Polycomb, pairing and PIWI--RNA silencing and nuclear interactions.

In Drosophila, the RNA interference (RNAi) genes participate in Polycomb (Pc)-mediated transgene silencing. Recently, the involvement of the RNAi genes in Pc silencing, pairing-sensitive silencing and long-range contacts among Pc-associated sequences has been explored. These Pc-associated sequences are involved with the control of the proper expression of developmental HOX genes.

Genetics and biochemistry of RNAi in Drosophila.

RNA interference (RNAi) is the technique employing double-stranded RNA to target the destruction of homologous messenger RNAs. It has gained wide usage in genetics. While having the potential for many practical applications, it is a reflection of a much broader spectrum of small RNA-mediated processes in the cell. The RNAi machinery was originally perceived as a defense mechanism against viruses and transposons. While this is certainly true, small RNAs have now been implicated in many other aspects of cell biology. Here we review the current knowledge of the biochemistry of RNAi in Drosophila and the involvement of small RNAs in RNAi, transposon silencing, virus defense, transgene silencing, pairing-sensitive silencing, telomere function, chromatin insulator activity, nucleolar stability, and heterochromatin formation. The discovery of the role of RNA molecules in the degradation of mRNA transcripts leading to decreased gene expression resulted in a paradigm shift in the field of molecular biology. Transgene silencing was first discovered in plant cells (Matzke et al. 1989; van der Krol et al. 1990; Napoli et al. 1990) and can occur on both the transcriptional and posttranscriptional levels, but both involve short RNA moieties in their mechanism. RNA interference (RNAi) is a type of gene silencing mechanism in which a double-stranded RNA (dsRNA) molecule directs the specific degradation of the corresponding mRNA (target RNA). The technique of RNAi was first discovered in Caenorhabditis elegans in 1994 (Guo and Kemphues 1994). Later the active component was found to be a dsRNA (Fire et al. 1998). In subsequent years, it has been found to occur in diverse eukaryotes

RNA silencing in Drosophila.

Knowledge of the role of RNA in affecting gene expression has expanded in the past several years. Small RNAs serve as homology guides to target messenger RNAs for destruction at the post-transcriptional level in the experimental technique known as RNA interference and in the silencing of some transgenes. These small RNAs are also involved in sequence-specific targeting of chromatin modifications for transcriptional silencing of transgenes, transposable elements, heterochromatin and some cases of Polycomb-mediated gene silencing. RNA silencing processes in Drosophila are described.

Quantitatively increased somatic transposition of transposable elements in Drosophila strains compromised for RNAi.

In Drosophila melanogaster, small RNAs homologous to transposable elements (TEs) are of two types: piRNA (piwi-interacting RNA) with size 23-29nt and siRNA (small interfering RNA) with size 19-22nt. The siRNA pathway is suggested to silence TE activities in somatic tissues based on TE expression profiles, but direct evidence of transposition is lacking. Here we developed an efficient FISH (fluorescence in Situ hybridization) based method for polytene chromosomes from larval salivary glands to reveal new TE insertions. Analysis of the LTR-retrotransposon 297 and the non-LTR retroposon DOC shows that in the argonaut 2 (Ago2) and Dicer 2 (Dcr2) mutant strains, new transposition events are much more frequent than in heterozygous strains or wild type strains. The data demonstrate that the siRNA pathway represses TE transposition in somatic cells. Nevertheless, we found that loss of one functional copy of Ago2 or Dcr2 increases somatic transpositions of the elements at a lower level depending on the genetic background, suggesting a quantitative role for RNAi core components on mutation frequency.

Identification of Inverse Regulator-a (Inr-a) as Synonymous with Pre-mRNA Cleavage Complex II Protein (Pcf11) in Drosophila.

A common modulation of gene expression in aneuploids is an inverse correlation of the monitored gene with the dosage of another segment of the genome. Such effects can be reduced to the action of single genes. One gene previously found to modulate leaky alleles of the white eye color gene in Drosophila is Inverse regulator-a (Inr-a). Heterozygotes of mutations increase the expression of white about 2-fold, and trisomic regions surrounding the gene reduce the expression to about two-thirds of the normal diploid level. Further cytological definition of the location of this gene on the second chromosome led to a candidate pre-mRNA cleavege complex II protein (Pcf11) as the only gene in the remaining region whose mutations exhibit recessive lethality as do alleles of Inr-a. The product of Pcf11 has been implicated in transcriptional initiation, elongation, and termination reactions. Four mutant alleles showed molecular lesions predicted to lead to nonfunctional products of Pcf11. The identification of the molecular lesion of Inr-a provides insight into the basis of this common aneuploidy effect.

Reflections on the inhibition of RNAi by cell death signaling.

Mutations and most transgenes that induce ectopic cell death in Drosophila will produce an inhibitory effect on RNA interference (RNAi) in adjacent cells. When extensive cell death is sporadically induced using a heat shock promoted-head involution defective (hs-hid) transgene, molecular attributes of this inhibition can be studied. For a Green Fluorescent Protein (GFP) RNAi construct, cell death causes a greater accumulation of the mature mRNA and the double stranded RNA with an accompanying reduction in the homologous siRNAs. Endogenous transposable element expression is increased and there is an overall reduction in their corresponding siRNAs. The implications of this finding for the conduct of RNAi and potential reasons for its existence are discussed.

Inhibition of RNA interference and modulation of transposable element expression by cell death in Drosophila.

RNA interference (RNAi) regulates gene expression by sequence-specific destruction of RNA. It acts as a defense mechanism against viruses and represses the expression of transposable elements (TEs) and some endogenous genes. We report that mutations and transgene constructs that condition cell death suppress RNA interference in adjacent cells in Drosophila melanogaster. The reversal of RNAi is effective for both the white (w) eye color gene and green fluorescent protein (GFP), indicating the generality of the inhibition. Antiapoptotic transgenes that reverse cell death will also reverse the inhibition of RNAi. Using GFP and a low level of cell death produced by a heat shock-head involution defective (hs-hid) transgene, the inhibition appears to occur by blocking the conversion of double-stranded RNA (dsRNA) to short interfering RNA (siRNA). We also demonstrate that the mus308 gene and endogenous transposable elements, which are both regularly silenced by RNAi, are increased in expression and accompanied by a reduced level of siRNA, when cell death occurs. The finding that chronic ectopic cell death affects RNAi is critical for an understanding of the application of the technique in basic and applied studies. These results also suggest that developmental perturbations, disease states, or environmental insults that cause ectopic cell death would alter transposon and gene expression patterns in the organism by the inhibition of small RNA silencing processes.

Re-evaluation of the function of the male specific lethal complex in Drosophila.

A set of proteins and noncoding RNAs, referred to as the male specific lethal (MSL) complex, is present on the male X chromosome in Drosophila and has been postulated to be responsible for dosage compensation of this chromosome - the up-regulation of its expression to be equal to that of two X chromosomes in females. This hypothesis is evaluated in view of lesser known aspects of dosage compensation such as the fact that metafemales with three X chromosomes also have equal expression to normal females, which would require a down-regulation of each gene copy. Moreover, when this complex is ectopically expressed in females or specifically targeted to a reporter in males, there is no increase in expression of the genes or targets with which it is associated. These observations are not consistent with the hypothesis that the MSL complex conditions dosage compensation. A synthesis is described that can account for these observations.

Phosphorylation regulates integration of the yeast Ty5 retrotransposon into heterochromatin.

The yeast Ty5 retrotransposon preferentially integrates into heterochromatin at the telomeres and silent mating loci. Target specificity is mediated by a small domain of Ty5 integrase (the targeting domain, TD), which interacts with the heterochromatin protein Sir4 and tethers the integration complex to target sites. Here we demonstrate that TD is phosphorylated and that phosphorylation is required for interaction with Sir4. The yeast cell, therefore, through posttranslational modification, controls Ty5's mutagenic potential: when TD is phosphorylated, insertions occur in gene-poor heterochromatin, thereby minimizing deleterious consequences of transposition; however, in the absence of phosphorylation, Ty5 integrates throughout the genome, frequently causing mutations. TD phosphorylation is reduced under stress conditions, specifically starvation for amino acids, nitrogen, or fermentable carbon. This suggests that Ty5 target specificity changes in response to nutrient availability and is consistent with McClintock's hypothesis that mobile elements restructure host genomes as an adaptive response to environmental challenge.

Global analysis of siRNA-mediated transcriptional gene silencing.

The RNAi machinery is not only involved with post-transcriptional degradation of messenger RNAs, but also used for targeting of chromatin changes associated with transcriptional silencing. Two recent papers determine the global patterns of gene expression and chromatin modifications produced by the RNAi machinery in fission yeast.(9, 10) The major sites include the outer centromere repeats, the mating-type locus and subtelomeric regions. By comparison, studies of Arabidopsis heterochromatin also implicate transposons as a major target for silencing. Analyses of siRNA libraries from Drosophila, nematodes and Arabidopsis indicate that major repeats at centromeres, telomeres and transposable elements are likely targets of RNAi. Also, intergenic regions are implicated as targets in Arabidopsis.