Alternative polyadenylation regulates acetyl-CoA carboxylase function in peanut

Abstract

Abstract Background Polyadenylation is an important mechanism by which mRNA molecules are terminated at their 3′-ends. Alternative polyadenylation (APA) can produce multiple transcripts from the same locus with different polyadenylation sites (PASs) and result in several 3′ untranslated regions (UTRs) varying by length and composition. APA affects approximately 60–70% of eukaryotic genes, with fundamental consequences on cell proliferation, differentiation, and tumorigenesis. Results In this study, we performed long-read, single-molecule sequencing of mRNA from peanut seeds, which revealed that more than half of all peanut genes have more than two PASs, with more PASs in older developing seeds, indicating that the PAS is highly tissue specific and plays an important role in peanut seed maturation. We identified four 3′ UTRs for the peanut acetyl-CoA carboxylase A1 (AhACCA1) gene, designated UTR1–4. RT-PCR analysis showed that UTR1-containing transcripts are expressed mainly in roots, leaves, and early developing seeds; transcripts with UTR2/3 accumulated mainly in roots, flowers, seeds; and transcripts harboring UTR4 were constitutively expressed. We transiently expressed all four UTRs in Nicotiana benthamiana leaves, which indicated that each UTR affects protein abundance but not subcellular location. We also transformed yeast cells with each UTR for functional verification. UTR2 promoted the expression level of AhACCA1 compared to a yeast transcription terminator, whereas UTR3 did not. We determined ACC gene structures from seven plant species, detecting 51 PASs for 15 ACC genes from four plant species, indicating that APA of the ACC gene family is universal in plants. Conclusion Our data reveal that APA is universal in peanut seeds and plays important role in peanut seed maturation. We identified four 3′ UTRs for AhACCA1 gene, each of them showed different tissue-specific expression pattern. Using subcellular location experiment and yeast transformation test, we identified that UTR2 had a stronger effect in gene expression than the other three ones.