Function and regulatory mechanism of cis-regulatory RNA structure family in key methylation gene MAT2A

Abstract

MAT2A influences S-adenosylmethionine (SAM) synthesis, impacting DNA, RNA and protein methylation. In our previous work, using our novel computational method, EvoFam which enables us to search for conserved RNA structures genome-wide, our group has discovered a novel post-transcriptional regulatory RNA structure consisting of a family of six long stem-loops (hairpin structures, which we denote by hp1-6 or hp A-F), in the 3' untranslated region (3'UTR) of MAT2A mRNA. Remarkably, this structure is conserved across the majority of vertebrates species from humans to fish and beyond. This highlights its essential role in regulating MAT2A. Strikingly, there is a short fully conserved sequence motif within the loops of each of these structures. Since our group's publication of these RNA structures, there have been reports indicating that the m6A methylation writer METTL16 can act as an RNA-binding protein (RBP) binding to the conserved loop motif of hp1 and can methylate an m6A site of hp1-6 to regulate MAT2A mRNA degradation. We hypothesize that there may be other RBPs that can target these hairpin structures, especially those that may have specific binding with hp2-6. In this PhD research program, we study this research question, with an aim to discover other possible interacting RBP's, and their role in this MAT2A structured RNA regulatory mechanism. We screen RBPs binding to MAT2A mRNA using RNA pull-down experiments followed by mass spectrometry (MS) identification and western blotting identification, discovering that a key enzyme controlling the folate cycle and the rate limiting step of the SAM cycle, MTHFD1, also acts as an RBP specifically binding to hp2-6 in the 3'UTR of MAT2A mRNA: a role that has not been previously described. We next constructed hp2-6-/-, MTHFD1+/- and hp2-6-/-MTHFD1+/- double knockout HepG2 cells using the CRISP-cas9 method. Based on experiments conducted on both wild-type (WT) HepG2 cells and genetically modified cells, we have verified that the MAT2A mRNA-MTHFD1 binding occurs in HepG2 cells, occurring in both the nucleus and cytoplasm, and requires hp2-6. Exposure to excess extra-cellular methionine enhances this binding, leading to increased m6A methylation of hp2-6, enhanced splicing of a MAT2A mRNA intron retention isoform (MAT2A-IR), and consequent inhibition of MAT2A expression. A switch from MAT1A to MAT2A has been reported to promote tumor progression. However, we found that knocking down MTHFD1 promotes the expression of MAT2A while inhibiting tumor progression, as evidenced by cell proliferation, migration, invasion assays, and tumor growth data from tumor-bearing mice. This indicates that, besides its role in suppressing MAT2A, MTHFD1 promotes tumorigenesis via other pathway effects.