CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - FrP-44)
Mass spectrometry – based identification of 5’-RNA caps

Anton Škríba 1, Hana Cahová 1 *

  1. Ústav organické chemie a biochemie AV ČR, v.v.i., Flemingovo náměstí 542/2, 160 00, Praha 6

Abstrakt

It is well known that most of the RNAs are post-transcriptionally modified on the nucleobases or ribose. Increased sensitivity of next-generation sequencing and especially mass-spectrometry based analysis revealed new class of modifications on 5’ end of RNA, which are called RNA caps [1]. They are mostly derived from metabolites, cofactors and dinucleotide polyphosphates [2,3]. It is believed that their presence can affect the RNA stability, cellular metabolism and even mRNA translation, however their exact role is still not well described. The physicochemical properties of these molecules (high hydrophilicity, acidic phosphate functional groups, nucleobases) makes their analysis very challenging. This work will present workflow for qualitative and quantitative analysis of (non)canonical 5’-RNA caps in bacteria and mammalian tissue cell cultures.
RNA was isolated from various in vitro and in vivo samples and analyzed by UHPLC (Acquity H-class, Waters) equipped with dual mode reversed phase-anion exchange column (Atlantis Premier BEH C18 AX, Waters) coupled to high resolution mass spectrometer (Xevo G2-XS qTOF, Waters).
We have developed a robust and reproductible workflow for RNA caps analysis. In all digested RNAs we have observed signals of different canonical and non-canonical caps such as NAD, CoA, dinucleotide polyphosphates and their methylated analogues. Their structure was validated based on the precise retention time, m/z ratio and compared to the commercial standards. In other cases, fragmentation spectra were acquired to confirm the identity of the cap.

* Korespondující autor: hana.cahova@uochb.cas.cz

Literatura

  1. Ramanathan, A., Robb, G.B. and Chan, S.H. (2016) mRNA capping: biological functions and applications, Nucleic Acid Res., 44, 7511-7526.
  2. Cahova, H., Winz, M. L., Hofer, K., Nubel, G. and Jaschke, A. (2015) NAD captureSeq indicates NAD as a bactgerial cap for subset of regulatory RNAs, Nature, 519, 374-377.
  3. Hudeček, O., Benoni, R., Reyes-Gutierrez, P.E., Culka, M., Šanderová, H., Hubálek, M., Rulíšek, L., Cvačka, J., Krásný, L. and Cahová, H. (2020) Dinucleoside polyphosphates act as 5’ – RNA caps in bacteria, Nature Comm., 11, 1052.

Poděkování:

European Research Council Executive Agency (ERCEA) under the European Union’s Horizon Europe Framework Programme for Research and Innovation (ERC Starting grant agreement No 101041374 - StressRNaction).


Partneři společnosti

LabRules LCMS LabRules GCMS

Partneři

Bruker HPST Merck Pragolab Amedis EastPort Shimadzu Waters