Crick’s core postulate of molecular biology states that information flows in a straight path from DNA to messenger RNA (mRNA) and onwards to proteins. The work of many subsequent
scientists collectively showed that RNA, in fact, passes through numerous stages of maturation, including capping, splicing, 3’-end processing, and editing.
Nucleotide changes are also frequently found in mRNAs of all organisms, and many studies showed the importance of such changes in gene expression. Of all the different modified residues discovered, the N6-methyladenosine (m6A) is regarded as the most abundant. Its deposition plays vital role in posttranscriptional gene regulation in eukaryotes.
The existence of RNA-binding proteins (RBPs) is another crucial component of the regulation of gene expression. 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 (Evolutionarily Conserved C-Terminal proteins), which were shown to have a role in post-embryonic development in A. thaliana.
Two orthogonal sequencing methods were applied to identify high-quality ECT2 and ECT3 target sets: iCLIP (individual nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing). It was shown that their target sets largely overlap, consistent with their redundant roles inferred from genetic analyses.
Three next-generation sequencing techniques were used, along with mass spectrometry, to investigate the effects of loss of ECT2/3 function 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. Since protection from endonucleolytic cleavage has been suggested to constitute a core mechanism of action of m6A in plants, the occurrence of endonucleolytic cleavage sites around m6A sites in the presence and absence of ECT2/3/4 was quantified. No evidence that ectopic cleavage underlies the repression of target sets in the absence of m6A-binding proteins was found.
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. It was tested that the differences were a genuine result of being a target. Possible other differences between targets and non-targets (GC content, expression levels, transcript and poly(A) tail length) could not account for the observed differences. This unexpected finding suggested that ECT2/3 targets are chemically different in ect2/3/4 compared to wild type, possibly relating to differences in the m6A content itself.