Diverse Pathways Generate MicroRNA-like RNAs and Dicer-Independent Small Interfering RNAs in Fungi

Lee, Heng-Chi; Li, Liande; Gu, Weifeng; Xue, Zhihong; Crosthwaite, Susan K.; Pertsemlidis, Alexander; Lewis, Zachary A.; Freitag, Michael; Selker, Eric U.; Mello, Craig C.; Liu, Yi

doi:10.1016/j.molcel.2010.04.005

Cited by 322 publications

(460 citation statements)

References 47 publications

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“…The Neurospora crassa Argonaute protein QDE-2 (Nc QDE-2) is a close sequence homolog of eukaryotic AGOs that act in the small interfering RNA (siRNA) and miR-NA pathways [e.g., sequence identities with Hs AGO2 and Dm AGO1 are 30% and 29.7%, respectively (Fig. S1) (22,23)]. We obtained diffracting crystals of a QDE-2 fragment containing the MID and PIWI domains (amino acids 506-938) and determined the structure at 3.65 Å resolution (crystal form I; Table S1).…”

Section: Resultsmentioning

confidence: 99%

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Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Boland

Huntzinger

Schmidt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

113

118

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Argonaute proteins (AGOs) are essential effectors in RNAmediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein.Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.P roteins of the Argonaute (AGO) family play essential roles in RNA-mediated gene silencing mechanisms in eukaryotes (1, 2). They are loaded with small noncoding RNAs to form the core of RNA-induced silencing complexes, which repress the expression of target genes at the transcriptional or posttranscriptional level (1, 2). The targets to be silenced are selected through base-pairing interactions between the loaded small RNA (also known as the guide RNA) and an mRNA target containing partially or fully complementary sequences (1-3).Thus far, structural information on full-length AGOs has been available only for the homologous proteins from Archaea and Eubacteria, which preferentially use DNA as a guide (4-10). These studies revealed that AGOs consist of four domains: the N-terminal domain; the PAZ domain, which binds the 3′ end of guide RNAs/DNAs; the MID domain, which provides a binding pocket for the 5′ phosphate of guide RNAs/DNAs; and the PIWI domain, which adopts an RNase H fold and has endonucleolytic activity in some, but not all, AGOs (4-11).For the eukaryotic AGO clade of Argonaute proteins, structural information is available only for the isolated PAZ domains of Drosophila melanogaster (Dm) AGO1 and AGO2, human AGO1 (12-16) and the MID domains of human AGO2 and Neurospora crassa (Nc) QDE-2 (17, 18). Structural information is also available for PAZ domains of the PIWI clade of AGOs (19,20). These studies showed that the PAZ and MID domains of eukaryotic AGOs adopt folds similar to the prokaryotic homologs and recognize the 3′-and 5′-terminal nucleotides of the guide strand, respectively, in a similar manner to their prokaryotic counterparts (12-18).Our previous structure of the isolate...

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Section: Resultsmentioning

confidence: 99%

“…To investigate the contribution of the PIWI domain to 5′-nucleotide binding in vitro, we used size exclusion chromatography. As a substrate, we chose a 10-mer guide RNA mimic that started with a 5′-phosphorylated uridine (23). We first showed that the MID domain alone could not bind the substrate (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Boland

Huntzinger

Schmidt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

113

118

View full text Add to dashboard Cite

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“…Consistent with their ability to mediate a cancer-specific function, MRPL3 is overexpressed in nonsmall cell lung cancers (14) and MALAT1 in non-small cell lung cancers and hepatocellular carcinomas (15, 16). Although we have not examined the mechanism by which these genes promote metastasis, MRPL3 promotes the translation of mRNAs encoding mitochondrial enzymes (17) and the biogenesis of miRNA-like small RNAs (miRNAs) (18), and MALAT1 is involved in alternative splicing (19) and transcriptional activation of growth-control genes (20). These results indicate that a gain-of-function cDNA screen in a mouse model of mammary-tumor progression enables the identification of single genes implicated in lung colonization.…”

Section: Significancementioning

confidence: 97%

Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation

Gao

Chakraborty²,

Lee-Lim³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a screening platform for the isolation of genetic entities involved in metastatic reactivation. Retroviral libraries of cDNAs from fully metastatic breast-cancer cells or pooled microRNAs were transduced into breast-cancer cells that become dormant upon infiltrating the lung. Upon inoculation in the tail vein of mice, the cells that had acquired the ability to undergo reactivation generated metastatic lesions. Integrated retroviral vectors were recovered from these lesions, sequenced, and subjected to a second round of validation. By using this strategy, we isolated canonical genes and microRNAs that mediate metastatic reactivation in the lung. To identify genes that oppose reactivation, we screened an expression library encoding shRNAs, and we identified target genes that encode potential enforcers of dormancy. Our screening strategy enables the identification and rapid biological validation of single genetic entities that are necessary to maintain dormancy or to induce reactivation. This technology should facilitate the elucidation of the molecular underpinnings of these processes.forward genetic screens | metastatic reactivation | cDNA library screen | shRNA library screen | microRNA library screen T he majority of cancer-related deaths are caused by metastatic relapse (1). The process through which cancer cells acquire metastatic capacity is complex. Unrestrained proliferation, resistance to proapoptotic insults, and invasion through tissue boundaries are not sufficient for metastasis. To colonize distant organs, cancer cells must also adapt to the local microenvironment of the target organ and finally outgrow (2, 3). Mathematical modeling of clinical data and experiments in mouse models suggest that cancer cells disseminating from prevalent cancers, such as those of the breast and prostate, undergo an extended period of dormancy at premetastatic sites (4). Insights into the mechanisms that enable disseminated cancer cells to survive during dormancy and then outgrow into life-threatening lesions may lead to the identification of novel therapeutic targets for the prevention or treatment of metastatic disease.Advances in genomics and mouse modeling have fostered a renaissance of studies on metastasis (2, 3). Current approaches to the study of metastasis can be divided in two categories. In the first, genetic methods are used to modify the function of a candidate gene in intact mice or in cells that are subsequently transplanted in mice (5, 6). In the second, genomic methods, such as DNA microarray analysis or array comparative genomic hybridization (aCGH), are used to identify a restricted number of candidate genes, which are then tested in appropriate mouse models (7,8). Although these approaches have been extremely successful, they are very laborious and do not necessarily yield biologically potent mediators of metastasis. Functional genetic screens can lead to the rapid identification of strong mediators of a selectable phenotype (9, 10). In agreement with this notion, recent studies have ...

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“…The investigators hypothesized that the observed burst of rDNA-related qiRNAs inhibits protein translation as a response to DNA damage. MicroRNA-like RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) have also been reported (Lee et al 2010b). 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-like domain-containing protein, MRPL3.…”

Section: Quellingmentioning

confidence: 99%

“…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-like domain-containing protein, MRPL3. In contrast, disiRNAs do not require the known RNAi components as they originate from loci producing overlapping sense and antisense transcripts (Lee et al 2010b). Notably, it was observed that the product of qde-1, QDE-1, has DNA-and RNA-dependent biochemical activities (Lee et al 2010a).…”

Section: Quellingmentioning

confidence: 99%

Neurospora crassa, a Model System for Epigenetics Research

Aramayo

Selker

2013

Cold Spring Harbor Perspectives in Biology

132

121

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SUMMARYThe filamentous fungus Neurospora crassa has provided a rich source of knowledge on epigenetic phenomena that would have been difficult or impossible to gain from other systems. Neurospora sports features found in higher eukaryotes but absent in both budding and fission yeast, including DNA methylation and H3K27 methylation, and also has distinct RNA interference (RNAi)-based silencing mechanisms operating in mitotic and meiotic cells. This has provided an unexpected wealth of information on gene silencing systems. One silencing mechanism, named repeat-induced point mutation (RIP), has both epigenetic and genetic aspects and provided the first example of a homology-based genome defense system. A second silencing mechanism, named quelling, is an RNAi-based mechanism that results in silencing of transgenes and their native homologs. A third, named meiotic silencing, is also RNAi-based but is distinct from quelling in its time of action, targets, and apparent purpose.Outline

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Diverse Pathways Generate MicroRNA-like RNAs and Dicer-Independent Small Interfering RNAs in Fungi

Cited by 322 publications

References 47 publications

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation

Neurospora crassa, a Model System for Epigenetics Research

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