Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

64To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward 65 genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana. This 66 effort generated a collection of sixteen mutants impaired in various splicing-related proteins, 67 many of which had not been recovered in any prior genetic screen or implicated in splicing in 68 plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP 69 biogenesis pathway, transcription, and mRNA transport. We have described eleven of the 70 mutants in recent publications. Here we present the final five mutants, which are defective, 71 respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME 72WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing 74 data and analyses of differential gene expression and alternative splicing patterns for the cbp80 75 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, 76 for which such information was not yet available. Sequencing of small RNAs from the cbp80 77 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) 78 precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing 79 factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss 80 the cumulative findings and their implications for the regulation of pre-mRNA splicing 81 efficiency and alternative splicing in plants. The mutant collection provides a unique resource for 82 further studies on a coherent set of splicing factors and their roles in gene expression, alternative 83 splicing and plant development. 84 85 86 87 88 89 90 91 92 93 94 95Splicing of pre-mRNAs by the excision of introns and ligation of flanking exons is a 96 prerequisite for the expression of most eukaryotic genes. Splicing entails two transesterification 97 reactions carried out by the spliceosome, a large and dynamic ribonucleoprotein (RNP) machine 98 located in the nucleus. At least six structurally and functionally distinct spliceosomal complexes 99 containing core spliceosomal proteins, transiently-associated factors and different combinations 100 of five different small nuclear (sn) RNAs -U1, U2, U4, U5 and U6 -act sequentially to execute 101 the two catalytic steps of the splicing process (Matera and Wang, 2014;Yan et al., 2017). The 102 spliceosome is able to carry out constitutive splicing, in which the same splice sites are always 103 used for a given intron, and alternative splicing, in which splice site usage for a given intron is 104 variable. Alternative splicing increases transcriptome and proteome diversity (Nilsen and 105 Graveley, 2010;Syed et al., 2012;Reddy et al., 2013) and is important for development and 106 stress adaptation in plants (Staiger and Brown, 2013; Filichkin et al., 2015; Szakonyi and Duque, 107 2018). 108Most information on spliceosome composition and the splicing mechanism has been derived 109 from genetic, bioc...

Highlights d Chloroplast retrograde signals control alternative splicing in Arabidopsis thaliana d TOR kinase has a central role in alternative splicing control in roots d Mitochondria mediate light/sucrose effects on alternative splicing in roots

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