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Related Experiment Videos

Protein Binding to mRNA 3' Isoforms.

Joseph V Geisberg1, Zarmik Moqtaderi1

  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.

Current Protocols in Molecular Biology
|September 11, 2019
PubMed
Summary

CLIP-READS maps messenger ribonucleoprotein (mRNP) binding to mRNA 3' isoforms genome-wide. This technique combines crosslinking and immunoprecipitation (CLIP) with 3' region extraction and deep sequencing (READS) for precise analysis.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Messenger ribonucleoprotein (mRNP) complexes play crucial roles in gene regulation.
  • Understanding the specific binding of mRNPs to different mRNA isoforms is essential for deciphering gene expression control.
  • Existing methods may lack the resolution to map binding across all mRNA 3' isoforms genome-wide.

Purpose of the Study:

  • To introduce CLIP-READS, a novel technique for mapping genome-wide mRNA 3' isoform binding by specific mRNPs.
  • To enable precise quantification of mRNP interactions with diverse mRNA 3' isoforms.

Main Methods:

  • CLIP-READS integrates crosslinking and immunoprecipitation (CLIP) with 3' region extraction and deep sequencing (READS).
  • Cells undergo UV crosslinking to stabilize RNA-protein interactions.
Keywords:
3′ mRNA isoformCLIPmRNPprotein−mRNA binding

Related Experiment Videos

  • Specific mRNPs are immunoprecipitated, followed by protein digestion and 3' READS library preparation.
  • Parallel processing of input samples allows for relative quantification of isoform-specific binding.

    • Main Results:

    • CLIP-READS successfully generates a genome-wide map of mRNA 3' isoform binding.
    • The technique allows for the relative quantification of mRNP binding to specific mRNA isoforms.
    • This provides a detailed view of mRNP interactions across the transcriptome.

    Conclusions:

    • CLIP-READS is an effective method for mapping mRNP binding to mRNA 3' isoforms genome-wide.
    • This technique advances the study of gene regulation by providing high-resolution insights into mRNP-mRNA interactions.
    • CLIP-READS facilitates a deeper understanding of post-transcriptional gene control mechanisms.