UMMS Affiliation

Department of Cell Biology

Publication Date

10-1-1993

Document Type

Article

Subjects

Actins; Base Sequence; *Cell Compartmentation; Cells, Cultured; Cytoplasm; Fibroblasts; Genes, Reporter; Genetic Vectors; In Situ Hybridization; Molecular Sequence Data; Muscles; Oligonucleotide Probes; RNA, Messenger; *Regulatory Sequences, Nucleic Acid; Transfection; beta-Galactosidase

Disciplines

Cell Biology | Genetics and Genomics

Abstract

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm.

Rights and Permissions

Citation: J Cell Biol. 1993 Oct;123(1):165-72.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of cell biology

PubMed ID

8408195

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