Abstract
Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.
Highlights
Serine–arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation
We designed a CLK1 construct containing a C-terminal RFP (CLK1-RFP) for direct fluorescence monitoring. We found that this construct localized exclusively to the nucleus of live HeLa cells, whereas one lacking the N terminus (CLK1(⌬N)-RFP) was present in both the cytoplasm and nucleus (Fig. 1B)
We mutated the charged residues to alanine in the full-length kinase and found that the RFP-tagged CLK1(⌬NLS3) localized to the nucleus of live HeLa cells, similar to the WT enzyme (Fig. 1E). These findings show that the N terminus does not have a short, classical NLS for CLK1 nuclear localization
Summary
To characterize residues that might constitute a classical NLS, we initially identified two monopartite sequences (NLS1 and NLS2) in the CLK1 N terminus using cNLS Mapper (Fig. 1A). We found that this construct localized exclusively to the nucleus of live HeLa cells, whereas one lacking the N terminus (CLK1(⌬N)-RFP) was present in both the cytoplasm and nucleus (Fig. 1B) We quantified these results in fractionation experiments showing that CLK1(⌬N)-RFP partitions 58% in the nucleus, whereas the fulllength CLK1 is 95% nuclear (Fig. 1C). We mutated the charged residues to alanine in the full-length kinase and found that the RFP-tagged CLK1(⌬NLS3) localized to the nucleus of live HeLa cells, similar to the WT enzyme (Fig. 1E). Overall, these findings show that the N terminus does not have a short, classical NLS for CLK1 nuclear localization
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