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Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
09:16

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Published on: May 3, 2014

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Analysis of RNA processing reactions using cell free systems: 3' end cleavage of pre-mRNA substrates in vitro

Joseph Jablonski1, Mark Clementz1, Kevin Ryan2

  • 1Department of Infectious Diseases, The Scripps Research Institute.

Journal of Visualized Experiments : Jove
|May 20, 2014
PubMed

Insights

Mammalian mRNA 3' end processing involves specific cleavage of precursor RNA by a protein complex, followed by polyadenylation. This study details an in vitro cleavage assay using radiolabeled RNA substrates to analyze this essential gene expression step.

Area of Science:

  • Molecular Biology
  • Gene Expression
  • RNA Processing

Background:

  • Messenger RNA (mRNA) 3' end formation in mammals is a complex process, not simple transcription termination.
  • RNA polymerase II (RNPII) transcribes past the mature mRNA sequence, requiring post-transcriptional processing.
  • Specific endonuclease activity is crucial for cleaving precursor mRNA at precise sites.

Purpose of the Study:

  • To detail an in vitro assay for studying mRNA 3' end cleavage.
  • To elucidate the mechanism of precursor RNA cleavage and subsequent polyadenylation.
  • To provide a method for analyzing 3' end processing, exemplified by HIV-1 mRNA.

Main Methods:

  • Utilizing cellular nuclear extracts containing the mRNA cleavage complex.
  • Employing specific, radiolabeled precursor RNA substrates for in vitro assays.
  • Assessing cleavage efficiency via gel electrophoresis and autoradiography to detect 5' cleaved products.

Main Results:

  • Demonstrated the feasibility of studying 3' end processing in vitro.
  • Quantified cleavage by detecting and measuring the shorter 5' product.
  • Established a detailed protocol using HIV-1 mRNA processing as a model.

Conclusions:

  • The 3' end processing of mRNA is an active, multi-step process involving specific protein complexes.
  • The described cleavage assay provides a robust method for investigating mRNA 3' end formation.
  • This assay is applicable to studying the processing of various RNA transcripts, including viral mRNAs.

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