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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

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Feed-forward microprocessing and splicing activities at a microRNA-containing intron

Maja M Janas1, Mehdi Khaled, Steffen Schubert

  • 1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

Plos Genetics
|October 27, 2011
PubMed

Insights

The study reveals that microRNA (miRNA) processing and messenger RNA (mRNA) splicing are linked. Spliceosome assembly on an intron helps process the intronic miRNA, demonstrating a cooperative mechanism.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Most mammalian microRNA (miRNA) genes are located within introns of other genes.
  • The mechanisms coordinating the processing of primary transcripts into mature miRNAs and spliced mRNAs are not well understood.

Purpose of the Study:

  • To investigate the interplay between microRNA processing and mRNA splicing at an intronic miRNA locus.
  • To elucidate the molecular mechanisms coordinating the biogenesis of intronic microRNAs and the splicing of host gene transcripts.

Main Methods:

  • Analysis of miR-211 processing and melastatin splicing in relation to Drosha activity.
  • Site-directed mutagenesis of intron 6 splice sites, branch point, and polypyrimidine tract.
  • Knockdown of U1 splicing factors to assess global impact on intronic miRNA expression.

Main Results:

  • Microprocessing of intronic miR-211 promotes the splicing of the melastatin exon 6-exon 7 junction, requiring Drosha RNase III activity.
  • Mutations in the 5' splice site (5'SS) of intron 6 impaired miR-211 biogenesis and Drosha recruitment, indicating 5'SS recognition is crucial.
  • Knockdown of U1 splicing factors led to reduced intronic miRNA expression, supporting a global role for splicing factors.

Conclusions:

  • Demonstrates a novel, mutually cooperative relationship between microprocessing of intronic miRNAs and mRNA splicing.
  • Suggests that the initiation of spliceosome assembly on an intron may promote the microprocessing of intronic miRNAs.

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