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.

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.

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.

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.

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).

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.