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

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.

Chromatin-associated YTHDC1 coordinates heat-induced reprogramming of gene expression.

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.

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

To 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.

Selective termination of lncRNA transcription promotes heterochromatin silencing and cell differentiation.

Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes.

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

Regularly positioned nucleosomes are a common feature of 5' ends of most eukaryotic genes. A series of three studies, Shim et al (2012) and Pointner et al (2012) in this issue of The EMBO Journal and Hennig et al (2012) in EMBO Reports, now show that in the fission yeast Schizosaccharomyces pombe this intragenic nucleosome positioning mostly requires two ATP-dependent remodellers of the CHD family, Hrp1 and Hrp3. Moreover, they suggest that Hrp1- and Hrp3-dependent nucleosome spacing contributes to the silencing of cryptic antisense transcription.

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.

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

RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast.

SMC5 and MMS21 are required for chromosome cohesion and mitotic progression.

Members of the structural maintenance of chromosome (SMC) protein family have essential functions during mitosis, ensuring chromosome condensation (SMC2/4) and cohesion (SMC1/3). The SMC5/6 complex has been implicated in a variety of DNA maintenance processes but unlike the other SMC proteins, SMC5/6 have not been attributed any role in mitosis. Here, we find that ablation of either SMC5 or the SUMO-ligase MMS21 leads to premature sister chromatid separation prior to anaphase. The failure of normal chromosome alignment activates the spindle assembly checkpoint and blocks mitotic progression. Interestingly, there is no similar mitotic response to ablation of SMC6. Further, we show that mitotic SMC5 co-elutes from column fractions that contain MMS21 but lack SMC6. Our results thus establish that SMC5 is crucial for mitotic progression and maintenance of sister chromatid cohesion during mitosis, and that this role of SMC5 seems to be independent of the SMC5/6 complex.

Common themes in siRNA-mediated epigenetic silencing pathways.

Small 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.