To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced GFPreporter gene in Arabidopsis thaliana. In wild-type plants, three major splice variants issue from the GFPgene but only one represents a translatable GFPmRNA. Compared to wild-type seedlings, which exhibit an intermediate level of GFPexpression, 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 prp8and 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 GFPmRNA, which arises from enhanced splicing of a U2-type intron with noncanonical AT–AC splice sites. Here we report three new hgfmutants 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 smu1and cwc16mutants have substantially increased levels of translatable GFPtranscript owing to preferential splicing of the U2-type AT–AC intron, suggesting that SMU1 and CWC16 influence splice site selection in GFPpre-mRNA. Genome-wide analyses of splicing in smu1and cwc16mutants 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.