Modulation of alternative pre-mRNA splicing in vivo by pinin.
Pre-mRNA splicing occurs in a large macromolecular RNA-protein complex called the spliceosome. The major components of the spliceosome include snRNP and SR proteins. We have previously identified an SR-like protein, pinin (pnn), which is localized not only in nuclear speckles but also at desmosomes. The nuclear localization of pnn is a dynamic process because pnn can be found not only with SR proteins in nuclear speckles but also in enlarged speckles following treatment of cells with RNA polymerase II inhibitors, DRB, and alpha-amanitin. Using adenovirus E1A and chimeric calcitonin/dhfr construct as a splicing reporter minigene in combination with cellular cotransfection, we found that pnn regulates alternative 5(') and 3(') splicing by decreasing the use of distal splice sites. Regulation of 5(') splice site choice was also observed for RNPS1, a general splicing activator that interacts with pnn in nuclear speckles. The regulatory ability of pnn in alternative 5(') splicing, however, was not dependent on RNPS1 and a pnn mutant, lacking the N-terminal 167 amino acids, behaved like a dominant negative species, inhibiting E1A splicing when applied in splicing assays. These results provide direct evidence that pnn functions as a splicing regulator which participates itself directly in splicing reaction or indirectly via other components of splicing machinery.
Wang
Ping
P
Epithelial Biology Laboratory, Department of Anatomy, Chang Gung University, Medical College, Guei-San, Taoyuan, Taiwan 333, ROC.
Lou
Pei-Jen
PJ
Leu
Steve
S
Ouyang
Pin
P
D020121:5' Untranslated Regions / Q000235:genetics; D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism; D017398:Alternative Splicing / Q000187:drug effects*; D000818:Animals; D019556:COS Cells; D002116:Calcitonin / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000378:metabolism* / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D022003:Cell Nucleus Structures / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism* / Q000494:pharmacology*; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D020121:5' Untranslated Regions; D017500:Adenovirus E1A Proteins; D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C102646:PNN protein, human; D012322:RNA Precursors; D016601:RNA-Binding Proteins; C084538:RNPS1 protein, human; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D002116:Calcitonin; D013762:Tetrahydrofolate Dehydrogenase