Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles. The molecular function of coilin, particularly of the substantial nucleoplasmic fraction, remains uncertain. We identified coilin loss-of-function mutations in a genetic screen for mutants showing either reduced or enhanced expression of an alternatively spliced GFP reporter gene in Arabidopsis thaliana. The coilin mutants feature enhanced GFP fluorescence and diminished Cajal bodies compared with wild-type plants. The amount of GFP protein is several-fold higher in the coilin mutants owing to elevated GFP transcript levels and more efficient splicing to produce a translatable GFP mRNA. Genome-wide RNA-sequencing data from two distinct coilin mutants revealed a small, shared subset of differentially expressed genes, many encoding stress-related proteins, and, unexpectedly, a trend toward increased splicing efficiency. These results suggest that coilin attenuates splicing and modulates transcription of a select group of genes. The transcriptional and splicing changes observed in coilin mutants are not accompanied by gross phenotypic abnormalities or dramatically altered stress responses, supporting a role for coilin in fine tuning gene expression. Our GFP reporter gene provides a sensitive monitor of coilin activity that will facilitate further investigations into the functions of this enigmatic protein.

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...

BackgroundDegradation is essential for RNA maturation, turnover, and quality control. RNA degradome sequencing that integrates a modified 5′-rapid amplification of cDNA ends protocol with next-generation sequencing technologies is a high-throughput approach for profiling the 5′-end of uncapped RNA fragments on a genome-wide scale. The primary application of degradome sequencing has been to identify the truncated transcripts that result from endonucleolytic cleavage guided by microRNAs or small interfering RNAs. As many pathways are involved in RNA degradation, degradome data should contain other RNA species besides the cleavage remnants of small RNA targets. Nevertheless, no systematic approaches have been established to explore the hidden complexity of plant degradome.ResultsThrough analyzing Arabidopsis and rice RNA degradome data, we recovered 11 short motifs adjacent to predominant and abundant uncapped 5′-ends. Uncapped ends associated with several of these short motifs were more prevalent than those targeted by most miRNA families especially in the 3′ untranslated region of transcripts. Through genome-wide analysis, five motifs showed preferential accumulation of uncapped 5′-ends at the same position in Arabidopsis and rice. Moreover, the association of uncapped 5′-ends with a CA-repeat motif and a motif recognized by Pumilio/Fem-3 mRNA binding factor (PUF) proteins was also found in non-plant species, suggesting that common mechanisms are present across species. Based on these motifs, potential sources of RNA ends that constitute degradome data were proposed and further examined. The 5′-end of small nucleolar RNAs could be precisely captured by degradome sequencing. Position-specific enrichment of uncapped 5′-ends was seen upstream of motifs recognized by several RNA binding proteins especially for the binding site of PUF proteins. False uncapped 5′-ends produced from capped transcripts through non-specific PCR amplification were common artifacts among degradome datasets.ConclusionsThe complexity of plant RNA degradome data revealed in this study may contribute to the alternative applications of degradome in RNA research.

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