Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination.

Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

HP1(Swi6) mediates the recognition and destruction of heterochromatic RNA transcripts.

HP1 proteins are major components of heterochromatin, which is generally perceived to be an inert and transcriptionally inactive chromatin structure. Yet, HP1 binding to chromatin is highly dynamic and robust silencing of heterochromatic genes can involve RNA processing. Here, we demonstrate by a combination of in vivo and in vitro experiments that the fission yeast HP1(Swi6) protein guarantees tight repression of heterochromatic genes through RNA sequestration and degradation. Stimulated by positively charged residues in the hinge region, RNA competes with methylated histone H3K9 for binding to the chromodomain of HP1(Swi6). Hence, HP1(Swi6) binding to RNA is incompatible with stable heterochromatin association. We propose a model in which an ensemble of HP1(Swi6) proteins functions as a heterochromatin-specific checkpoint, capturing and priming heterochromatic RNAs for the RNA degradation machinery. Sustaining a functional checkpoint requires continuous exchange of HP1(Swi6) within heterochromatin, which explains the dynamic localization of HP1 proteins on heterochromatin.

RNAi keeps Atf1-bound stress response genes in check at nuclear pores.

RNAi pathways are prevalent throughout the eukaryotic kingdom and are well known to regulate gene expression on a post-transcriptional level in the cytoplasm. Less is known about possible functions of RNAi in the nucleus. In the fission yeast Schizosaccharomyces pombe, RNAi is crucial to establish and maintain centromeric heterochromatin and functions to repress genome activity by a chromatin silencing mechanism referred to as cotranscriptional gene silencing (CTGS). Mechanistic details and the physiological relevance of CTGS are unknown. Here we show that RNAi components interact with chromatin at nuclear pores to keep stress response genes in check. We demonstrate that RNAi-mediated CTGS represses stress-inducible genes by degrading mRNAs under noninduced conditions. Under chronic heat stress conditions, a Dicer thermoswitch deports Dicer to the cytoplasm, thereby disrupting CTGS and enabling expression of genes implicated in the acquisition of thermotolerance. Taken together, our work highlights a role for nuclear pores and the stress response transcription factor Atf1 in coordinating the interplay between the RNAi machinery and the S. pombe genome and uncovers a novel mode of RNAi regulation in response to an environmental cue.

Anhydrous Hydrogen Fluoride Cleavage in Boc Solid Phase Peptide Synthesis.

Solid phase peptide synthesis using tert-butyloxycarbonyl/benzyl chemistry (Boc-SPPS) is important for producing peptides for fundamental research as well as for clinical use. During Boc-SPPS, liquid anhydrous hydrogen fluoride (HF) is used to remove the side chain protecting groups of the assembled peptide and to release it from the resin. Here, we provide a detailed protocol for "HF cleavage," aiming to improve accessibility and the use of this valuable and well-validated technique.

Nuclear organisation and RNAi in fission yeast.

Over the last decade, the fission yeast Schizosaccharomyces pombe has been used extensively for investigating RNA interference (RNAi)-mediated heterochromatin assembly. However, only recently have studies begun to shed light on the 3D organisation of chromatin and the RNAi machinery in the fission yeast nucleus. These studies indicate association of repressive and active chromatin with different regions of the nuclear periphery, similar to other model organisms, and clustering of functionally related genomic features. Unexpectedly, RNAi factors were shown to associate with nuclear pores and were implicated in the regulation of genomic features outside of the well-studied heterochromatic regions. Nuclear organisation is likely to contribute to substrate specificity of the RNAi pathway. However, further studies are required to elucidate the exact mechanisms and functional importance of this nuclear organisation.

Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe.

In the fission yeast S. pombe, the RNA interference (RNAi) pathway is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic silencing of centromeric repeats. Here, we demonstrate that RNAi also functions to repress genomic elements other than constitutive heterochromatin. Using DNA adenine methyltransferase identification (DamID), we show that the RNAi proteins Dcr1 and Rdp1 physically associate with some euchromatic genes, noncoding RNA genes and retrotransposon long terminal repeats, and that this association is independent of the Clr4 histone methyltransferase. Physical association of RNAi with chromatin is sufficient to trigger a silencing response but not to assemble heterochromatin. The mode of silencing at the newly identified RNAi targets is consistent with a co-transcriptional gene silencing model, as proposed earlier, and functions with trace amounts of siRNAs. We anticipate that similar mechanisms could also be operational in other eukaryotes.