Abstract
ABSTRACTGemin4 is a member of the Survival Motor Neuron (SMN) protein complex, which is responsible for the assembly and maturation of Sm-class small nuclear ribonucleoproteins (snRNPs). In metazoa, Sm snRNPs are assembled in the cytoplasm and subsequently imported into the nucleus. We previously showed that the SMN complex is required for snRNP import in vitro, although it remains unclear which specific components direct this process. Here, we report that Gemin4 overexpression drives SMN and the other Gemin proteins from the cytoplasm into the nucleus. Moreover, it disrupts the subnuclear localization of the Cajal body marker protein, coilin, in a dose-dependent manner. We identified three putative nuclear localization signal (NLS) motifs within Gemin4, one of which is necessary and sufficient to direct nuclear import. Overexpression of Gemin4 constructs lacking this NLS sequestered Gemin3 and, to a lesser extent Gemin2, in the cytoplasm but had little effect on the nuclear accumulation of SMN. We also investigated the effects of Gemin4 depletion in the laboratory mouse, Mus musculus. Gemin4 null mice die early in embryonic development, demonstrating that Gemin4 is an essential mammalian protein. When crossed onto a severe SMA mutant background, heterozygous loss of Gemin4 failed to modify the early postnatal mortality phenotype of SMA type I (Smn−/−;SMN2+/+) mice. We conclude that Gemin4 plays an essential role in mammalian snRNP biogenesis, and may facilitate import of the SMN complex (or subunits thereof) into the nucleus.
Highlights
Pre-mRNA splicing is a central feature of the eukaryotic gene expression program
Mislocalization of the tagged constructs was not due to the presence of the tag itself, as a construct containing a deletion of the nuclear localization signal (NLS) (GFP-Gemin4ΔNLS2; Fig. 2) was completely excluded from the nucleus, with the exception of a very small number of cells that had distinct myc-Gemin4ΔNLS2 nuclear foci that colocalized with Survival Motor Neuron (SMN) (Fig. 6A)
This rare targeting to Cajal bodies suggests that a small fraction of myc-Gemin4ΔNLS2
Summary
Pre-mRNA splicing is a central feature of the eukaryotic gene expression program. The removal of intronic sequences from premRNAs is catalyzed by a macromolecular machine called the spliceosome. Key components of spliceosomes include the small. Received 3 January 2018; Accepted 5 January 2018 nuclear ribonucleoproteins (snRNPs). Each of these snRNPs contains a common set of seven RNA binding factors, called Sm proteins, that forms a heptameric ring around the snRNA, known as the Sm core. Biogenesis of the Sm core is carried out by another macromolecular assemblage called the Survival Motor Neuron (SMN) complex, consisting of at least nine proteins (Gemins 2-8, unrip and SMN) (reviewed in Battle et al, 2006a; Matera et al, 2007; Matera and Wang, 2014)
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