Table 1. Mutants identified in a forward genetic screen based on an alternatively-spliced GFP reporter gene.
| Hyper-GFP (hgf) mutant | Name | AGI number | Predicted function in splicing | No. of alleles | Effect of mutation on development | Reference |
|---|---|---|---|---|---|---|
| hgf1 | coilin | At1g13030 | marker protein for Cajal bodies, which facilitate snRNP maturation | 12a | negligible | Kanno et al., 2016 |
| hgf2 | CWC16a | At1g25682 | step I factor | 3a | negligible | Kanno et al., 2017a |
| hgf3 | SMU1 | At1g73720 | recruited prior to B* complex formation; | 1 | negligible | Kanno et al., 2017a |
| hgf4 | SMFA | At4g30220 | small nuclear ribonucleoprotein | 1 | negligible | Kanno et al., 2017a |
| hgf5 | PRP39A | At1g04080 | U1 snRNP component | 5a | negligible | Kanno et al., 2017b |
| hgf6 | RBP45D | At5g19350 | U1 snRNP component | 2 | negligible | this study |
| hgf7 | DG CR14-related | At3g07790 | spliceosomal C complex | 2 | negligible | this study |
| hgf8 | CDKG2 | At1g67580 | splicing-related protein kinase | 1 | early flowering | this study |
| hgf9 | IWS1 | At1g32130 | transcription elongation | 2 | negligible | this study |
| gfw1 | AtRTF2 | At5g58020 | contributes to ubiquitin-based regulation of the spliceosome? | 2 | embryo lethal | Sasaki et al., 2015; Kanno et al., 2017a |
| gfw2 | PRP8A | At1g80070 | U5 snRNP component; acts at catalytic core of spliceosome | 3 | embryo lethal | Sasaki et al., 2015; Kanno et al., 2017a |
| gfw3 | RBM25 | At1g60200 | U1 snRNP component | 2 | low seed set | Kanno et al., 2017b |
| gfw4 | PRP18A | At1g03140 | step II factor | 1 | short roots, small siliques | Kanno et al., 2018a |
| gfw5 | PRP4KA | A3g25840 | recruited prior to B* complex formation; needed for catalytic activation of spliceosome | 5 | broad rosettes, late flowering, tall stature, low seed set | Kanno et al., 2018b |
| gfw6 | SAC3A | At2g39340 | mRNA export factor | 5 | negligible | Kanno et al., 2018b |
| gfw7 | CBP80 | At2g13540 | multiple | 1 | Serrated leaves, early flowering | this study |
| T line (WT) | n.a. | n.a. | Wild-type line expressing GFP reporter gene; used for EMS mutagenesis | n.a. | normal |
The mutants retrieved in a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis (Figure 1) include a predicted core spliceosomal protein (SMFa); putative components of the U1 snRNP (PRP39a, RBM25, RBP45d) and U5 snRNP (PRP8); putative step I and step II factors transiently associated with the spliceosome (CWC16a and PRP18a, respectively); a predicted complex C protein (DGCR14); putative splicing regulatory proteins (RTF2, SMU1, PRP4ka, CDKG2); one structural protein presumed to be important for snRNP maturation (coilin), putative mRNA export factors (SAC3a, CBP80) and a predicted transcription elongation factor (IWS1). Developmental phenotypes are primarily observed in six (of seven) identified gfw mutations, two of which are embryo-lethal.
Further screening of the M2 population after publication of the first alleles of coilin, PRP39a and CWC16a has identified three new alleles of coilin (R9H; first intron, 3′ splice site; second intron, 5′ splice site), one new prp39a allele (R226*) and two new cwc16a alleles (W18*; fifth intron, 3′ splice site). These unpublished alleles are counted in the number of alleles shown here.
Abbreviation: SRA, Sequence Read Archive (NCBI); ABRC, Arabidopsis Biological Resource Center; T (or ST) refers to the WT T line harboring the alternatively-spliced GFP reporter gene. If the sequencing data from T line has a separate SRA number, it is noted in the table; n.d., not done; n.a., not available.