Plants perceive environmental light conditions and optimize their growth and development accordingly by regulating gene activity at multiple levels. Photoreceptors are important for light sensing and downstream gene regulation. Phytochromes, red/far-red light receptors, are believed to regulate light-responsive alternative splicing, but little is known about the underlying mechanism. Alternative splicing is primarily regulated by transacting factors, such as splicing regulators, and by cis-acting elements in precursor mRNA. In the moss Physcomitrella patens, we show that phytochrome 4 (PpPHY4) directly interacts with a splicing regulator, heterogeneous nuclear ribonucleoprotein F1 (PphnRNP-F1), in the nucleus to regulate light-responsive alternative splicing. RNA sequencing analysis revealed that PpPHY4 and PphnRNP-F1 coregulate 70% of intron retention (IR) events in response to red light. A repetitive GAA motif was identified to be an exonic splicing silencer that controls red light-responsive IR. Biochemical studies indicated that PphnRNP-F1 is recruited by the GAA motif to form RNA-protein complexes. Finally, red light elevates PphnRNP-F1 protein levels via PpPHY4, increasing levels of IR. We propose that PpPHY4 and PphnRNP-F1 regulate alternative splicing through an exonic splicing silencer to control splicing machinery activity in response to light.
Plant photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although gene expression is modulated by photoreceptors at various levels, the regulatory mechanism at the pre-mRNA splicing step remains unclear. Alternative splicing, a widespread mechanism in eukaryotes that generates two or more mRNAs from the same pre-mRNA, is largely controlled by splicing regulators, which recruit spliceosomal components to initiate pre-mRNA splicing. The red/ far-red light photoreceptor phytochrome participates in light-mediated splicing regulation, but the detailed mechanism remains unclear. Here, using protein-protein interaction analysis, we demonstrate that in the moss Physcomitrella patens, phytochrome4 physically interacts with the splicing regulator heterogeneous nuclear ribonucleoprotein H1 (PphnRNP-H1) in the nucleus, a process dependent on red light. We show that PphnRNP-H1 is involved in red light-mediated phototropic responses in P. patens and that it binds with higher affinity to the splicing factor pre-mRNA-processing factor39-1 (PpPRP39-1) in the presence of red light-activated phytochromes. Furthermore, PpPRP39-1 associates with the core component of U1 small nuclear RNP in P. patens. Genome-wide analyses demonstrated the involvement of both PphnRNP-H1 and PpPRP39-1 in lightmediated splicing regulation. Our results suggest that phytochromes target the early step of spliceosome assembly via a cascade of protein-protein interactions to control pre-mRNA splicing and photomorphogenic responses.
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