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Functional association of the Microprocessor complex with the spliceosome

Naoyuki Kataoka1, Megumi Fujita, Mutsuhito Ohno

  • 1Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. kataoka.mtt@mri.tmd.ac.jp

Molecular and Cellular Biology
|April 8, 2009
PubMed

Insights

Most human microRNAs (miRNAs) originate within introns. A new in vitro system reveals Drosha-mediated miRNA cropping can precede spliceosome-mediated splicing and trans-splicing of intronic sequences.

Area of Science:

  • Molecular Biology
  • Genetics
  • RNA Biology

Background:

  • The majority of human microRNAs (miRNAs) are encoded within introns of protein-coding genes.
  • A previous model proposed that Drosha processes pre-miRNAs from introns, followed by trans-splicing of the remaining intronic fragments.
  • The molecular mechanisms underlying this intronic miRNA processing pathway remained unelucidated.

Purpose of the Study:

  • To investigate the molecular mechanism of intronic microRNA (miRNA) processing.
  • To elucidate the relationship between pre-miRNA processing and messenger RNA (mRNA) splicing.
  • To provide molecular evidence for a pathway involving Drosha, the spliceosome, and trans-splicing of intronic miRNAs.

Main Methods:

  • Development of an in vitro system to simultaneously detect pre-miRNA processing and mRNA splicing.
  • Glycerol gradient sedimentation to assess co-fractionation of pre-miRNA with the spliceosome.
  • Co-immunoprecipitation with an anti-Drosha antibody to identify associated splicing intermediates.

Main Results:

  • Pre-miRNA cropping from pre-mRNA was found to occur kinetically faster than splicing.
  • A portion of pre-miRNA co-fractionated with the spliceosome.
  • Drosha was shown to associate with pre-miRNA cropping products, a Y-shaped branch intron, and a Y-shaped splicing intermediate.

Conclusions:

  • The Microprocessor complex (including Drosha) associates with the spliceosome.
  • Pre-miRNA cropping precedes spliceosome-mediated splicing and subsequent trans-splicing of intronic fragments.
  • These findings provide a molecular basis for a novel pathway of intronic miRNA biogenesis.

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