Let it shine: light regulation of alternative splicing in plants

GODOY HERZ, MICAELA A; PETRILLO, EZEQUIEL; FUCHS, ARMIN; BARTA, ANDREA; KALYNA, MARIA; KORNBLIHTT, ALBERTO

Simposio; RNA 3' ends: Mechanism and biological function in eukaryotic genomes; 2013

Light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. We use here the At-RS31 (a plant-specific SR protein) transcriptional unit as a model. At-RS31 has three alternative splicing isoforms. Among them, mRNA1 is the only isoform encoding a full-length protein. In seedlings, light causes an increase of mRNA1. Since changes in the relative abundance of At-RS31 mRNA isoforms could be caused by differences in their stability, we wanted to demonstrate that the dark/light effect resulted from alternative splicing regulation and not from differential mRNA degradation. For this purpose we used three different approaches of independent nature. First, the transcription inhibitor actinomycin D greatly inhibits At-RS31 alternative splicing changes observed in response to light/dark transitions, suggesting that, once synthesized, the stability of At-RS31 mRNA isoforms is not affected by light/dark conditions. Second, the effects on At-RS31 alternative splicing are still observed in upf mutants that are defective in the mRNA degradation pathway known as nonsense-mediated decay (NMD), ruling out a putative regulation of NMD as the source of pattern change. Third, transformation of Arabidopsis-derived protoplasts with a plasmid expressing the constitutive splicing factor U2AF65 (U2 snRNP auxiliary factor 65-kDa subunit) mimics the effects of light on At-RS31 alternative splicing by increasing the usage of the stronger distal 3?ss leading to the mRNA1 isoform. To investigate the light transducing system involved in the regulation of alternative splicing, we used two kinds of approaches: the use of drugs that affect the electronic photosynthetic transport in whole plants and the use of Arabidopsis protoplasts. DCMU and DBMIB block the photosynthetic electron transport at different steps. DCMU interferes with the electron transfer from photosystem II to the plastoquinone pool and DBMIB prevents the electron transfer from the plastoquinone pool to the cytochrome b6/f complex. Surprisingly, while DCMU inhibits, DBMIB mimics the effect of light on At-RS31 alternative splicing. This not only indicates that chloroplast function is necessary for the signaling pathway that modulates At-RS31 alternative splicing but that reduced plastoquinones are essential to trigger the response to light. To gain further insight into the nature of this retrograde signal, we performed experiments with Arabidopsis protoplasts. Light/dark conditions affect alternative splicing in protoplasts, like was previously observed in Arabidopsis seedlings. Furthermore, protoplasts can be incubated in the light or in the dark for a certain time, and then centrifuged to collect conditioned medium. Conditioned medium obtained in the light added to protoplasts that are kept in the dark produces the same effect on alternative splicing as light incubation. This effect has been observed in a subset of Arabidopsis transcripts and suggests that protoplasts produce a signal during light incubation that can accumulate in the conditioned medium and then modulate alternative splicing. This new protoplast system will allow us to perform experiments with the conditioned medium to get closer to the nature of the signal and the mechanism that modulate alternative splicing in the light