A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression |
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Authors: | Tanja Dorothe R?sel-Hillg?rtner Lee-Hsueh Hung Ekaterina Khrameeva Patrick Le Querrec Mikhail S. Gelfand Albrecht Bindereif |
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Affiliation: | 1.Institute of Biochemistry, Justus Liebig University of Giessen, Giessen, Germany;2.Kharkevich Institute for Information Transmission Problems, RAS, Moscow, Russia;3.Lomonosov Moscow State University, Department of Bioengineering and Bioinformatics, Moscow, Russia;University of California San Francisco, United States of America |
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Abstract: | The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function. |
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