Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes-more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca(2+) signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes

The finding that small non-coding RNAs (ncRNAs) are able to control gene expression in a sequence specific manner has had a massive impact on biology. Recent improvements in high throughput sequencing and computational prediction methods have allowed the discovery and classification of several types of ncRNAs. Based on their precursor structures, biogenesis pathways and modes of action, ncRNAs are classified as small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs), promoter associate RNAs (pRNAs), small nucleolar RNAs (snoRNAs) and sno-derived RNAs. Among these, miRNAs appear as important cytoplasmic regulators of gene expression. miRNAs act as post-transcriptional regulators of their messenger RNA (mRNA) targets via mRNA degradation and/or translational repression. However, it is becoming evident that miRNAs also have specific nuclear functions. Among these, the most studied and debated activity is the miRNA-guided transcriptional control of gene expression. Although available data detail quite precisely the effectors of this activity, the mechanisms by which miRNAs identify their gene targets to control transcription are still a matter of debate. Here, we focus on nuclear functions of miRNAs and on alternative mechanisms of target recognition, at the promoter lavel, by miRNAs in carrying out transcriptional gene silencing.

In plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. This phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. Transgene-induced gene silencing has been termed quelling in Neurospora crassa and co-suppression in plants. Quelling-defective (qde) mutants of N. crassa, in which transgene-induced gene silencing is impaired, have been isolated. Here we report the cloning of qde-1, the first cellular component of the gene-silencing mechanism to be isolated, which defines a new gene family conserved among different species including plants, animals and fungi. The qde-1 gene product is similar to an RNA-dependent RNA polymerase found in the tomato. The identification of qde-1 strongly supports models that implicate an RNA-dependent RNA polymerase in the post-transcriptional gene-silencing mechanism. The presence of qde-1 homologues in a variety of species of plants and fungi indicates that a conserved gene-silencing mechanism may exist, which could have evolved to preserve genome integrity and to protect the genome against naturally occurring transposons and viruses.

The molecular mechanisms involved in transgene‐induced gene silencing (‘quelling’) in Neurospora crassa were investigated using the carotenoid biosynthetic gene albino‐1 (al‐1) as a visual marker. Deletion derivatives of the al‐1 gene showed that a transgene must contain at least approximately 132 bp of sequences homologous to the transcribed region of the native gene in order to induce quelling. Transgenes containing only al‐1 promoter sequences do not cause quelling. Specific sequences are not required for gene silencing, as different regions of the al‐1 gene produced quelling. A mutant defective in cytosine methylation (dim‐2) exhibited normal frequencies and degrees of silencing, indicating that cytosine methylation is not responsible for quelling, despite the fact that methylation of transgene sequences frequently is correlated with silencing. Silencing was shown to be a dominant trait, operative in heterokaryotic strains containing a mixture of transgenic and non‐transgenic nuclei. This result indicates that a diffusable, trans‐acting molecule is involved in quelling. A transgene‐derived, sense RNA was detected in quelled strains and was found to be absent in their revertants. These data are consistent with a model in which an RNA‐DNA or RNA‐RNA interaction is involved in transgene‐induced gene silencing in Neurospora.

We report the isolation of 15 Neurospora crassa mutants defective in ''quelling'' or transgene-induced gene silencing. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. We show that when qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and͞or targeting͞degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.