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Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
11:32

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Published on: December 4, 2010

Nuclear export of metazoan replication-dependent histone mRNAs is dependent on RNA length and is mediated by TAP

Judith A Erkmann1, Ricardo Sànchez, Nathalie Treichel

  • 1Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

RNA (New York, N.Y.)
|December 22, 2004
PubMed

Insights

Histone mRNAs, unique in lacking polyadenylation, are efficiently exported from the nucleus. This process relies on the export receptor TAP, not the stem-loop binding protein (SLBP).

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Replication-dependent histone mRNAs are unique metazoan transcripts lacking polyadenylation, terminating in a conserved stem-loop.
  • Histone pre-mRNAs undergo nuclear processing via a single cleavage step to generate mature 3' ends.

Purpose of the Study:

  • To systematically investigate the molecular requirements for nuclear export of mouse histone mRNA.
  • To determine the roles of 3' end processing, stem-loop binding protein (SLBP), and export receptor TAP in histone mRNA export.

Main Methods:

  • Utilized the Xenopus oocyte system for microinjection and export assays of mature and mutated histone mRNAs.
  • Employed competition assays with constitutive transport element (CTE) RNA to probe involvement of export receptors.
  • Performed RNA interference (RNAi) in Drosophila cells to deplete TAP and assess its effect on histone mRNA localization.

Main Results:

  • Mature histone mRNAs were efficiently exported from oocytes, indicating 3' end cleavage is not essential for export factor binding.
  • Histone mRNA export was independent of the stem-loop binding protein (SLBP), as mutations preventing SLBP binding did not impede export.
  • Export efficiency correlated with the length of the region upstream of the stem-loop, not its sequence.
  • Competition with CTE RNA blocked histone mRNA export, implicating the export receptor TAP.
  • TAP depletion in Drosophila cells resulted in nuclear retention of mature histone mRNAs.

Conclusions:

  • Nuclear export of histone mRNAs is independent of SLBP and 3' end cleavage.
  • The mRNA export receptor TAP plays a crucial role in the nuclear export of histone mRNAs.
  • The length of the upstream region, rather than its specific sequence, influences histone mRNA export efficiency.

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