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Related Concept Videos

Alternative RNA Splicing02:18

Alternative RNA Splicing

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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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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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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
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Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
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Identification of Alternative Splicing and Polyadenylation in RNA-seq Data.

Gunjan Dixit1, Ying Zheng1, Brian Parker2

  • 1Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University.

Journal of Visualized Experiments : Jove
|July 12, 2021
PubMed
Summary

RNA sequencing (RNA-Seq) analysis can reveal differential gene expression and uncover complex regulatory mechanisms like alternative splicing and polyadenylation site usage, providing deeper biological insights beyond whole gene analysis.

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Using the E1A Minigene Tool to Study mRNA Splicing Changes
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Area of Science:

  • Transcriptomics
  • Molecular Biology
  • Bioinformatics

Background:

  • RNA sequencing (RNA-Seq) is typically used for differential gene expression (DGE) analysis.
  • Gene expression regulation occurs at multiple levels, including post-transcriptional modifications.
  • Alternative splicing and polyadenylation generate transcript isoforms, impacting gene function and expression.

Purpose of the Study:

  • To demonstrate detailed analyses for identifying and visualizing differential exon usage in RNA-Seq data.
  • To showcase methods for detecting differential polyadenylation site usage across biological conditions.
  • To highlight the importance of exon-level analysis for a comprehensive understanding of gene regulation.

Main Methods:

  • Utilized Bioconductor packages and functions for RNA-Seq data analysis.
  • Applied DEXSeq for differential exon usage analysis.
  • Employed diffSplice (Limma package) and rMATS for alternative splicing and polyadenylation site analysis.

Main Results:

  • Provided step-by-step protocols for complex RNA-Seq analyses.
  • Demonstrated the identification of differential exon and polyadenylation site usage.
  • Visualized regulatory events at the exon level.

Conclusions:

  • RNA-Seq data analysis can extend beyond DGE to explore exon-level regulatory mechanisms.
  • Alternative splicing and polyadenylation are critical layers of gene regulation missed by whole-gene analysis.
  • The presented methods enable a more comprehensive understanding of transcriptomic diversity and gene regulation.