Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis

Abstract

Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences, and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations. In this study, we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene. Cloning of OsRAD1 cDNAs revealed three transcript variants, named as OsRAD1.1, OsRAD1.2 and OsRAD1.3, respectively, which were derived from alternative splicing of the last intron. Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain. The three transcripts exhibited similar expression patterns in various tissues, but the expression level of OsRAD1.1 was the highest. Specific knockout of OsRAD1.1 led to sterility, while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility. Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility. Yeast two-hybrid assay showed that OsRAD1.1, but not OsRAD1.2 and OsRAD1.3, interacted with the three other meiosis proteins OsHUS1, OsRAD9 and OsRAD17, suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.