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author:("Xue, zhikong")
1.  Convergent Transcription Induces Dynamic DNA Methylation at disiRNA Loci 
PLoS Genetics  2013;9(9):e1003761.
Cytosine methylation of DNA is an important epigenetic gene silencing mechanism in plants, fungi, and animals. In the filamentous fungus Neurospora crassa, nearly all known DNA methylations occur in transposon relics and repetitive sequences, and DNA methylation does not depend on the canonical RNAi pathway. disiRNAs are Dicer-independent small non-coding RNAs that arise from gene-rich part of the Neurospora genome. Here we describe a new type of DNA methylation that is associated with the disiRNA loci. Unlike the known DNA methylation in Neurospora, disiRNA loci DNA methylation (DLDM) is highly dynamic and is regulated by an on/off mechanism. Some disiRNA production appears to rely on pol II directed transcription. Importantly, DLDM is triggered by convergent transcription and enriched in promoter regions. Together, our results establish a new mechanism that triggers DNA methylation.
Author Summary
DNA methylation in eukayrotes refers to the modification of cytidines at 5th position with methyl group (5mC). Though absent in some species, DNA methylation is conserved across fungi, plants and animals and plays a critical role in X chromosome inactivation, genomic imprinting, transposon silencing etc. In addition, DNA methylation also occurs at the promoter sequence to regulate gene expression. Filamentous fungus Neurospora crassa has a well-known mechanism of DNA methylation for genomic defense. During sexual stage repetitive sequences (e.g. transposons) are recognized and point mutations are introduced. During vegetative stage these mutations serve as signals for establishing static DNA methylation to silence all copies of the sequences. In this study, we report a new type of DNA methylation in Neurospora. It is tightly linked to a type of non-coding small RNA termed dicer-independent siRNA (disiRNA) and therefore was termed disiRNA loci DNA methylation (DLDM). DLDM is dynamic regulated and shows an on/off pattern, i.e. most alleles contain no 5mC but some are densely methylated. Interestingly, DLDM can be triggered by convergent transcription and is accumulated at promoter regions. In summary, our findings demonstrate a new type of dynamic DNA methylation.
PMCID: PMC3764098  PMID: 24039604
2.  Reconstitution of an Argonaute-dependent small RNA biogenesis pathway reveals a hand-over mechanism involving the RNA exosome and the exonuclease QIP 
Molecular Cell  2012;46(3):299-310.
Argonaute proteins are required for the biogenesis of some small RNAs (sRNAs), including the PIWI-interacting RNAs and some microRNAs. How Argonautes mediate maturation of sRNAs independent of their slicer activity is not clear. The maturation of the Neurospora miRNA-like sRNA, milR-1, requires the Argonaute protein QDE-2, Dicer, and QIP. Here, we reconstitute this Argonaute-dependent sRNA biogenesis pathway in vitro and discover that the RNA exosome is also required for milR-1 production. Our results demonstrate that QDE-2 mediates milR-1 maturation by recruiting exosome and QIP and by determining the size of milR-1. The exonuclease QIP first separates the QDE-2-bound pre-milR-1 duplex and then mediates 3’ to 5’ trimming and maturation of pre-milRNA together with exosome using a hand-over mechanism. In addition, exosome is also important for the decay of sRNAs. Together, our results establish a biochemical mechanism of an Argonaute-dependent sRNA biogenesis pathway and critical roles of exosome in sRNA processing.
PMCID: PMC3351553  PMID: 22516970
3.  Transcription of the Major Neurospora crassa microRNA–Like Small RNAs Relies on RNA Polymerase III 
PLoS Genetics  2013;9(1):e1003227.
Most plant and animal microRNAs (miRNAs) are transcribed by RNA polymerase II. We previously discovered miRNA–like small RNAs (milRNAs) in the filamentous fungus Neurospora crassa and uncovered at least four different pathways for milRNA production. To understand the evolutionary origin of milRNAs, we determined the roles of polymerases II and III (Pol II and Pol III) in milRNA transcription. Our results show that Pol III is responsible for the transcription of the major milRNAs produced in this organism. The inhibition of Pol III activity by an inhibitor or by gene silencing abolishes the production of most abundant milRNAs and pri–milRNAs. In addition, Pol III associates with these milRNA producing loci. Even though silencing of Pol II does not affect the synthesis of the most abundant milRNAs, Pol II or both Pol II and Pol III are associated with some milRNA–producing loci, suggesting a regulatory interaction between the two polymerases for some milRNA transcription. Furthermore, we show that one of the Pol III–transcribed milRNAs is derived from a tRNA precursor, and its biogenesis requires RNase Z, which cleaves the tRNA moiety to generate pre–milRNA. Our study identifies the transcriptional machinery responsible for the synthesis of fungal milRNAs and sheds light on the evolutionary origin of eukaryotic small RNAs.
Author Summary
microRNAs (miRNAs) are small RNAs that are used by many organisms to regulate a wide variety of molecular, developmental, and physiological activities. In higher eukaryotes, such as animals and plants, the majority of the independent transcribed miRNAs are produced by RNA polymerase II (Pol II), an enzyme that is also responsible for the production of most of the messenger RNAs. On the other hand, only a few tRNA–associated miRNAs are known to be produced by RNA polymerase III (Pol III), an enzyme that is responsible for the production of small sized RNAs such as tRNAs and 5s rRNA. We previously identified the first fungal miRNAs by identifying the small RNAs associated with an Argonaute protein in the filamentous fungus Neurospora crassa. In this study, we examined the role of Pol II and Pol III in the production of Neurospora miRNAs. We showed that, unlike in plants and animals, Pol III appears to be a major RNA polymerase responsible for miRNA production in this fungus. Our study identified the transcriptional machinery responsible for the synthesis of fungal miRNAs and shed light on the evolutionary origin of miRNAs.
PMCID: PMC3547838  PMID: 23349642
4.  RNA Interference in Fungi: Pathways, Functions, and Applications ▿ 
Eukaryotic Cell  2011;10(9):1148-1155.
Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora, and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.
PMCID: PMC3187057  PMID: 21724934
5.  Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi 
Molecular cell  2010;38(6):803-814.
A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.
PMCID: PMC2902691  PMID: 20417140

Results 1-5 (5)