GSBS Student Publications

Title

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

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Graduate School of Biomedical Sciences; Program in Molecular Medicine

Date

8-1-1994

Document Type

Article

Medical Subject Headings

Actins; Animals; Base Sequence; Chick Embryo; Cytoplasm; Fibroblasts; Humans; Immunohistochemistry; In Situ Hybridization; Microscopy, Immunoelectron; Oligonucleotide Probes; Poly A; RNA, Messenger; Transcription, Genetic

Disciplines

Life Sciences | Medicine and Health Sciences

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 alpha-actinin, and the elongation factor, EF-1 alpha (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.

Rights and Permissions

Citation: J Cell Biol. 1994 Aug;126(4):863-76.

Related Resources

Link to article in PubMed

Journal Title

The Journal of cell biology

PubMed ID

7914201