Sequencing of the amplified products revealed that intron retention occurs more frequently in rgh3 mutants at seven of these loci ( Fig. Localized differences in read coverage depth between rgh3 and WT libraries were used to design semiquantitative RT-PCR assays for nine of the genes identified as differentially spliced ( Fig. Cufflinks ( 20) predicted 46 genes had altered isoform use in rgh3 ( Table S1). We determined the genome-wide effect of rgh3 on mRNA splicing with RNA-seq and isoform expression analysis from homozygous rgh3 and WT sibling root and shoot tissues. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates.
The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. We found that most U12-type introns are retained or misspliced in rgh3. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects.
The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants.
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