3' End Sequencing Library Preparation with A-seq2

Martín, Georges; Schmidt, Ralf; Gruber, Andreas J.; Ghosh, Souvik; Keller, W.O.; Zavolan, Mihaela

doi:10.3791/56129-v

Cited by 1 publication

(2 citation statements)

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“…A-seq2 was performed for two independent biological replicate samples from ect2/3/4 and WT as described previously [ 46 ]. Briefly, 500 ng poly(A) + RNA from 12-day-old seedlings per sample was purified using and fragmented in alkaline fragmentation buffer for 7 min at 95 °C and 650 rpm.…”

Section: Methodsmentioning

confidence: 99%

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m6A readers ECT2/ECT3/ECT4 enhance mRNA stability through direct recruitment of the poly(A) binding proteins in Arabidopsis

et al. 2023

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Background RNA N6-methyladenosine (m6A) modification is critical for plant growth and crop yield. m6A reader proteins can recognize m6A modifications to facilitate the functions of m6A in gene regulation. ECT2, ECT3, and ECT4 are m6A readers that are known to redundantly regulate trichome branching and leaf growth, but their molecular functions remain unclear. Results Here, we show that ECT2, ECT3, and ECT4 directly interact with each other in the cytoplasm and perform genetically redundant functions in abscisic acid (ABA) response regulation during seed germination and post-germination growth. We reveal that ECT2/ECT3/ECT4 promote the stabilization of their targeted m6A-modified mRNAs, but have no function in alternative polyadenylation and translation. We find that ECT2 directly interacts with the poly(A) binding proteins, PAB2 and PAB4, and maintains the stabilization of m6A-modified mRNAs. Disruption of ECT2/ECT3/ECT4 destabilizes mRNAs of ABA signaling-related genes, thereby promoting the accumulation of ABI5 and leading to ABA hypersensitivity. Conclusion Our study reveals a unified functional model of m6A mediated by m6A readers in plants. In this model, ECT2/ECT3/ECT4 promote stabilization of their target mRNAs in the cytoplasm.

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Section: Methodsmentioning

confidence: 99%

“…The A-seq2 data analysis was followed as previously described [ 31 , 46 ]. For statistical analysis of PAC shift events, genes with at least two PACs were selected for analysis of APA shift.…”

Section: Methodsmentioning

confidence: 99%