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Related Experiment Videos

Beyond the 3' end: experimental validation of extended transcript isoforms.

Virginie Moucadel1, Fabrice Lopez, Takeshi Ara

  • 1INSERM ERM206, Université de la Méditerranée, Luminy case 928, Marseille cedex 09, France.

Nucleic Acids Research
|March 7, 2007
PubMed
Summary
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Researchers experimentally validated numerous alternative polyadenylation sites in mouse tissues, confirming their biological relevance. These findings suggest conserved regulatory functions for alternative 3' end transcript variations in mammals.

Area of Science:

  • Molecular Biology
  • Genomics
  • Transcriptomics

Background:

  • High-throughput sequencing reveals extensive 3' end variations in mammalian transcripts.
  • The biological significance of these variations is debated, with potential contributions from transcription or polyadenylation leakage.

Purpose of the Study:

  • To experimentally validate conserved, unannotated tandem polyadenylation (poly(A)) sites in mouse tissues.
  • To confirm the exon structure and transcriptional unit integrity of alternative 3' isoforms with significant size variations.

Main Methods:

  • Selection of tandem poly(A) sites conserved between human and mouse, exhibiting unusual size features and absent in genome databases.
  • Utilized a specialized reverse transcription-polymerase chain reaction (RT-PCR) strategy for individual poly(A) site confirmation.

Related Experiment Videos

  • Employed long-distance RT-PCR to validate exon structure between distant tandem poly(A) sites and stop codons.

    • Main Results:

    • 84 out of 86 tested poly(A) sites from 44 genes were experimentally confirmed.
    • Long transcripts spanning large distances (up to 4.5 kb or more) between poly(A) sites or stop codons and poly(A) sites were detected.
    • Confirmation that these distant tandem sites belong to the same transcription unit.

    Conclusions:

    • The experimental validation supports the biological relevance of numerous alternative polyadenylation sites.
    • Conserved alternative 3' ends suggest potential roles in gene regulation, influenced by transcription start site location.