Discovery of highly reactive self-splicing group II introns within the mitochondrial genomes of human pathogenic fungi

Abstract

Fungal pathogens represent an expanding global health threat for which treatment options are limited. Self-splicing group II introns have emerged as promising drug targets, but their development has been limited by a lack of information on their distribution and architecture in pathogenic fungi. To meet this challenge, we developed a bioinformatic workflow for scanning sequence data to identify unique RNA structural signatures within group II introns. Using this approach, we discovered a set of ubiquitous introns within thermally dimorphic fungi (genera of Blastomyces, Coccidioides and Histoplasma). These introns are the most biochemically reactive group II introns ever reported, and they self-splice rapidly under near-physiological conditions without protein cofactors. Moreover, we demonstrated the small molecule targetability of these introns by showing that they can be inhibited by the FDA-approved drug mitoxantrone in vitro. Taken together, our results highlight the utility of structure-based informatic searches for identifying riboregulatory elements in pathogens, revealing a striking diversity of reactive self-splicing introns with great promise as antifungal drug targets.

In Silico Discovery of Group II Intron RNA Splicing Inhibitors

“…Inhibitors that can function across group II intron families would be of interest given that self-splicing introns are required for metabolism in pathogenic fungi, for which new antimicrobials represent a major unmet medical need. 21 To evaluate pan-splicing capability of the compounds, we tested several inhibitors of the O.i. intron in a self-splicing reaction of the Saccharomyces cerevisiae (S.c.) group IIB ai5γ intron, which is a genetically and biochemically well-characterized system.…”

“… , Given these parallels, we asked whether group IIC intron splicing inhibitors can also inhibit splicing of a group IIB intron. Inhibitors that can function across group II intron families would be of interest given that self-splicing introns are required for metabolism in pathogenic fungi, for which new antimicrobials represent a major unmet medical need . To evaluate pan-splicing capability of the compounds, we tested several inhibitors of the O.i.…”

Structural insights into intron catalysis and dynamics during splicing

“…The radiolabelled intron precursor RNA for the splicing assay was prepared using the previously described body-labelling protocol 39 . Briefly, the intron precursor RNA transcripts were body-labelled using inhouse-prepared T7 RNA polymerase and 50 μCi of [α- 32 P]-UTP (PerkinElmer) in a transcription buffer containing 40 mM Tris-HCl pH 8.0, 10 mM NaCl, 15 mM MgCl 2 , 2 mM spermidine, 0.01% Triton X-100, 10 mM dithiothreitol and 3.6 mM each rNTP (except UTP, which was at 1 mM) and 5 μg of linearized pCZ26 plasmid.…”