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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.

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