Arginine methylation of SM-LIKE PROTEIN 4 antagonistically affects alternative splicing during Arabidopsis stress responses.

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process.

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.