Biogenesis of small nuclear ribonucloceoproteins (snRNPs) is biphasic. Small nuclear RNAs (snRNAs) are exported to the cytoplasm for assembly into pre-snRNPs where they are hypermethylated, forming a trimethylguanosine (TMG) cap, and then transported back into the nucleus via the import adaptor, snurportin1 (SPN) and the import receptor importin-β. I have identified CG42303 as dSNUP, the Drosophila orthologue of human SPN (hSPN). Strikingly, the importin-β binding (IBB) domain, which is essential for SPN-mediated snRNP import in humans, is not conserved in dSNUP. Consistent with the lack of an IBB, dSNUP did not interact with the Drosophila importin-β orthologue, Ketel. Despite this fact, dSNUP localized to the nucleus, indicating that there is an alternative dSNUP import pathway or that dSNUP is imported indirectly through importin-β bound snRNPs. I excluded the latter possibility since, in contrast to human cells, snRNPs did not associate with importin-β in Drosophila cells. Previous results suggested that hSPN interacts indirectly with a known import receptor, importin-7. I tested the possibility that the Drosophila orthologue of importin-7, known as Moleskin (Msk), interacts with dSNUP and snRNPs. I discovered that Msk physically associates with both dSNUP and U snRNPs, while snRNP components failed to bind importin-β. Furthermore, Msk null mutant larvae had a significant in vivo reduction of the snRNP component survival motor neuron (SMN), and the snRNP specific nuclear Cajal body marker coilin. Additionally, Msk null mutants exhibited cytoplasmic accumulation of TMG cap signal in the Malpighian tubules, indicating that the import of TMG capped snRNAs is inhibited in the absence of Msk. The reduction of SMN protein was dramatic enough to be detected by western blotting, suggesting a vital role for Msk in the stability of SMN. Interestingly, Msk also localized to snRNP specific nuclear Cajal bodies. In sum, these data indicate that importin-β does not play a role in snRNP import in Drosophila and implicate a crucial function for Msk in fruit fly snRNP biogenesis. Future experiments will be needed to determine the precise function of importin-7/Moleskin in both fruit fly and human snRNP biogenesis.Doctor of Philosoph
