An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana

Alternative splicing (AS) of pre-mRNAs is an important regulatory mechanism shaping the transcriptome. In plants, only few RNA-binding proteins are known to affect AS. Here, we show that the glycine-rich RNA-binding protein AtGRP7 influences AS in Arabidopsis thaliana. Using a high-resolution RT-PCR-based AS panel, we found significant changes in the ratios of AS isoforms for 59 of 288 analyzed AS events upon ectopic AtGRP7 expression. In particular, AtGRP7 affected the choice of alternative 5' splice sites preferentially. About half of the events are also influenced by the paralog AtGRP8, indicating that AtGRP7 and AtGRP8 share a network of downstream targets. For 10 events, the AS patterns were altered in opposite directions in plants with elevated AtGRP7 level or lacking AtGRP7. Importantly, RNA immunoprecipitation from plant extracts showed that several transcripts are bound by AtGRP7 in vivo and indeed represent direct targets. Furthermore, the effect of AtGRP7 on these AS events was abrogated by mutation of a single arginine that is required for its RNA-binding activity. This indicates that AtGRP7 impacts AS of these transcripts via direct interaction. As several of the AS events are also controlled by other splicing regulators, our data begin to provide insights into an AS network in Arabidopsis.