Mutational analysis of an essential RNA stem-loop structure in a minimal RNA substrate specifically cleaved by Leishmania RNA virus 1-4 (LRV1-4) capsid endoribonuclease

Youngtae Ro, Jean L. Patterson

    Research output: Contribution to journalArticle

    1 Scopus citations

    Abstract

    The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5′ untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5′ 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5′ 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg2+ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA-cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg2+ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca2+ and Mn 2+ ions, among the reagents tested, could change the mobility of the substrate RNA 5′ 249-342 on a gel similarly to that of Mg2+ ions, but only Ca2+ ions identically showed the stabilizing effect of Mg2+ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg2+ or Ca2+) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

    Original languageEnglish (US)
    Pages (from-to)239-247
    Number of pages9
    JournalJournal of Microbiology
    Volume41
    Issue number3
    StatePublished - Sep 1 2003

    Keywords

    • Endoribonuclease
    • Leishmaniavirus
    • Metal ion
    • RNA cleavage
    • RNA stem-loop structure

    ASJC Scopus subject areas

    • Microbiology
    • Applied Microbiology and Biotechnology

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