MECHANISM OF ACTIN MRNA LOCALIZATION

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.