Optimizing RNA Interference in Cryptococcus neoformans

Initially regarded as a phenomenon unique to a select number of plants, post-transcriptional gene silencing (PTGS), a naturally occurring process that inhibits the transcription of specific genes, is now one of the hottest topics in molecular biology. Though further research has made it clear that PTGS occurs in both plants and animals, with roles in viral defense and transposon silencing mechanisms, perhaps most exciting is the emergence of RNA interference—PTGS initiated by the experimental introduction of double stranded RNA (dsRNA)—as a tool to alter expression of specific genes in a variety of organisms. Use of RNAi allows researchers to successfully turn off or “knock out” genes without manipulating a cell’s DNA.

Current models of the RNAi mechanism suggest that when dsRNA corresponding to the coding sequence of a particular gene is introduced to a cell producing that gene product, the gene product is no longer produced. RNA interference (RNAi) is being harnessed as a tool for the study of the pathogenic fungus Cryptococcus neoformans. C. neoformans is a distant relative of yeast, and as such, can be readily found in the natural environment. Humans are in regular contact with this organism, but only those who are immunocompromised are susceptible to the disease caused by this opportunistic pathogen. C. neoformans begins by infecting the lungs of its host, and can spread to cause fatal meningitis. Current methods of treatment are severely inadequate in controlling infection, and as some strains of C. neoformans have become drug resistant, research efforts to understand this pathogen have heightened. By applying the technique of RNAi to C. neoformans, members of the Doering Lab have begun to study capsule synthesis and are moving toward identifying essential genes of the organism.

Already a successfully applied technique in C. neoformans, RNAi has yet to be fully optimized as a direct method for altering gene expression. This project took the first steps towards the optimization of this tool in C. neoformans by examining the optimal size of the introduced dsRNA. As a test case the ADE2 gene, which encodes a protein required for adenine biosynthesis in the organism, was used. If successfully interfered with in this pathogen, cells of the organism, which are normally cream colored, will appear pink due to the accumulation of red pigment associated with the buildup of intracellular purines.

To use RNAi to alter gene expression, dsRNA must be introduced into the organism of study. For this project, a plasmid was used which contained opposing promoters, so that transcription of RNA could occur from either direction. The goal was to use variable length inserts in this plasmid to test the effectiveness of long or short dsRNA fragments for triggering RNAi. Over the course of experimentation, these plasmids would be introduced to C. neoformans cells by the method of electroporation. Afterwards, the appearance of pink cells would indicate the success of the experiment.

While in the lab, success was made in amplifying four inserts of different lengths for ligation into the plasmid vector of choice. Due to the limitations of time and required troubleshooting, this project remains a work in progress. The next steps for this experiment will be to ligate these inserts into vector plasmids, transform these into E. coli cells to generate large amounts of plasmids, and use electroporation to transfer the resulting DNA into cells of the pathogen. Observation of electroporated C. neoformans cells would then help to determine the extent of dsRNA expression in these cells from the various vectors.

As there remains much to know and understand about the mechanisms of RNAi in Cryptococcus neoformans, future experiments will continue to work toward the optimization of this useful technique and the discovery of more of its applications.