Practical Synthesis of Cap‐4 RNA

Abstract

Eukaryotic mRNAs possess 5′ caps that are determinants for their function. A structural characteristic of 5′ caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the common cap‐0 (m7GpppN) shows a rather simple methylation pattern, the Trypanosoma cap‐4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap‐4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom‐made nucleoside phosphoramidite building blocks for m62Am and m3Um, their incorporation into short RNAs, the efficient construction of the 5′‐to‐5′ triphosphate bridge to guanosine by using a solid‐phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much‐needed cap‐4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps.

1H, 13C, and 15N backbone chemical shift assignments of m7GTP cap-bound Leishmania initiation factor 4E-1

“…All chemical shifts were deposited in the BioMagResBank (www.bmrb.wisc.edu) under accession number 50250. This work paves the way to understanding how LeishIF4E-1 binds the trypanosomatid cap-4, now that two studies have reported its efficient chemical synthesis (Reolon et al 2019;Leiter et al 2020). Summary of the data establishing the sequence-specific resonance assignments of LeishIF4E-1 bound to m 7 GTP.…”

“…These parasites encode six highly diverged cap-binding protein isoforms (LeishIF4E-1 through −6) that significantly differ among themselves and from their orthologs in mammalian cells (Yoffe et al 2004 , 2006 , 2009 ; Zinoviev et al 2011 ). Trypanosomatids also contain a specific hypermethylated cap structure at the 5′ end of their mRNAs, called cap-4 (Reolon et al 2019 ; Leiter et al 2020 ). In the human developmental stage of the parasite’s life cycle, LeishIF4E-1 is the only isoform that is highly expressed and maintains cap-binding activity, suggesting that it is the functional cap-binding protein in the human infective stage (Zinoviev et al 2011 ).…”

Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1

“…To test this, we had to develop an assay for in situ methylation of long capped mRNAs and detection of translation output. To this point, we could show that Ecm1 can be used to introduce the guanine N7 methylation to produce a cap-4 RNA, which displays seven distinguished additional methylations within the first four nucleotides 35 and modified dinucleotide caps ( e.g. 1b ), which can be applied for in vitro transcription.…”

“…To achieve this, we chose the guanine N7 MTase Ecm1 that can be efficiently produced in E. coli and that we used previously to generate functional 5 0 caps. 30,[32][33][34][35] We sought to inhibit the activity of Ecm1 by incorporating sterically demanding but photo-cleavable groups in the active site. To generate such "caged" proteins, the genetic code can be expanded, allowing for the site-specic incorporation of non-natural "caged" amino acids into proteins.…”

Quantification of mRNA cap-modifications by means of LC-QqQ-MS

“…Here, synthetic standards might not be commercially available. Synthesis of modified nucleosides is more straightforward than synthesizing the respective cap-dinucleotide, which is made by only few specialists [ 22 – 26 ]. Additionally, we observed that in LC-QqQ-MS the detection of nucleosides is more sensitive than of caps.…”