Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts.

• Type of Material: Article
• Authors: Gary Bassell, Emory University; Christina M. Powers, University of Massachusetts; Krishan L. Taneja, University of Massachusetts; Robert H. Singer, University of Massachusetts
• Language: English
• Date: 1994-08-01
• Publisher: Rockefeller University Press
• Publication Version: Final Published Version
• Copyright Statement: © 1994 Rockefeller University Press
• Final Published Version (URL): http://dx.doi.org/10.1083/jcb.126.4.863
• Title of Journal or Parent Work: Journal of Cell Biology
• ISSN: 0021-9525
• Volume: 126
• Issue: 4
• Start Page: 863
• End Page: 876
• Keywords: POLY(A) RNA; HELA-CELLS; INSITU HYBRIDIZATION; Cell Biology; LEUKEMIA L5178Y CELLS; BINDING-PROTEIN; CELL BIOLOGY; PROTEIN-SYNTHESIS; MESSENGER-RNA; Life Sciences & Biomedicine; Science & Technology; NUCLEAR MATRIX; ELECTRON-MICROSCOPIC EVIDENCE; CYTOSKELETAL FRAMEWORK
• Research Categories: Biology, Cell; Biology, Molecular; Health Sciences, General

Abstract

Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize 'individual' poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (<10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and α-actinin, and the elongation factor, EF-1α (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a 'microdomain' where mRNA is attached and functionally expressed.