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dc.contributor.authorKislauskis, Edward H.
dc.contributor.authorLi, Zhifang
dc.contributor.authorSinger, Robert H.
dc.contributor.authorTaneja, Krishan L.
dc.date2022-08-11T08:10:05.000
dc.date.accessioned2022-08-23T16:55:01Z
dc.date.available2022-08-23T16:55:01Z
dc.date.issued1993-10-01
dc.date.submitted2008-10-31
dc.identifier.citationJ Cell Biol. 1993 Oct;123(1):165-72.
dc.identifier.issn0021-9525 (Print)
dc.identifier.pmid8408195
dc.identifier.urihttp://hdl.handle.net/20.500.14038/42626
dc.description.abstractWe 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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=8408195&dopt=Abstract">Link to Article in PubMed</a>
dc.subjectActins
dc.subjectBase Sequence
dc.subject*Cell Compartmentation
dc.subjectCells, Cultured
dc.subjectCytoplasm
dc.subjectFibroblasts
dc.subjectGenes, Reporter
dc.subjectGenetic Vectors
dc.subjectIn Situ Hybridization
dc.subjectMolecular Sequence Data
dc.subjectMuscles
dc.subjectOligonucleotide Probes
dc.subjectRNA, Messenger
dc.subject*Regulatory Sequences, Nucleic Acid
dc.subjectTransfection
dc.subjectbeta-Galactosidase
dc.subjectCell Biology
dc.subjectGenetics and Genomics
dc.titleIsoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments
dc.typeJournal Article
dc.source.journaltitleThe Journal of cell biology
dc.source.volume123
dc.source.issue1
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1956&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/957
dc.identifier.contextkey659138
refterms.dateFOA2022-08-23T16:55:01Z
html.description.abstract<p>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.</p>
dc.identifier.submissionpathoapubs/957
dc.contributor.departmentDepartment of Cell Biology
dc.source.pages165-72


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