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Review
, 13 (5), 625-42

Cis-acting Determinants of Asymmetric, Cytoplasmic RNA Transport

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Review

Cis-acting Determinants of Asymmetric, Cytoplasmic RNA Transport

Ashwini Jambhekar et al. RNA.

Abstract

Asymmetric subcellular distribution of RNA is used by many organisms to establish cell polarity, differences in cell fate, or to sequester protein activity. Accurate localization of RNA requires specific sequence and/or structural elements in the localized RNA, as well as proteins that recognize these elements and link the RNA to the appropriate molecular motors. Recent advances in biochemistry, molecular biology, and cell imaging have enabled the identification of many RNA localization elements, or "zipcodes," from a variety of systems. This review focuses on the mechanisms by which various zipcodes direct RNA transport and on the known sequence/structural requirements for their recognition by transport complexes. Computational and experimental methods for predicting and identifying zipcodes are also discussed.

Figures

FIGURE 1.
FIGURE 1.
Schematics of zipcode identification strategies. (A) in vivo GFP-RNA tagging (after Bertrand et al. 1998). The RNA of interest is expressed fused to a reporter containing six tandem repeats of the MS2 stem–loop sequence. An MS2-coat protein–GFP fusion is coexpressed, resulting in a GFP-marked RNA. (B) Molecular beacons (after Bratu et al. 2003). A hairpin oligonucleotide containing sequences complementary to the target RNA is conjugated to a fluorophore at one end and a quencher at the other. In the hairpin conformation, the fluorophore is quenched. Upon injection of the molecular beacon into cells and subsequent hybridization to the target RNA, the quencher is removed from the proximity of the fluorophore and the RNA can be tracked based on a signal from the fluorophore. (C) CLIP (after Ule et al. 2005). RNA–protein complexes are cross-linked with UV, isolated, and treated with Rnase. Protein-bound RNA regions are protected from digestion. A linker is ligated to the 3′ end, and intact complexes are isolated by immunoprecipitation of the RNA-binding protein, followed by SDS-PAGE purification. The protein is then removed by digestion. A second linker is ligated to the 5′ end and the isolated RNA is amplified by reverse transcription and PCR.

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