Carlotta Porcelli:
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Nucleotide changes are frequently found in mRNAs of all organisms, and many studies showed the importance of such changes in gene expression. Of all the modified residues discovered, the N6-methyladenosine (m6A) is the most abundant. Its deposition plays a vital role in post-transcriptional gene regulation in eukaryotes. Another critical aspect of gene expression regulation on the RNA level is RNA-binding proteins (RBPs). RBPs are involved in post-transcriptional functions in all eukaryotes, ranging from mRNA translation, decay, and splicing regulation. They can also recognize specific mRNA nucleotide modifications and bind to them thereby playing a role in regulating the bound transcript. It is thus important to identify mRNA targets bound by RBPs when decoding the molecular mechanisms guided by these proteins-RNA interactions.
This project aims to identify and characterize the set of mRNA targets bound on their m6A marks by RBPs in the model organism Arabidopsis thaliana. The focus was set on the RBPs ECT2, ECT3 and ECT4, which were shown to have a role in embryonic development in A. thaliana. Two orthogonal sequencing methods were applied to identify high-quality ECT2 and ECT3 target sets: iCLIP and HyperTRIBE. Three next-generation sequencing techniques were used, along with mass spectrometry, to investigate the effects of ECT2/3 functional loss on target mRNA expression levels. The implementation of downstream bioinformatics analyses allowed us to highlight a pattern of reduced expression levels of the target sets, mirrored in the changes in protein abundance and characterize the set of small RNAs changing in expression levels upon loss of ECT2/3/4.
Additionally, the occurrence of endonucleolytic cleavage sites around m6A sites in the presence and absence of ECT2/3/4 was quantified. The differential gene expression analysis results carried out with two different mRNA-seq library construction methods, Smart-seq2 and regular RNA-Seq, yield qualitatively similar but quantitatively different results. SMART-seq2 consistently returned greater fold change differences between ECT2/3 target mRNAs in ect2/ect3/ect4 mutants compared to wild type. Possible differences between targets and non-targets could not account for the observed differences, thus suggesting that ECT2/3 targets are chemically different in ect2/3/4 compared to wild type.