Summary Protecting the genome from transposable element (TE) mobilization is critical for germline development. In Drosophila, Piwi proteins and their bound small RNAs (piRNAs) provide a potent defense against TE activity. TE targeting piRNAs are processed from TE-dense heterochromatic loci termed ‘piRNA clusters’. While piRNA biogenesis from cluster precursors is beginning to be understood, little is known about piRNA cluster transcriptional regulation. Here we show that deposition of histone 3 lysine 9 by the methyltransferase dSETDB1 (egg) is required for piRNA cluster transcription. In the absence of dSETDB1, cluster precursor transcription collapses in germline and somatic gonadal cells and TEs are activated, resulting in germline loss and a block in germline stem cell differentiation. We propose that heterochromatin protects the germline by activating the piRNA pathway.
Compact but non-native intermediates have been implicated in the hierarchical folding of several large RNAs, but there is little information on their structure. In this article, ribonuclease and hydroxyl radical cleavage protection assays showed that base pairing of core helices stabilize a compact state of a small group I ribozyme from Azoarcus pre-tRNA ile . Base pairing of the ribozyme core requires 10-fold less Mg 2؉ than stable tertiary interactions, indicating that assembly of helices in the catalytic core represents a distinct phase that precedes the formation of native tertiary structure. Tertiary folding occurs in <100 ms at 37°C. Such rapid folding is unprecedented among group I ribozymes and illustrates the association between structural complexity and folding time. A 3D model of the Azoarcus ribozyme was constructed by identifying homologous sequence motifs in rRNA. The model reveals distinct structural features, such as a large interface between the P4 -P6 and P3-P9 domains, that may explain the unusual stability of the Azoarcus ribozyme and the cooperativity of folding.RNA modeling ͉ RNA structure ͉ metal ions ͉ hydroxyl radical footprinting T he assembly of RNA into functional structures underlies many steps in gene expression and regulation. Recent work has outlined the Mg 2ϩ -dependent folding pathways of large ribozymes (1, 2). Experimental and theoretical results suggest that the initial association of divalent cations induces collapse of the extended RNA chain into more compact structures that favor formation of tertiary interactions (3-8). The structure of the compact intermediates and the extent to which these interactions lead to the native conformation are not yet characterized.On the one hand, individual domains of the Tetrahymena group I ribozyme and the catalytic domain of RNase P fold on a time scale (10-100 ms) similar to the initial collapse transitions of the ribozyme (6, 9, 10). On the other hand, nonspecific collapse results in an ensemble of native and non-native conformations. Many larger RNAs, such as the Tetrahymena group I and Bacillus subtilis RNase P ribozymes, fold slowly in vitro because a large fraction of the RNA population becomes trapped in misfolded intermediates (11,12). An important question is whether RNAs with a simpler 3D architecture are more likely to fold directly to the native structure, as expected from theoretical models (4). Furthermore, we want to know whether the likelihood of misfolding can be predicted from the specificity of counterion-induced collapse.We investigated the Mg 2ϩ -dependent equilibrium folding pathway and constructed a 3D model of the Azoarcus group I intron. The 205-nt group IC3 intron in the pre-tRNA ile of the Azoarcus bacterium is the smallest known self-splicing group I intron (13). It retains the conserved catalytic core common to all group I introns, but lacks the peripheral domains that stabilize folding intermediates of the larger Tetrahymena intron (14).We observed two macroscopic folding transitions in the Azoarcus ribozyme...
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