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

Abstract

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.

Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses

“…Studies on the impact of a small number of SFs on the transcriptome have mainly been performed under normal growth conditions with little variation in conditions (Simpson et al, 2008;Raczynska et al, 2010;Rühl et al, 2012;Streitner et al, 2012). In the future, it will be important to identify SFs and their gene targets systematically and to understand how dynamic changes in AS during stress contribute to both short-term responses and long-term adjustment or acclimation.…”

“…Generation of robust quantitative data on AS among ecotypes may aid the identification of key splicing regulatory elements and the position of binding sites. Quantitative differences in AS between Col and C24 using a limited number of genes/AS events revealed that 28% of the AS events showed significant changes between the two ecotypes, whereas more than 70% were not affected (Streitner et al, 2012).…”

“…GRP7 regulates a number of AS events, in particular those involving alternative 59 splice sites. A number of the target transcripts coimmunoprecipitate with GRP7 in plant extracts, suggesting that GRP7 regulates AS of these transcripts by direct binding in vivo (Streitner et al, 2012). The sr45-1 mutant defective in the noncanonical SR45 protein (see above) is also hypersensitive to ABA (Carvalho et al, 2010).…”

“…At-GRP7 negatively autoregulates via AS: Elevated levels promote the use of a cryptic 59splice site in the intron, leading to a switch to a PTC-containing transcript isoform that rapidly decays via NMD. At-GRP7 also affects AS of numerous downstream targets some of which are rhythmic themselves (Streitner et al, 2012). …”

Complexity of the Alternative Splicing Landscape in Plants

“…These two proteins are controlled by the circadian clock, and their protein levels are regulated similarly to PTBs by AS and the production of NMDsensitive transcripts (Carpenter et al, 1994;Heintzen et al, 1994;Staiger, 2001;Staiger et al, 2003). Using a high-resolution RT-PCR panel, it was shown that GRP7 affects 59 events from a total of 288 AS events tested (Streitner et al, 2012). RIP experiments confirmed binding of GRP7 to seven targets that was dependent on the RRM domain.…”