Functional Genomics and G-Protein Signaling in Neurospora crassa
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Functional Genomics and G-Protein Signaling in Neurospora crassa

  • Author(s): Carrillo, Alexander
  • Advisor(s): Borkovich, Katherine
  • et al.
Creative Commons 'BY' version 4.0 license
Abstract

Functional genomics is a powerful tool for identifying genes that function in the same pathway in an organism. Environmental sensing mechanisms, and G protein signaling in particular, are important for relaying information from outside the cell to allow it to generate the proper response in eukaryotes. The main objectives of this dissertation are to 1. Generate and utilize phenotypic data that may reveal potential gene pathways in N. crassa, 2. Decipher the ways in which G-protein signaling is regulated and 3. Determine the role G-protein signaling has in regulating metabolism.In Chapter 2, N. crassa transcription factors were annotated and characterized for growth and development phenotypes. Publicly available RNAseq datasets were mined to determine possible correlations between transcription factor phenotype and transcript abundance during sexual development. We identified a total of 312 transcription factors in N. crassa. Complete phenotypic data were obtained for 242 strains using a combination of publicly available data and new analysis of gene deletion mutants generated during the study. Of the 242 transcription factor gene deletion strains, 64% had at least one defect in growth and development. The combination of RNAseq analysis with phenotypic data revealed several transcription factor genes with sexual development phenotypes that correlated with transcript abundance in wild type. In Chapter 3, I took all available phenotypic data that has been generated for gene deletion mutants in N. crassa (data for nearly 1,300 strains) and tested the ability of several statistical clustering methods to group mutants based on their growth and development phenotypes, with the goal of identifying potential pathways. Analysis of several clustering methods showed that using a weighted partitioning around medoids approach generated the most biologically relevant grouping of mutants. Publicly available RNAseq datasets were used to determine if there is any correlation between gene expression and phenotype. Most phenotypic clusters contained multiple expression profiles, suggesting that co-expression is not generally observed for genes with shared phenotypes. Yeast ortholog data for genes that co-clustered with MAPK signaling cascade genes were mined and revealed potential networks of interacting proteins in N. crassa. In Chapter 4, we investigated genetic interactions between the Receptor for Activated C Kinase-1 (RACK1) homolog cpc-2, the Gβ subunit gnb-1 and other G protein signaling components in N. crassa. We showed that CPC-2 is a cytosolic protein via cell fractionation and fluorescent microscopy. We observed genetic epistasis between cpc-2 and gna-2 for basal hyphae growth rate and aerial hyphae height. Mutational activation of gna-3 alleviated the submerged conidiation defect observed in the Δcpc-2 mutant. cpc-2 and gnb-1 exhibited a largely synergistic relationship, with mutants lacking both genes showing more severe defects for all phenotypic traits. In Chapter 5, I used a combination of RNAseq and liquid chromatography-mass spectrometry to profiles the transcriptomes and metabolomes of wild type, Δgna-1, Δgna-3 and Δric8 N. crassa strains. We observed large transcriptional differences between mutants and wild type. Many of the differentially expressed genes encode metabolic enzymes, and the electron transport chain was impacted some strains. Metabolome analysis revealed changes in levels of several primary metabolites for all mutants. Comparing the RNAseq and metabolomics data provided evidence for both transcriptional and post-transcriptional regulation of certain metabolic proteins in the various mutants.

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