Hexanucleotide motifs mediate recruitment of the RNA elimination machinery to silent meiotic genes

The selective elimination system blocks the accumulation of meiosis-specific mRNAs during the mitotic cell cycle in fission yeast. These mRNAs harbour a region, the determinant of selective removal (DSR), which is recognized by a YTH-family RNA-binding protein, Mmi1. Mmi1 directs target transcripts to destruction in association with nuclear exosomes. Hence, the interaction between DSR and Mmi1 is crucial to discriminate mitosis from meiosis. Here, we show that Mmi1 interacts with repeats of the hexanucleotide U(U/C)AAAC that are enriched in the DSR. Disruption of this ‘DSR core motif’ in a target mRNA inhibits its elimination. Tandem repeats of the motif can function as an artificial DSR. Mmi1 binds to it in vitro. Thus, a core motif cluster is responsible for the DSR activity. Furthermore, certain variant hexanucleotide motifs can augment the function of the DSR core motif. Notably, meiRNA, which composes the nuclear Mei2 dot required to suppress Mmi1 activity during meiosis, carries numerous copies of the core/augmenting motifs on its tail and is indeed degraded by the Mmi1/exosome system, indicating its likely role as decoy bait for Mmi1

Nucleosome positioning and transcription: fission yeast CHD remodellers make their move

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Common themes in siRNA-mediated epigenetic silencing pathways.

International audienceSmall interfering RNAs (siRNAs) act through RNA interference (RNAi) pathways to silence gene expression either at the transcriptional or post-transcriptional level. Here, we review mechanisms and functions of siRNA-mediated silencing pathways that promote chromatin modifications in the fission yeast Schizosaccharomyces pombe, plants and animals. In fission yeast, siRNAs are involved in heterochromatin formation and key aspects of the underlying siRNA-dependent pathway have been uncovered. Two RNAi complexes, the RNA-Induced Transcriptional Silencing complex (RITS), which contains a siRNA bound to an Argonaute protein, and the RNA-Directed RNA polymerase Complex (RDRC) are critical components of the pathway. In addition, this pathway implicates non-coding nascent transcripts synthesized by RNA polymerase II (RNApII) and the RNApII itself. In Arabidopsis thaliana, the RNA-directed DNA methylation (RdDM) pathway appears to share a similar set of proteins and enzymatic activities, suggesting that, beyond certain aspects that are specific to each pathway, part of the siRNA-mediated epigenetic silencing mechanisms are conserved between fission yeast and plants. Moreover, in both organisms the pathways target repetitive DNA sequences. This conservation of mechanisms and genomic targets might actually extend to animals as recent investigations revealed the existence of endogenous siRNA-based pathways directed against repetitive DNA sequences in flies and mammals

Protocol to study the role of a human nuclear m6A RNA reader on chromatin-associated RNA targets

International audienceHere, we present a detailed protocol to study the role of human YTHDC1, a nuclear m6A RNA reader, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins

Protocol to study the role of a human nuclear m6A RNA reader on chromatin-associated RNA targets

Summary: Here, we present a detailed protocol to study the role of a human nuclear m6A RNA reader, YTHDC1, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins.For complete details on the use and execution of this protocol, please refer to Timcheva et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex

International audienceTo eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1 - Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture

Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast

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