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A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana.

ABSTRACT: To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana In wild-type plants, three major splice variants issue from the GFP gene but only one represents a translatable GFP mRNA. Compared to wild-type seedlings, which exhibit an intermediate level of GFP expression, mutants identified in the screen feature either a "GFP-weak" or "Hyper-GFP" phenotype depending on the ratio of the three splice variants. GFP-weak mutants, including previously identified prp8 and rtf2, contain a higher proportion of unspliced transcript or canonically spliced transcript, neither of which is translatable into GFP protein. In contrast, the coilin-deficient hyper-gfp1 (hgf1) mutant displays a higher proportion of translatable GFP mRNA, which arises from enhanced splicing of a U2-type intron with noncanonical AT-AC splice sites. Here we report three new hgf mutants that are defective, respectively, in spliceosome-associated proteins SMU1, SmF, and CWC16, an Yju2/CCDC130-related protein that has not yet been described in plants. The smu1 and cwc16 mutants have substantially increased levels of translatable GFP transcript owing to preferential splicing of the U2-type AT-AC intron, suggesting that SMU1 and CWC16 influence splice site selection in GFP pre-mRNA. Genome-wide analyses of splicing in smu1 and cwc16 mutants revealed a number of introns that were variably spliced from endogenous pre-mRNAs. These results indicate that SMU1 and CWC16, which are predicted to act directly prior to and during the first catalytic step of splicing, respectively, function more generally to modulate splicing patterns in plants.

SUBMITTER: Kanno T

PROVIDER: S-EPMC5473141 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana.

Kanno Tatsuo T Lin Wen-Dar WD Fu Jason L JL Matzke Antonius J M AJM Matzke Marjori M

RNA (New York, N.Y.) 20170403 7

To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana In wild-type plants, three major splice variants issue from the GFP gene but only one represents a translatable GFP mRNA. Compared to wild-type seedlings, which exhibit an intermediate level of GFP expression, mutants identified in the screen feature either a "GFP-weak" or "Hyper-GFP" phenotype ...[more]

PMID: 28373290

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Project description:BackgroundSeveral recent studies indicate that alternative splicing in Arabidopsis and other plants is a common mechanism for post-transcriptional modulation of gene expression. However, few analyses have been done so far to elucidate the functional relevance of alternative splicing in higher plants. Representing a frequent and universal subtle alternative splicing event among eukaryotes, alternative splicing at NAGNAG acceptors contributes to transcriptome diversity and therefore, proteome plasticity. Alternatively spliced NAGNAG acceptors are overrepresented in genes coding for proteins with RNA-recognition motifs (RRMs). As SR proteins, a family of RRM-containing important splicing factors, are known to be extensively alternatively spliced in Arabidopsis, we analyzed alternative splicing at NAGNAG acceptors in SR and SR-related genes.ResultsIn a comprehensive analysis of the Arabidopsis thaliana genome, we identified 6,772 introns that exhibit a NAGNAG acceptor motif. Alternative splicing at these acceptors was assessed using available EST data, complemented by a sequence-based prediction method. Of the 36 identified introns within 30 SR and SR-related protein-coding genes that have a NAGNAG acceptor, we selected 15 candidates for an experimental analysis of alternative splicing under several conditions. We provide experimental evidence for 8 of these candidates being alternatively spliced. Quantifying the ratio of NAGNAG-derived splice variants under several conditions, we found organ-specific splicing ratios in adult plants and changes in seedlings of different ages. Splicing ratio changes were observed in response to heat shock and most strikingly, cold shock. Interestingly, the patterns of differential splicing ratios are similar for all analyzed genes.ConclusionNAGNAG acceptors frequently occur in the Arabidopsis genome and are particularly prevalent in SR and SR-related protein-coding genes. A lack of extensive EST coverage can be compensated by using the proposed sequence-based method to predict alternative splicing at these acceptors. Our findings indicate that the differential effects on NAGNAG alternative splicing in SR and SR-related genes are organ- and condition-specific rather than gene-specific.

Dataset Information

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana.

Publications

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in <i>Arabidopsis thaliana</i>.

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