MECHANISM OF ACTIN MRNA LOCALIZATION

Project: Research project

Project Details

Description

The objective of this work is to investigate the mechanism of actin mRNA
localization. We have shown that actin MRNA is concentrated in the
leading edge of motile myoblasts. This sequestering of the message is
independent of protein synthesis, so that the nucleic acid contains cis-
regulatory elements controlling localization. The MRNA must be
transported from the site of exit from the nucleus to the distal part of
the cell, and anchored there. Both of these processes require actin
filaments. We have fused the actin MRNA to beta-galactosidase and have
been able to localize the heterologous message, not ordinarily localized.
We wish now to isolate the sequences responsible for the localization of
the fusion message and dissect its fine structure using deletional
analysis and point mutations. There must be a protein or proteins which
recognize this site and transports and/or anchors the message within the
cell. We propose to isolate and characterize this protein(s) using RNA
affinity columns, gel retardation analysis and UV crosslinking combined
with RNAse protection. We then will purify immunogenic quantities for
antibody production and use these antibodies, or microsequencing, to
clone the protein. Possibly this protein will interact with or be, an
actin binding protein. These reagents can then be used to disrupt the
localization of the endogenous actin MRNA to investigate its function.
Differentiating systems such as muscle may use localization sequences on
isoforms of messages such as actin, myosin, tropomyosin, etc. to organize
macromolecular complexes such as the sarcomere by assembling the proteins
close to their sites of synthesis. Stable transfectants of the myogenic
quail cell line, QM7, which has no endogenous alpha-actin will be used to
assess the sorting of beta- and alpha-actin messages within a common
cytoplasm.
StatusFinished
Effective start/end date3/3/922/28/93

Funding

  • National Institute of Arthritis and Musculoskeletal and Skin Diseases

ASJC

  • Genetics
  • Molecular Biology
  • Biophysics
  • Structural Biology
  • Cell Biology

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