Related Experiment Video
Updated: Dec 15, 2025
Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans
Published on: March 19, 2012
Transcriptomic Analysis of Extracellular RNA Governed by the Endocytic Adaptor Protein Cin1 of Cryptococcus
Muxing Liu1, Zhengguang Zhang1, Chen Ding2
1Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
Abstract:
Membrane vesicles are considered virulence cargoes as they carry capsular and melanin components whose secretory transport is critical for the virulence of the human fungal pathogen Cryptococcus species. However, other components of the vesicles and their function in the growth and virulence of the fungus remain unclear. We have previously found that the cryptococcal intersectin protein Cin1 governs a unique Cin1-Wsp1-Cdc42 endocytic pathway required for intracellular transport and virulence. Using RNA sequencing, we compared the profiles of extracellular RNA (exRNA), including microRNA (miRNA), small interference RNA (siRNA), long noncoding RNA (lncRNA), and messenger RNA (mRNA) between the wild-type (WT), and derived Δcin1 mutant strains of Cryptococcus deneoformans. Seven hundred twelve miRNAs and 88 siRNAs were identified from WT, whereas 799 miRNAs and 66 siRNAs were found in Δcin1. Also, 572 lncRNAs and 7,721 mRNAs were identified from WT and 584 lncRNAs and 7,703 mRNAs from Δcin1. Differential expression analysis revealed that the disruption of CIN1 results in many important cellular changes, including those in exRNA expression, transport, and function. First, for miRNA target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that cellular processes, components, and macromolecular functions are the most affected pathways. A higher number of genes were involved in the intracellular transport of endocytosis. Second, the results of GO term and KEGG analysis of differentially expressed lncRNA target genes and mRNA genes were consistent with those of miRNA targets. In particular, protein export is the topmost affected pathway among lncRNA target genes and one of the affected pathways among mRNA genes. The result of quantitative real-time reverse transcription PCR (qRT-PCR) from 12 mRNAs tested is largely agreeable with that of RNA-Seq. Taken together, our studies provide a comprehensive reference that Cryptococcus secretes abundant RNAs and that Cin1 plays a critical role in regulating their secretion. Given the growing clinical importance of exRNAs, our studies illuminate the significance of exploring this cutting-edge technology in studies of cryptococcal pathogenesis for the discovery of novel therapeutic strategies.
Insights
Cryptococcus fungal pathogens secrete abundant extracellular RNAs (exRNAs), including microRNAs and lncRNAs. The Cin1 protein is crucial for regulating the secretion of these RNAs, impacting fungal growth and virulence.
Area of Science:
- Mycology
- Molecular Biology
- Genomics
Background:
- Membrane vesicles are critical virulence factors in Cryptococcus species, carrying capsular and melanin components.
- The precise function of other vesicle components and their role in fungal growth and virulence remain largely unknown.
- The intersectin protein Cin1 regulates an endocytic pathway essential for intracellular transport and virulence in Cryptococcus.
Purpose of the Study:
- To comprehensively analyze and compare the extracellular RNA (exRNA) profiles, including miRNA, siRNA, lncRNA, and mRNA, between wild-type and Δcin1 mutant strains of Cryptococcus deneoformans.
- To investigate the impact of CIN1 disruption on exRNA expression, transport, and function.
- To elucidate the role of Cin1 in regulating the secretion of exRNAs in Cryptococcus.
Main Methods:
- RNA sequencing was employed to profile extracellular RNAs (miRNA, siRNA, lncRNA, mRNA) from wild-type and Δcin1 mutant Cryptococcus deneoformans strains.
- Differential gene expression analysis was performed to identify changes in RNA profiles upon CIN1 disruption.
- Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on target genes of differentially expressed RNAs.
Main Results:
- Significant differences in the abundance of miRNAs, siRNAs, lncRNAs, and mRNAs were observed between wild-type and Δcin1 mutant strains.
- Disruption of CIN1 led to widespread changes in cellular processes, macromolecular functions, and particularly intracellular transport and endocytosis pathways, as indicated by miRNA target gene analysis.
- Analysis of lncRNA and mRNA targets revealed consistent impacts, with protein export being a significantly affected pathway.
Conclusions:
- Cryptococcus species secrete a substantial amount of diverse extracellular RNAs.
- The Cin1 protein plays a pivotal role in governing the secretion of these extracellular RNAs.
- Understanding Cin1-mediated exRNA secretion is crucial for deciphering Cryptococcus pathogenesis and developing novel therapeutic strategies.
Related Concept Videos
02:28Leaky Scanning
02:24Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
03:21RNA-seq
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...