Gene Silencing Via RNA Interference in Cryptococcus

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 May 17

PMID 38758313

Authors

Indrani Bose

Affiliations

Soon will be listed here.

Abstract

RNA interference (RNAi) is a molecular biology technique for silencing specific eukaryotic genes without altering the DNA sequence in the genome. The silencing effect occurs because of decreased levels of mRNA that then result in decreased protein levels for the gene. The specificity of the silencing is dependent upon the presence of sequence-specific double-stranded RNA (dsRNA) that activates the cellular RNAi machinery. This chapter describes the process of silencing a specific target gene in Cryptococcus using a dual promoter vector. The plasmid, pIBB103, was designed with two convergent GAL7 promoters flanking a ura5 fragment that acts as a reporter for efficient RNAi. The target gene fragment is inserted between the promoters to be transcribed from both directions leading to the production of dsRNA in cells that activate the RNAi pathway.

References

Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C . Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391(6669):806-11. DOI: 10.1038/35888. View

Ghildiyal M, Zamore P . Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009; 10(2):94-108. PMC: 2724769. DOI: 10.1038/nrg2504. View

Lax C, Tahiri G, Patino-Medina J, Canovas-Marquez J, Perez-Ruiz J, Osorio-Concepcion M . The Evolutionary Significance of RNAi in the Fungal Kingdom. Int J Mol Sci. 2020; 21(24). PMC: 7763443. DOI: 10.3390/ijms21249348. View

Nakayashiki H, Kadotani N, Mayama S . Evolution and diversification of RNA silencing proteins in fungi. J Mol Evol. 2006; 63(1):127-35. DOI: 10.1007/s00239-005-0257-2. View

Malone C, Hannon G . Small RNAs as guardians of the genome. Cell. 2009; 136(4):656-68. PMC: 2792755. DOI: 10.1016/j.cell.2009.01.045. View

Dumesic P, Natarajan P, Chen C, Drinnenberg I, Schiller B, Thompson J . Stalled spliceosomes are a signal for RNAi-mediated genome defense. Cell. 2013; 152(5):957-68. PMC: 3645481. DOI: 10.1016/j.cell.2013.01.046. View

Yadav V, Sun S, Billmyre R, Thimmappa B, Shea T, Lintner R . RNAi is a critical determinant of centromere evolution in closely related fungi. Proc Natl Acad Sci U S A. 2018; 115(12):3108-3113. PMC: 5866544. DOI: 10.1073/pnas.1713725115. View

Ma X, Li C, Ma L, Zhao X, Liu Y, Hao X . RNAi machinery regulates nutrient metabolism and fluconazole resistance in the pathogenic fungus Cryptococcus deneoformans. Med Mycol. 2022; 61(2). DOI: 10.1093/mmy/myac095. View

Zamore P, Tuschl T, Sharp P, Bartel D . RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000; 101(1):25-33. DOI: 10.1016/S0092-8674(00)80620-0. View

10.

Hammond S, Bernstein E, Beach D, Hannon G . An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000; 404(6775):293-6. DOI: 10.1038/35005107. View

11.

Rivas F, Tolia N, Song J, Aragon J, Liu J, Hannon G . Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol. 2005; 12(4):340-9. DOI: 10.1038/nsmb918. View

12.

Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T . Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 2002; 110(5):563-74. DOI: 10.1016/s0092-8674(02)00908-x. View

13.

Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T . Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell. 2004; 15(2):185-97. DOI: 10.1016/j.molcel.2004.07.007. View

14.

Burke J, Longhurst A, Natarajan P, Rao B, Liu J, Sales-Lee J . A Non-Dicer RNase III and Four Other Novel Factors Required for RNAi-Mediated Transposon Suppression in the Human Pathogenic Yeast . G3 (Bethesda). 2019; 9(7):2235-2244. PMC: 6643885. DOI: 10.1534/g3.119.400330. View

15.

Choi J, Kim K, Jeon J, Wu J, Song H, Asiegbu F . funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi. BMC Genomics. 2014; 15 Suppl 9:S14. PMC: 4290597. DOI: 10.1186/1471-2164-15-S9-S14. View

16.

Janbon G, Maeng S, Yang D, Ko Y, Jung K, Moyrand F . Characterizing the role of RNA silencing components in Cryptococcus neoformans. Fungal Genet Biol. 2010; 47(12):1070-80. PMC: 3021383. DOI: 10.1016/j.fgb.2010.10.005. View

17.

Bose I, Doering T . Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformans. J Microbiol Methods. 2011; 86(2):156-9. PMC: 3163904. DOI: 10.1016/j.mimet.2011.04.014. View

18.

Zhang H, Gao J, Chen J, Peng Y, Han Z . RNA-dependent RNA polymerase could extend the lasting validity period of exogenous dsRNA. Pest Manag Sci. 2022; 78(11):4569-4578. DOI: 10.1002/ps.7076. View

19.

Sijen T, Fleenor J, Simmer F, Thijssen K, Parrish S, Timmons L . On the role of RNA amplification in dsRNA-triggered gene silencing. Cell. 2001; 107(4):465-76. DOI: 10.1016/s0092-8674(01)00576-1. View

20.

Lipardi C, Wei Q, Paterson B . RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell. 2001; 107(3):297-307. DOI: 10.1016/s0092-8674(01)00537-2. View