Session: Session 2

Characterization of two RNA modification enzymes involved in human pathologies using LC-HRMS and MALDI-FTICR

RNA modification enzymes are highly involved in maintaining the efficiency, fidelity and regulation of protein synthesis by chemically modifying key factors (eg. tRNA, mRNA, rRNA). In particular, ALKBH8 is a tRNA modification enzyme mutated in patients suffering from intellectual disorders. TRMT9B is a putative tumor suppressor protein with a strong structural similarity to ALKBH8. It is proposed to also modify RNA, however, its substrate and therefore function in disease is still unknown. The goals are firstly to quantify modified nucleosides produced by ALKBH8 and pathogenic mutants using LC-HRMS and secondly, to identify the substrate of TRMT9B and the modification it catalyses using MALDI-FTICR.



Firstly, ALKBH8 wild-type and variants were incubated with RNA substrates which were fully digested into nucleosides, then quantified by LC-HRMS (timsTOF). We have also validated the use of MALDI-FTICR (SolariX XR, 9.4 T) to localize modifications to specific oligonucleotides for partially digested tRNA. By constructing specific knockouts in human cell lines, we can also use these methods to study the effect of pathogenic ALKBH8 variants and TRMT9B on RNA modifications in cellulo.



After LC-HRMS method optimization, modified nucleosides were quantified with limit of quantification down to the ng injected. LC-HRMS and HRMS/MS offers the unique opportunity to search for unexpected modifications compared to classical targeted LC-MS/MS experiments based on TQ instrument. Secondly, the ultra-high resolution of the FT-ICR instrument offers the unique opportunity to separate isobaric species for which isotopic pattern overlapping was previously observed with classical MALDI-TOF experiments. Both approaches offer new avenues for the fine characterization of RNA bearing chemical modifications.