Nucleocytoplasmic Shuttling of the Splicing Factor SIPP1

Abstract

SIPP1 (splicing factor that interacts with PQBP1 and PP1) is a widely expressed protein of 70 kDa that has been implicated in pre-mRNA splicing. It interacts with protein Ser/Thr phosphatase-1 (PP1) and with the polyglutamine-tract-binding protein 1 (PQBP1), which contributes to the pathogenesis of X-linked mental retardation and neurodegenerative diseases caused by polyglutamine tract expansions. We show here that SIPP1 is a nucleocytoplasmic shuttling protein. Under basal circumstances SIPP1 was largely nuclear, but it accumulated in the cytoplasm following UVor X-radiation. Nuclear import was mediated by two nuclear localization signals. In addition, SIPP1 could be piggy-back transported to the nucleus with its ligand PQBP1. In the nucleus SIPP1 and PQBP1 formed inclusion bodies similar to those detected in polyglutamine diseases. SIPP1 did not function as a nuclear targeting subunit of PP1 but re-localized nuclear PP1 to storage sites for splicing factors. The C-terminal residues of SIPP1, which do not conform to a classic nuclear export signal, were required for its nuclear export via the CMR-1 pathway. Finally, SIPP1 activated pre-mRNA splicing in intact cells, and the extent of splicing activation correlated with the nuclear concentration of SIPP1. We conclude that SIPP1 is a positive regulator of pre-mRNA splicing that is regulated by nucleocytoplasmic shuttling. These findings also have potential implications for a better understanding of the pathogenesis of X-linked mental retardation and polyglutamine-linked neurodegenerative disorders. SIPP1,2 also known as SNP70 or Npw38BP, is a polypeptide of 70 kDa that appears to be expressed in all metazoa and higher plants (1-3). It is a largely nuclear protein but has also been reported to be bound to vimentin-containing intermediate filaments in the cytoplasm (2, 3). Within the nucleus SIPP1 is associated with both the speckles (2, 3), which represent storage/assembly sites for splicing factors, and the spliceosomes (3-6), the nuclear protein-RNA complexes that catalyze pre-mRNA splicing. In further agreement with a role in pre-mRNA splicing, SIPP1 was shown to interact directly with single-stranded RNA (2), the polyglutamine-tract-binding protein PQBP1/Npw38 (1), and protein phosphatase-1 (3), all established components of the spliceosomes (Fig. 1A, see also Refs. 6 and 7). RNA binds to the N terminus of SIPP1 (2), the WW-domain of PQBP1 interacts with two proline-rich regions of SIPP1 (1), and the binding of protein phosphatase-1 (PP1) is mediated by two motifs in the central domain of SIPP1 (3). SIPP1 also contains a coiled-coil domain and an Asp-rich domain but the function of these domains is still unknown (1-3).Although the above data strongly suggest that SIPP1 has a role in pre-mRNA splicing, there are no in vivo data to support this contention. A further complication is that the exact function of the protein ligands of SIPP1 in splicing is not yet known and that these ligands have also functions outside pre-mRNA splicing. Indeed, PP1 plays a role in ...

Multiple regions in dengue virus capsid protein contribute to nuclear localization during virus infection

“…Furthermore, recombinant C protein lacking aa 73-100 or 85-100 did not localize to the nucleus of transfected HeLa cells and residues 85-100 were able to mediate nuclear localization of multiple copies of green fluorescent protein (Wang et al, 2002). Alternatively, the C protein may interact directly with components of the nuclear-pore complex, as observed with Stat1 (signal transducer and activator of transcription factor 1; Marg et al, 2004), or 'piggyback' on other cellular proteins that are transported actively to the nucleus, as in the cases of the mouse DNA primase p46 subunit (Mizuno et al, 1996) and the splicing factor SIPP1 (Llorian et al, 2005). Intriguingly, a number of other viral proteins, such as dengue virus NS5 (Brooks et al, 2002), adeno-associated virus capsid (Grieger et al, 2006) andinfluenza A virus NS1 proteins (Melén et al, 2007), also possess more than one NLS, and some of these NLS sequences are utilized in other protein-protein or protein-DNA/RNA interactions.…”

Proline-rich Sequence Recognition

“…NpwBP (25,40) is not stably integrated into the hPrp19-CDC5 complex after formation of the activated complex B (32), and it represents a likely interaction partner of CD2BP2/52K. Based on the literature of the individual proteins, a complex consisting of CD2BP2/52K, U5-15K, NpwBP, and PQBP-1 could be formed (45,46). PRS hubs, proteins with numerous PRD binding sites, are present in several spliceosomal subcomplexes.…”

Y65C Missense Mutation in the WW Domain of the Golabi-Ito-Hall Syndrome Protein PQBP1 Affects Its Binding Activity and Deregulates Pre-mRNA Splicing

“…The buffer used was phosphate-buffered saline (20 mM phosphate, pH 7.4, 5 mM KCl, and 100 mM NaCl). EGFP-WBP11 was obtained as described (20). In parallel, we obtained WBP11 cDNA from Open Biosystems.…”

“…We also chose a peptide from the WBP11 protein (WBP11-PR peptide ϭ PPGPPPRGPPPR (NCBI accession number NM_016312)), which was shown as a cognate partner of PQBP1 in several unbiased protein interaction and functional assays (2, 37, 41, 42). Functionally, WBP11 acts as a nucleocytoplasmic shuttle that regulates pre-mRNA splicing (20). These peptides were synthesized and further analyzed for their binding potential to the WT and Y65C mutant WW domains of PQBP1 using ITC.…”