Nuclear export assays for poly(A) RNAs.
Nuclear export of mRNAs is a central step in eukaryotic gene expression. A defect in bulk poly(A) RNA export can be caused either by a direct disruption of the mRNA export machinery or by an indirect effect on mRNA biogenesis. One example of interference with the mRNA export pathway is viral-host interactions involving mRNA export factors. VSV M protein binds the mRNA export factor Rae1 that is in complex with Nup98, resulting in nuclear retention of mRNAs. To study regulation of mRNA export, we review here two useful methodologies, which include a reporter gene assay and oligo(dT) in situ hybridization. In a reporter gene assay one can assess up-regulation or down-regulation of gene expression that can occur at different levels, including transcription, mRNA processing, mRNA export, and translation. An effect on mRNA export can then be identified by determining the intracellular distribution of poly(A) RNA using oligo(dT) in situ hybridization. Reporter gene assays are quick, relatively simple and can thus be used in primary highthroughput screenings. To further pinpoint disruption of mRNA export, oligo(dT) in situ hybridization can be used. Since it is a more laborious methodology it is more suitable for a secondary screening. We also review here a combination of oligo(dT) in situ hybridization with immunofluorescence for simultaneous localization of endogenous or ectopically expressed proteins. Altogether, these assays are valuable tools for identifying major regulatory effects on mRNA nuclear export.
Chakraborty
Papia
P
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039, USA.
Satterly
Neal
N
Fontoura
Beatriz M A
BM
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D001681:Biological Assay; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology
D028861:Nuclear Pore Complex Proteins; D012333:RNA, Messenger; C093350:nuclear pore complex protein 98; D011061:Poly A; D008156:Luciferases