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  • Three dimensional conformational changes in the

    2019-08-13

    Three-dimensional conformational changes in the cargo protein’s NES, caused by protein phosphorylation, dephosphorylation, or mutation, can regulate CRM1 binding (Craig et al., 2002, Vogt et al., 2005). Additional protein modifications such as sumoylation, ubiquitination, acetylation, and/or the binding of protein-specific co-factors can also influence the affinity of the NES for CRM1 binding (Turner et al., 2012). When CRM1 is bound, it forms a trimer with RanGTP in the nucleus, and exits through the nuclear pore complex into the brincidofovir (Fig. 1A and B). The binding of CRM1 to either RanGTP or to the NES of a cargo protein itself is weak. However, when both RanGTP and the cargo bind to CRM1 simultaneously, affinity to both cargo and Ran GTP is strengthened by 500- to 1000-fold (Dong et al., 2009a, Dong et al., 2009b, Monecke et al., 2009). In the cytoplasm, the hydrolysis of GTP to GDP by the RanGTP-activating protein promotes the release of the substrate from CRM1 (Kau et al., 2004) (Fig. 1C). Crystallography has illustrated that this increase in CRM1 affinity is caused by a change in the global conformation of the protein. This change in conformation occurs only when both the cargo substrate and RanGTP bind to CRM1 (Dong et al., 2009a, Dong et al., 2009b, Monecke et al., 2009). Ran-GTP and CRM1 are subsequently returned/recycled through the nuclear pore complex back into the nucleus (Turner et al., 2012) (Fig. 1D). Accumulating evidence suggests that CRM1 plays an important role in ribosomal biogenesis. Ribosomal biogenesis involves multiple, coordinated steps including the synthesis and processing of ribosomal RNA (rRNA), the synthesis of ribosomal proteins and their import into the nucleus, the assembly of ribosomal subunits, and the transport of the mature 40S (small subunit) and 60S (large subunit) subunits into the cytoplasm. The first step in rRNA biogenesis is transcription of 47S rRNA precursor by RNA polymerase I, followed by extensive processing, modification and maturation to 18S and 28S rRNAs (Fatica and Tollervey, 2002, Granneman and Baserga, 2005). During synthesis, the rRNAs are assembled into 90S ribosome precursors and processed further to 40S and 60S pre-ribosomes (Granneman & Baserga, 2005). Nuclear export of the 40S and 60S ribosomal subunits is dependent on CRM1 in eukaryotic cells, and mediated by the binding of the 60S ribosomal export protein NMD3 adaptor protein in RanGTP-dependent manner (Thomas & Kutay, 2003) (Fig. 1F). The CRM1-NMD3 complex has been detected in nucleoli, which are membraneless nuclear structures involved in ribosome biogenesis (Bai et al., 2013). Nucleolar localization of the CRM1-NMD3 complex is induced by inhibition of RNA polymerase I transcription with actinomycin D or by inhibiting RNA polymerase catalytic activity with siRNA (Bai et al., 2013). On the other hand, leptomycin B, thus CRM1 inhibition, blocked the processing of 28S rRNA. Furthermore, synthesis of pre-47S rRNA was inhibited by the depletion of NMD3 and inhibition of CRM1 (Bai et al., 2013). However, nucleolar disintegration, a hallmark of RNA polymerase I transcriptional stress (Raska et al., 2004) and pyrimidine depletion (Linke et al., 1996, Khutornenko et al., 2010), was not observed by inhibition of NMD3 and CRM1. This would suggest that the CRM1-NMD3 complex functions in pathways of rRNA synthesis and processing, probably by adjusting the rRNA synthesis rate through rRNA processing and export, not by regulating transcription. It has also been reported that CRM1, as well as other nuclear import receptors like importin-7, is regulated positively by MYC and negatively by p53 (Golomb et al., 2012), resulting in the modulation of ribosomal biogenesis. This is not surprising, since MYC is a known master regulator of ribosome biogenesis. As such, it can coordinate protein synthesis through the transcriptional control of RNA and the protein components of ribosomes, the gene products required for the processing of ribosomal RNA, the nuclear export of ribosomal subunits, and the initiation of mRNA translation (van Riggelen et al., 2010). MYC seems to transcriptionally upregulate XPO1 as part of a broad range of transcriptional program that also includes numerous ribosomal protein genes (Wu et al., 2008). The burden on the nuclear transport machinery is supposed to be increased once ribosomal protein levels become higher by MYC, so the dual induction of importin-7 and CRM1 presumably plays a pivotal role for a cell to overcome this burden.