transcription and translation.
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RNA Structure01:19

RNA Structure

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The basic structure of RNA consists of a string of ribonucleotides attached by phosphodiester bonds. Although most RNA is single-stranded, it can form complex secondary and tertiary structures. Such structures play essential roles in the regulation of transcription and translation.
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Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
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Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC
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Revealing nascent RNA processing dynamics with nano-COP.

Heather L Drexler1, Karine Choquet1, Hope E Merens1

  • 1Department of Genetics, Harvard Medical School, Boston, MA, USA.

Nature Protocols
|January 30, 2021
PubMed
Summary
This summary is machine-generated.

We developed nanopore analysis of co-transcriptional processing (nano-COP) to study RNA maturation. This method reveals real-time intron removal and transcription-splicing coupling in single RNA molecules.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Eukaryotic precursor RNAs undergo essential processing, including splicing and polyadenylation, during maturation.
  • Understanding the precise timing and patterns of these events is crucial for gene expression regulation.

Purpose of the Study:

  • To introduce nanopore analysis of co-transcriptional processing (nano-COP), a novel method for analyzing RNA processing.
  • To investigate the temporal dynamics and patterns of RNA processing events during transcription.

Main Methods:

  • nano-COP is an extension of native elongating transcript sequencing, utilizing long-read nanopore sequencing of nascent RNA.
  • Nascent RNA is purified using 4-thiouridine labeling and cellular fractionation.
  • Direct nanopore sequencing avoids reverse transcription and amplification, preserving native RNA context.

Main Results:

  • nano-COP enables genome-wide observation of global RNA processing patterns.
  • The method identifies active transcription sites and splice isoforms of single RNA molecules during synthesis.
  • It provides insights into intron removal patterns and the physical coupling between transcription and splicing.

Conclusions:

  • nano-COP offers a powerful tool to study the intricacies of co-transcriptional RNA processing.
  • The protocol yields data rapidly, within 3 days.
  • This method advances our understanding of RNA maturation and its regulation.