Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract.

Mild heat treatment of HeLa cell nuclear extracts (NE) selectively inhibits pre-mRNA splicing. Heat-inactivated extracts can be complemented by a small amount of untreated NE. Utilizing this complementation assay and a combination of ion-exchange, affinity, and hydrophobic chromatography, a heat reversal factor (HRF) was purified from NE that is required to rescue pre-mRNA splicing from a heat-inactivated extract. This activity in its most purified form consistently copurified in a fraction containing two 70-kDa proteins and a minor polypeptide of approximately 100 kDa. It was free of the major small nuclear RNAs, sensitive to protease, and required to rescue spliceosome formation from a heat-inactivated nuclear extract. These results suggest that this factor is a protein that may be an important component in pre-mRNA splicing, or alternatively, it may be involved in renaturation of a heat-sensitive splicing factor.     

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.     

Nucleocytoplasmic shuttling of the splicing factor SIPP1

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 UV- or 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.