Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization

Transcription of the major neurospora crassa microRNA-like small RNAs relies on RNA polymerase III.

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

RNAi Factors Are Present and Active in Human Cell Nuclei

RNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here, we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP, and TRNC6A/GW182 are in the human nucleus and associate together in multiprotein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription

Youth, Gender and Pornography - A Qualitative Study in Sweden

Introduction and objectives: The visibility and accessibility of pornography in public space has increased dramatically over the last decade. In many Western societies, among them Sweden, there is a wide-spread concern about the implications and consequences of this development, especially for young people. However, seldom are young people’s own voices being heard in this debate. Our research tries to remedy this by asking teenagers about their experiences, views and relationships to pornography. Methods: Data were collected in 2006 through qualitative research interviews and focus groups with young people; 73 informants between 14 and 20 years of age are included in the study, 36 girls and 37 boys. Results: The increasing accessibility of pornography has contributed to a process of normalization with regard to young people’s attitudes and behaviours in relation to pornography. This change, however, is related to both age and gender, which allows us to talk about gender specific pornography careers. Our study also confirms the influence and growing importance of the pornographic script as a frame of reference or behavioural code that more or less explicitly prescribes how to look and what to do. However, it seems that most of our interviewees have acquired the necessary skills in how to navigate in the pornographic landscape in a sensible and reflective manner. Most of them seem to have the ability to distinguish between pornographic fantasies and narratives on the one hand, and real life sexual interaction and relationships on the other. Conclusions: Growing up in a society with an easily accessible pornography both lead to a defused view on sexuality and to a critical and reflective outlook. The impact of the so-called pornographic script is clear. However, at the same time the script brings to the fore an ambivalence towards sexuality, and to pornography specifically. It contains both pleasure and harmfulness in a way that seems to be both tempting and frightening

DNA methylation is induced in the promoter region of an artificial convergent transcription construct upon induction of transcription.

<p>(A and B) MeDIP results showing the DNA methylation status of the <i>ccg-1</i> promoter, <i>luc</i> gene body, and the <i>qa-2</i> promoter of the indicated construct. The artificial construct <i>Pqa-2:cul:1-gccP</i> (A) or <i>Pqa-2:cul</i> (B) resides at the <i>his-3</i> locus. The top cartoon of each panel depicts the architecture of the construct and black bars indicate the approximate location of primer sets. Three independent repeats were performed. Values are mean ± s.d. (C) Distribution of DNA methylation at the endogenous location of the <i>qa-2</i> promoter of the <i>Pqa-2:cul:1-gccP</i> strain. (D) Distribution of DNA methylation around the <i>Pqa-2:cul:1-gccP</i> construct at the <i>his-3</i> locus with/without the activation of the <i>qa-2</i> promoter. In panels A-D, QA+ and QA- indicate presence or absence of quinic acid (QA), respectively. DNA methylation was measured with MeDIP. (E) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#s2" target="_blank">Results</a> of bisulfite PCR in the region upstream of the <i>qa-2</i> promoter of the <i>Pqa-2:cul:1-gccP</i> construct. Two aliquots of genomic DNA from <i>Pqa-2:cul:1-gccP</i> strain, one digested with <i>Bfu</i>CI and one untreated, were subject to bisulfite treatment and sequencing, respectively (strategy 2 of bisulfite sequencing, primer sequences in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#pgen.1003761.s012" target="_blank">table S4</a>). Each row of circles represents the order and number of cytosines in the subcloned sequence. Opened and filled circles indicate unmethylated and methylated cytosine, respectively.</p

DNA methylation and disiRNA in <i>disi-47</i> locus requires transcription.

<p>(A) The disiRNA distribution at <i>disi-47</i> locus in wild-type and <i>wc-2^KO</i> strain. Arrows indicate the transcription start sites. dLRE and pLRE are the two WC complex binding sites. The red bars indicate approximate primer set locations (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#pgen.1003761.s009" target="_blank">Table S1</a> for primer sequences). (B) RT-qPCR analyses of <i>frq</i> mRNA. (C) RT-qPCR analyses of the transcripts in the <i>frq</i> promoter region from a wild-type strain grown in constant light (LL) or constant darkness (DD) conditions and the <i>wc-2^KO</i> strain grown in LL. (D) MeDIP results of the dLRE region in the wild-type (LL and DD conditions) and the <i>wc-2^KO</i> strains (LL). In (B), (C) and (D), three independent repeats were performed. Values are mean ± s.d.</p