Citation

  • Authors: Toczydlowska-Socha, D., Zielinska, M. M., Kurkowska, M., Astha,, Almeida, C. F., Stefaniak, F., Purta, E., Bujnicki, J. M.
  • Year: 2018
  • Journal: Philos Trans R Soc Lond B Biol Sci 373
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK-293-F
    Description: Human embryonic kidney Fibroblast
    Known as: FreeStyle 293-F, HEK293-F, 293-F

Method

FreeStyle 293-F cells were grown in suspension in FreeStyleTM 293 Expression Medium in 8% CO2 at 378C. Transient transfectionswere performed with FectoPro (Polyplus-transfection) reagent using 0.5 mg of plasmid DNA per 1 ml of suspension culture. Cells were harvested 48 h post-transfection.

Abstract

The 5'-cap structure, characteristic for RNA polymerase II-transcribed RNAs, plays important roles in RNA metabolism. In humans, RNA cap formation includes post-transcriptional modification of the first transcribed nucleotide by RNA cap1 methyltransferase (CMTr1). Here, we report that CMTr1 activity is hindered towards RNA substrates with highly structured 5' termini. We found that CMTr1 binds ATP-dependent RNA DHX15 helicase and that this interaction, mediated by the G-patch domain of CMTr1, has an advantageous effect on CMTr1 activity towards highly structured RNA substrates. The effect of DHX15 helicase activity is consistent with the strength of the secondary structure that has to be removed for CMTr1 to access the 5'-terminal residues in a single-stranded conformation. This is, to our knowledge, the first demonstration of the involvement of DHX15 in post-transcriptional RNA modification, and the first example of a molecular process in which DHX15 directly affects the activity of another enzyme. Our findings suggest a new mechanism underlying the regulatory role of DHX15 in the RNA capping process. RNAs with highly structured 5' termini constitute a significant fraction of the human transcriptome. Hence, CMTr1-DHX15 cooperation is likely to be important for the metabolism of RNA polymerase II-transcribed RNAs.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

Pubmed