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Updated: Jul 17, 2026

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

Subnuclear localization and dynamics of the Pre-mRNA 3' end processing factor mammalian cleavage factor I 68-kDa

Stefano Cardinale1, Barbara Cisterna, Paolo Bonetti

  • 1Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milan, Italy.

Molecular Biology of the Cell
|February 3, 2007
PubMed

Insights

Mammalian cleavage factor I (CF Im) is crucial for pre-mRNA 3' end processing. This study reveals CF Im68 dynamically localizes within the nucleus, suggesting paraspeckles are functional sites for RNA metabolism.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Mammalian cleavage factor I (CF Im) plays a vital role in pre-mRNA 3' end processing.
  • Understanding the subnuclear localization and dynamics of CF Im is essential for elucidating its function.

Purpose of the Study:

  • To characterize the subnuclear distribution and mobility of CF Im68.
  • To investigate the relationship between CF Im68, transcription, and RNA processing.
  • To determine the dynamic nature of paraspeckles in relation to CF Im68.

Main Methods:

  • Fluorescence microscopy to visualize CF Im68 localization.
  • Analysis of synchronized cells to observe cell cycle-dependent distribution.
  • Ultrastructural analysis and colocalization studies with markers of transcription and splicing (bromouridine, RNA polymerase II, SC35).
  • Fluorescence recovery after photobleaching (FRAP) to assess mobility.

Main Results:

  • CF Im68 localizes to paraspeckles and partially overlaps with nuclear speckles, with cell cycle-dependent variations.
  • CF Im68 associates with perichromatin fibrils (active transcription sites) and interchromatin granules-associated zones.
  • CF Im68 colocalizes with bromouridine, RNA polymerase II, and SC35.
  • CF Im68 association with perichromatin fibrils is transcription-dependent, but it remains in interchromatin granules-associated zones.
  • FRAP analysis shows dynamic movement of CF Im68 within paraspeckles.

Conclusions:

  • 3' end processing, like splicing, likely occurs cotranscriptionally.
  • Paraspeckles represent a dynamic and functional nuclear compartment involved in RNA metabolism.

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