Browsing by Subject "Gene Expression Regulation, Fungal"
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Item The Biogenesis of Small Interfering RNA in Neurospora Crassa(2013-01-16) Chang, Shwu-Shin; Liu, YiRNA interference is a well-conserved gene silencing mechanism in eukaryotes. It regulates various biological processes including development, genome defense and heterochromatin formation. RNAi is initiated by the production of dsRNA, which is processed by Dicer to produce small interfering RNA (siRNA). In the filamentous fungus, Neurospora crassa, two types of siRNA have been characterized. One is involved in transgene-induced silencing, termed quelling; the other type is induced by DNA damage and functions to slow down protein translation after DNA damage. Both of these siRNAs originate from repetitive sequences in the Neurospora genome. We show that the components of the homologous recombination (HR) machinery are required to generate these types of small RNA specifically at repetitive regions. Furthermore, chromatin remodeling and DNA replication enzymes are required for efficient HR activity and small RNA production. Lastly, we show that the two small RNA pathways are mechanistically similar by demonstrating that quelling-induced siRNA can also be induced upon DNA damage. Our results suggest that the small RNA biogenesis machinery is recruited specifically to the repetitive loci after homologous recombination, which may result in the formation of aberrant DNA structures. dsRNA not only triggers the RNAi pathway, but also initiates a signaling cascade that results in activating the transcription of ~60 genes, including the RNAi components, in Neurospora. The function of the dsRNA activated genes suggests that RNAi is part of a broad ancient host defense response against viral and transposon infection. A genetic screen has been designed to identify the components involved in this dsRNA triggered transcriptional response; several mutants have been identified and characterized.Item The Role of Codon Usage in Regulating Protein Expression, Structure and Function(2014-06-10) Zhou, Mian; Tu, Benjamin; Liu, Yi; Takahashi, Joseph; Zinn, Andrew R.Codon usage bias has been observed in the genomes of almost all organisms and is thought to result from selection for efficient translation of highly expressed genes. Many genes, however, exhibit little codon usage bias. It's not clear whether the lack of codon bias for a gene is due to lack of selection for mRNA translation or it has some biological significance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. However, unlike most genes inNeurospora, frq exhibits non-optimal codon usage across its entire open reading frame (ORF). Optimization of frq codon usage results in the abolition of both overt and molecular circadian rhythms. Codon optimization not only increases FRQ expression level but surprisingly, also results in conformational changes in FRQ protein, impaired FRQ phosphorylation, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for maintaining circadian rhythmicity in Neurospora. Interestingly, there is a correlation between codon usage score and FRQ protein structure: the regions that are predicted to be disordered preferentially uses more non-optimal codons. This negative correlation is also found in the proteasome of Neurospora, as well in yeast, Drosophila, C. elegans and E. coli. By making a series of Neurospora strains with frq optimized in different regions, we find that codon optimizations in the predicted disordered regions of FRQ have more prominent effects on FRQ activity and structure. Furthermore, codon optimization of disordered regions in several other Neurospora genes results in altered protein degradation rates, suggesting structural changes by codon optimization. Together, these results suggest that codon usage adapts to protein structures and there is a "code" within genetic codons that allow optimal co-translational protein folding.