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Abstract. We have identified a protein named pinin that is associated with the mature desmosomes of the epithelia (Ouyang, P., and S.P. Sugrue. 1992. J. Cell

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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.In eukaryotic cells, the step to remove the intron sequences from the pre-mRNA is carried out by the spliceosome, a macromolecular complex consisting of small nuclear ribonucleoproteins (snRNPs) and a number of protein factors (22). A family of Ser/Arg-dipeptide-rich proteins (SR proteins) play essential roles in constitutive splicing and/or can modulate alternative splice site selection (13). Atypical SR protein RNPS1 was previously characterized with a splicing activity that promotes utilization of distal alternative 3Ј splice sites (32). However, recombinant RNPS1 instead synergizes with prototypical SR proteins to activate both constitutive and alternative pre-mRNA splicing, suggesting the role of RNPS1 as a general splicing activator (32). On the other hand, RNPS1 associates with SAP18 and acinus proteins to form the apoptosis and splicing-associated protein (ASAP) complex, which inhibits in vitro splicing and promotes apoptosis (42). It appears that RNPS1 functions to activate or suppress splicing by forming complexes with different regulatory proteins.During the pre-mRNA splicing process, a multiprotein complex is deposited on the spliced mRNP (24,25). This complex occupies a region 20 to 24 nucleotides (nt) upstream of the splice junctions of mature mRNA and is thus termed the exonexon junction complex (EJC) (25). RNPS1 has been determined as a component of the EJC (25), which is consistent with the observation that RNPS1 specifically associates with spliced mRNAs in vitro (32). The EJC is thought to function as an adaptor platform that provides multiple postsplicing functions (26). This notion is apparently held true in the case of nonsense-mediated decay (NMD), which subjects aberrant mRNAs with premature termination co...

SummaryPinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts.