Browsing by Subject "Neurospora crassa"
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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 Mechanism of Double-Stranded RNA Response in Neurospora(2009-01-09) Choudhary, Swati; Liu, YiIn eukaryotic cells, recognition of double-stranded RNA (dsRNA) by the enzyme Dicer initiates the RNA interference (RNAi) pathway, resulting in post-transcriptional gene silencing. Argonaute proteins play a critical role in this conserved pathway, which is present in protists, fungi, plants and animals. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that the production of dsRNA triggers the transcriptional induction of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for efficient gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 post-transcriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. A genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes (DRAGs) in Neurospora. Our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections. In order to understand the signaling mechanisms underlying this dsRNA response, we undertook a study of the dsRNA response elements (dsREs) in the promoter regions of qde-2 and other DRAGs. We demonstrate that different regions of the qde-2 promoter orchestrate early and late transcriptional induction in response to dsRNA. In the qde-2 promoter, a GC-rich element and downstream CAAT repeats were found to be important for the early response. In addition, the GC-rich dsRE was found in the promoters of other DRAGs, and was sufficient for dsRNA-induced transcriptional response. These results suggest that these DRAGs share the transcriptional induction pathway triggered by dsRNA. Finally, we demonstrate that QDE-2 contains an additional 10KDa N-terminal RGG domain, which is important for binding small interfering RNAs (siRNAs) and therefore required for its stability as well as efficient RNAi.Item The Mechanism of RNA Interference in Neurospora(2007-08-08) Maiti, Mekhala; Liu, YiIn the canonical RNA interference (RNAi) pathway, small-interfering RNA (siRNA) duplexes generated by Dicer are incorporated into the RNA-induced-silencing complex (RISC), and subsequently converted to single-stranded siRNA. Generation of single stranded siRNA is a pre-requisite for recognition and cleavage of the target mRNA by Argonaute. In biochemical experiments, Argonaute generates single-stranded siRNA by cleaving the passenger strand of the siRNA duplex. Mutational analysis of Neurospora homologue of Argonaute-2, known as Quelling Deficient -2 (QDE-2), revealed that the endonuclease activity of QDE-2 is required for the generation of singlestranded siRNA in vivo. Further biochemical studies to understand the mechanism for removal of the nicked passenger strand from siRNA duplex, led to the identification of a novel QDE-2 interacting protein (QIP) with a putative exonuclease domain. Disruption of qip led to the impairment of RNAi and most of the siRNAs were accumulated in nickedduplex form. Furthermore, QIP functions as an exonuclease to remove the cleaved passenger strand in a QDE-2 dependent manner. Thus, the cleavage of the passenger strand by QDE2 and its subsequent removal by QIP are critical biochemical steps in Neurospora RNAi pathway. Quelling, an RNAi related phenomenon in Neurospora, is induced by multiple copies of transgene. It was proposed that QDE-1 (a RNA dependent RNA polymerase, RdRp) and QDE-3 (a RecQ helicase) functions in quelling pathway by generating double-stranded RNA (dsRNA) from transgenes. To further understand the importance of QDE-1 and QDE-3, quelling assays were performed in the qde-1ko and qde-3ko strains. In contrast to previous results, the requirement of QDE-1 and QDE-3 was bypassed when the transgene copy number was high. Moreover, gene silencing analyses using strains lacking all potential RdRps suggested that unlike in C.elegans and Arabidopsis, the amplification of secondary dsRNA or siRNA is largely absent in Neurospora. The search for potential regulatory mechanisms of RNAi components in Neurospora led to the identification of a dsRNA response pathway. Two key components of the Neurospora RNAi pathway, qde2 and dicer like protein-2 (dcl-2), are induced by dsRNA at transcriptional and posttranscriptional level. The induction of QDE-2 is required for efficient gene silencing, indicating the importance of this regulatory mechanism in RNAi pathway.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.Item The Roles of Small and Long Non-Coding RNAs in Regulating Gene Expression(2014-01-23) Xue, Zhihong; McKnight, Steven L.; Liu, Yi; Liu, Qinghua; Conrad, NicholasRecent studies have revealed that a large proportion of a eukaryotic genome is transcribed into non-coding RNAs (ncRNAs). Based on size, these RNAs can be classified as small non-coding RNAs (sRNAs) and large non-coding RNAs (lncRNAs). The ncRNA regulatory networks control various levels of gene expression and play significant roles in diverse biological processes. Argonaute proteins, the core proteins in RNAi pathways, are required for the biogenesis of some sRNAs, including the PIWI-interacting RNAs and some microRNAs. How Argonautes mediate maturation of sRNAs independent of their slicer activity was not clear.The maturation of the Neurospora miRNA-like sRNA, milR-1, requires the Argonaute protein QDE-2, Dicer, and exonuclease QIP. Here, I reconstituted this Argonaute-dependent sRNA biogenesis pathway in vitro, and demonstrated that QDE-2 mediates milR-1 maturation by recruiting exosome and QIP, and by determining the size of milR-1. QIP first separates the QDE-2-bound duplex milR-1 precursor and then mediates 3’ to 5’ trimming and maturation of milR-1 precursor together with exosome using a hand-over mechanism. Our results establish a biochemical mechanism of an Argonaute-dependent sRNA biogenesis pathway and critical roles of exosome in sRNA processing. Natural antisense RNAs, which are mostly lncRNAs, are widely found in eukaryotic organisms and have been implicated in diverse physiological processes. The physiological importance of antisense RNAs and how they regulate sense RNAs are not clear. frequency (frq) encodes a core component of the Neurospora circadian oscillator. Here, I demonstrated that the simultaneous transcription of qrf, the long non-coding frq antisense RNA, represses frq transcription by inducing RNA polymerase II collision-triggered premature transcription termination and chromatin modifications. The expression of frq also inhibits the expression of qrf and surprisingly, drives the antiphasic rhythm of the qrf transcripts in the dark. The mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for robust and sustained circadian rhythmicity. Our results establish antisense transcription as an essential feature in a eukaryotic circadian system and demonstrate the importance and mechanism of antisense RNA action. Together, the studies described in this dissertation shed light on the mechanisms of gene expression regulated by sRNAs and lncRNAs.