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In JoVE (2)
- Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
- Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Other Publications (2)
Articles by Xianying A. Cui in JoVE
JoVE General
Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
Department of Biochemistry, University of Toronto
Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.
JoVE General
Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Department of Biochemistry, University of Toronto
Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.
Other articles by Xianying A. Cui on PubMed
Molecular Characterization of Chinese Hamster Cells Mutants Affected in Adenosine Kinase and Showing Novel Genetic and Biochemical Characteristics
Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms.
P180 Promotes the Ribosome-independent Localization of a Subset of MRNA to the Endoplasmic Reticulum
In metazoans, the majority of mRNAs coding for secreted and membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation. Using a mass spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism, including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 over-expression can enhance the association of generic mRNAs with the ER. We then show that p180 contains a lysine-rich region that can directly interact with RNA in vitro. Finally, we demonstrate that p180 is required for the efficient ER-anchoring of bulk poly(A) and of certain transcripts, such as placental alkaline phosphatase and calreticulin, to the ER. In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.
