Using Retroviruses To Study the Nuclear Export of mRNA

Abstract

Several non-exclusive approaches can be taken to identify and then characterize cellular proteins involved in the nucleocytoplasmic transport of protein or RNA molecules. One approach that has been extremely useful is the genetic identification of factors required for the nucleocytoplasmic transport of a particular macromolecule, e.g. nuclear export of poly(A)⁺ RNA, by screening yeast conditional mutants for, in this case, nuclear accumulation of poly(A)⁺ RNA at the restrictive temperature. While this kind of approach has led to the identification of gene products that play a direct or indirect role in several transport pathways, including mRNA export (Amberg et al. 1992; Kadowaki et al. 1994), it can be difficult to subsequently discern the biochemical role played by a particular genetically identified gene product. An alternative strategy is to use protein binding affinity, either in vitro or using the yeast two-hybrid protein:protein interaction assay, to identify factors that specifically bind to a relevant protein target. For example, the G-protein Ran, in its GTP-βound form, is a critical cofactor for nuclear transport mediated by all members of the extensive importin-β/karyopherin-β family of transport factors (Melchior et al. 1993; Moore and Blobel 1993; Izaurralde et al. 1997). The identification of human proteins that can bind to the GTP-, but not GDP-, bound form of Ran in vitro has therefore facilitated the identification of several nuclear transport factors, including CAS and exportin t (Kutay et al. 1997, 1998). Conversely, use of the two-hybrid assay allowed the initial identification of transportin/ karyopherin-β2, the import factor specific for the unusual type of nuclear localization signal (NLS) present in the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1; Pollard et al. 1996; Fridell et al. 1997).