Related Experiment Videos
Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament
G J Bassell1, C M Powers, K L Taneja
1Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655-0106.
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.
Insights
Messenger RNA (mRNA) associates with cellular cytoskeleton filaments, primarily F-actin networks. This interaction suggests cytoskeletal intersections act as mRNA receptors for localized gene expression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Messenger RNA (mRNA) is known to associate with the cellular cytoskeleton.
- This association is a potential mechanism for mRNA sorting and spatial regulation of gene expression.
- Precise structural interactions between mRNA and the cytoskeleton remain undefined.
Purpose of the Study:
- To visualize individual poly(A) mRNA molecules in situ using electron microscopy.
- To determine the relationship between mRNA and individual cytoskeletal filaments.
- To identify specific cytoskeletal components involved in mRNA localization.
Main Methods:
- Simultaneous visualization of poly(A) RNA and filaments using antibodies.
- Electron microscopy of human fibroblasts.
- Identification of mRNA colocalization with actin, vimentin, microtubules, and associated proteins.
- In situ cDNA synthesis using a labeled oligo dT probe and reverse transcriptase to visualize individual mRNA molecules.
Main Results:
- 72% of poly(A) mRNA localized within 5 nm of F-actin filaments.
- Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (<10%).
- mRNA localization predominantly occurred at cytoskeletal filament intersections.
- mRNA colocalized with actin crosslinking proteins (filamin, alpha-actinin) and elongation factor EF-1 alpha.
- Evidence confirmed single mRNA molecules at filament intersections.
Conclusions:
- Cytoskeletal intersections serve as receptors for mRNA.
- These intersections function as microdomains for mRNA attachment and functional expression.
- The actin cytoskeleton is a major component involved in mRNA localization and regulation.