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Related Concept Videos

Ribosome Profiling02:24

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.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
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RNA-seq03:21

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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. 
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Related Experiment Video

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3' End Sequencing Library Preparation with A-seq2
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Updated Pseudo-seq Protocol for Transcriptome-Wide Detection of Pseudouridines.

Yi Pan1, Hironori Adachi1, Xueyang He1

  • 1University of Rochester Medical Center, Department of Biochemistry and Biophysics, Center for RNA Biology, Rochester, NY, USA.

Bio-Protocol
|May 13, 2024
PubMed
Summary

This study updates Pseudo-seq, a method for precisely mapping pseudouridine (Ψ) sites in messenger RNAs (mRNAs). The enhanced technique allows for genome-wide identification and quantification of Ψ modifications in various human cell and tissue types.

Keywords:
Illumina NextSeqNext-generation sequencingPseudo-seqTranscriptome-wide pseudouridine mappingmRNA modification

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Pseudouridine (Ψ) is the most abundant modification in cellular RNAs, found in various RNA types including messenger RNAs (mRNAs).
  • Existing methods for Ψ identification face limitations with current sequencing technologies due to reagent incompatibility.
  • Accurate mapping of pseudouridylation sites is crucial for understanding RNA regulation and function.

Purpose of the Study:

  • To present an updated Pseudo-seq technique for genome-wide identification of pseudouridylation sites with single-nucleotide precision.
  • To provide a comprehensive protocol for Pseudo-seq adaptable to diverse cell and tissue types.
  • To enable discovery of novel pseudouridylation sites and facilitate functional studies.

Main Methods:

  • Development and description of an updated Pseudo-seq protocol.
  • Includes optimized RNA isolation from human cells, efficient library preparation using current materials, and detailed data analysis.
  • Methodology is designed for high-quality mRNA isolation and compatibility with modern sequencing platforms.

Main Results:

  • The updated Pseudo-seq method accurately identifies pseudouridine (Ψ) sites on mRNAs.
  • Provides precise positional and quantitative information regarding Ψ modifications.
  • Demonstrates efficient library preparation compatible with the latest sequencing technologies.

Conclusions:

  • The updated Pseudo-seq is a valuable tool for genome-wide pseudouridylation site identification.
  • The methodology is adaptable and facilitates the discovery of novel Ψ sites in various biological contexts.
  • This technique is essential for advancing the understanding of pseudouridine's role in RNA regulation and function.