ABSTRACT
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 light-mediated 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.
Subject(s)
Alternative Splicing/radiation effects , Bryopsida/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Photoreceptors, Plant/metabolism , Phytochrome/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , Alternative Splicing/genetics , Bryopsida/genetics , Bryopsida/radiation effects , Gene Ontology , Genome-Wide Association Study , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics , Light , Phytochrome/radiation effects , Protein Binding/radiation effects , Protein Interaction Mapping , RNA Precursors/metabolism , RNA Splicing Factors/metabolism , RNA, Messenger/metabolism , Ribonucleoprotein, U1 Small Nuclear/genetics , Spliceosomes/metabolismABSTRACT
The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA. Prp8 directly binds over the 5'SS and the branch site (BS) for the first catalytic step, and the 5'SS and 3'SS for the second step. Switching the Prp8 interaction from the BS to the 3'SS requires Slu7, which interacts dynamically with pre-mRNA first, and then interacts stably with the 3'-exon after Prp16-mediated spliceosome remodeling. Our results suggest that Prp8 plays a key role in positioning the 5'SS and 3'SS, facilitated by Slu7 through interactions with Prp8 and substrate RNA to advance exon ligation. We also provide evidence that Prp16 first docks on the intron 3' tail, then translocates in the 3' to 5' direction on remodeling the spliceosome.
