The 5′ stem‐loop and its role in mRNA stability in maize S cytoplasmic male sterility

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Abstract
The co-transcribed orf355-orf77 region of the mitochondrial genome is associated with S cytoplasmic male sterility (CMS-S) in maize; the amounts of its 1.6- and 2.8-kb transcripts were previously shown to be greatly reduced in fertility-restored microspores relative to the amounts in sterile plants. To investigate the mechanism underlying this reduction, detailed analysis of the 5' and 3' termini of these transcripts was conducted. Using 3' RACE analysis, the polyadenylation sites of the 1.6- and 2.8-kb transcripts were mapped adjacent to a 3' stem-loop, which may play an important role in stabilizing their 3' ends. No difference was found between the polyadenylation sites in sterile and fertility-restored microspores that could account for the differences in orf355-orf77 transcript levels. The 5' terminus of the 1.6-kb transcript was further studied by primer extension; the result revealed that there was a deletion of nine nucleotides only in fertility-restored microspores, and that this deletion eliminated a 5' stem-loop sequence. We propose that the elimination of the 5' stem-loop in the fertility-restored microspores could be the cause of the degradation of the 1.6-kb transcript. Because the 2.8-kb transcript can be cleaved to generate the 1.6-kb transcript, the amount of the 2.8-kb transcript is also reduced in fertility-restored microspores.