Dataset Information

A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana.

ABSTRACT: To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications. Here we present the final five mutants, which are defective, respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME CRITICAL REGION 14 (dgcr14), CYCLIN-DEPENDENT KINASE G2 (cdkg2), INTERACTS WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing data and analyses of differential gene expression and alternative splicing patterns for the cbp80 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, for which such information was not yet available. Sequencing of small RNAs from the cbp80 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss the cumulative findings and their implications for the regulation of pre-mRNA splicing efficiency and alternative splicing in plants. The mutant collection provides a unique resource for further studies on a coherent set of splicing factors and their roles in gene expression, alternative splicing and plant development.

SUBMITTER: Kanno T

PROVIDER: S-EPMC7263676 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana.

Kanno Tatsuo T Venhuizen Peter P Wu Ming-Tsung MT Chiou Phebe P Chang Chia-Liang CL Kalyna Maria M Matzke Antonius J M AJM Matzke Marjori M

G3 (Bethesda, Md.) 20200601 6

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, tra ...[more]

PMID: 32265287

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Project description:Ubiquitin-like proteins play important roles in the regulation of many biological processes. UBL5 (Ubiquitin-like protein 5)/Hub1 (Homologous to ubiquitin 1), a member of the ubiquitin family, acts as a ubiquitin-like modifier on a specific target, the spliceosomal protein Snu66, in yeast and human cells. The 22nd aspartic acid (Asp22) is involved in the attachment of Hub1 to the Hub1 interaction domain (HIND) of Snu66 in yeast to modulate spliceosomal activity. Hub1 differs from other modifiers which interact covalently with their targets. It modulates pre-mRNA splicing by binding to Snu66 non-covalently in both yeast and human cells. However, the molecular mechanisms of Hub1-mediated pre-mRNA splicing in plant systems remains unclear. To better understand the function of Hub1 in plants, we examined the role of this ubiquitin-like modifier in Arabidopsis thaliana, which has two Hub1 homologues. Arabidopsis UBL5/Hub1(UBL5) is highly conserved at the amino acid level, compared to eukaryotic homologues in both plants and animals. In this study, phenotypic analysis of A. thaliana with reduced UBL5 gene expression, generated by RNA interference of AtUBL5a and AtUBL5b were performed. Interestingly, knock down plants of AtUBL5 showed abnormalities in root elongation, plant development, and auxin response. AtUBL5b is highly expressed in the vascular tissue of the leaf, stem, and root tissue. Yeast two-hybrid analysis revealed that AtUBL5a and AtUBL5b interact with the putative splicing factor AtPRP38 through its C-terminal domain (AtPRP38C). Knock down of AtUBL5b resulted in a pattern of insufficient pre-mRNA splicing in several introns of AtCDC2, and in introns of IAA1, IAA4, and IAA5. Defects of pre-mRNA splicing in an AtPRP38 mutant resulted in an insufficient pre-mRNA splicing pattern in the intron of IAA1. Based on these results, we showed that AtUBL5b positively regulates plant root elongation and development through pre-mRNA splicing with AtPRP38C in A. thaliana.

Dataset Information

A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana.

Publications

A Collection of Pre-mRNA Splicing Mutants in <i>Arabidopsis thaliana</i>.

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