Chemical synthesis and binding activity of the trypanosomatid cap-4 structure

Magdalena Lewdorowicz, Yael Yoffe, Joanna Zuberek, Jacek Jemielity, Janusz Stepinski, Ryszard Kierzek, Ryszard Stolarski, Michal Shapira, Edward Darzynkiewicz

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5′ end of all mRNAs, thus donating a 5′ cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5′-5′-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5′ cap includes 2′-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7Gpppm 36,6,2′ Apm2 Apm2 Cpm 23,2′ U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5′ terminal mRNA tetranucleotide fragment (pm36,6,2′ Apm2′ Apm2′ Cpm2 3,2′ U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5′-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.

Original languageEnglish
Pages (from-to)1469-1478
Number of pages10
JournalRNA
Volume10
Issue number9
DOIs
StatePublished - 1 Sep 2004

Keywords

  • Cap-4
  • Chemical synthesis
  • EIF4E
  • Fluorescence
  • Leishmania

ASJC Scopus subject areas

  • Molecular Biology

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