Monika Gullerova

Cohesin Complex Promotes Transcriptional Termination between Convergent Genes in S. pombe

Transcription analyses reported in these studies reveal that convergent genes in S. pombe generate overlapping transcripts in the G1 phase of the cell cycle. We show that this double-strand (ds) RNA induces localized RNAi (Dicer and RITS) dependent transient heterochromatin structures including histone H3 lysine 9 trimethylation marks and Swi6 association. Consequently cohesin is recruited to these chromosomal positions through interaction with Swi6. In G2, localized cohesin is further concentrated into the intergenic regions of the convergent genes tested. This results in a block to further dsRNA formation by promoting gene-proximal transcription termination between the convergent genes. Cohesin release at mitosis leads to a new G1 phase with repeated dsRNA formation, transient heterochromatin, and cohesin recruitment. Our results uncover a hitherto unanticipated role for cohesin and further suggest a widespread role for the selective formation of dsRNA, heterochromatin, and subsequent cohesin recruitment in regulated transcriptional termination.

Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA

Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-stranded RNA (dsRNA), which in turn leads to induction of the interferon-response pathway and consequent cell death. Our results suggest that Pol II–associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units. Failure to do so has catastrophic effects on cell function.

Swiss army knives: non-canonical functions of nuclear Drosha and Dicer

The RNase III enzymes Drosha and Dicer are essential for the production of small non-coding RNAs (ncRNAs). In canonical RNAi, microRNAs (miRNAs) regulate gene expression by post-transcriptional gene silencing. In non-canonical RNAi, nuclear RNAi factors generate small ncRNAs that are essential for transcriptional gene silencing. Recent evidence points to the existence of additional non-canonical nuclear RNAi functions in various organisms, including in genome maintenance and editing, as well as in DNA repair. Drosha and Dicer directly regulate gene expression and RNA metabolism at different stages, such as transcriptional initiation and termination, and the processing of various RNA species, including pre-mRNAs. Furthermore, Dicer isoforms were recently discovered and attributed with roles in apoptosis, development and disease.

Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells.

We show that convergent transcription induces transcriptional gene silencing (TGS) in trans for both fission yeast and mammalian cells. This method has advantages over existing strategies to induce gene silencing. Previous studies in fission yeast have characterized TGS as a cis-specific process involving RNA interference that maintains heterochromatic regions such as centromeres. In contrast, in mammalian cells, gene silencing is known to occur through a post-transcriptional mechanism that uses exogenous short interfering RNAs or endogenous microRNAs to inactivate mRNA. We now show that the introduction of convergent transcription plasmids into either Schizosaccharomyces pombe or mammalian cells allows the production of double-stranded RNA from inserted gene fragments, resulting in TGS of endogenous genes. We predict that using convergent transcription to induce gene silencing will be a generally useful strategy and allow for a fuller molecular understanding of the biology of TGS.

Autoregulation of convergent RNAi genes in fission yeast

RNAi plays a central role in the regulation of eukaryotic genes. In Schizosaccharomyces pombe fission yeast, RNAi involves the formation of siRNA from dsRNA that acts to establish and maintain heterochromatin over centromeres, telomeres, and mating loci. We showed previously that transient heterochromatin also forms over S. pombe convergent genes (CGs). Remarkably, most RNAi genes are themselves convergent. We demonstrate here that transient heterochromatin formed by the RNAi pathway over RNAi CGs leads to their autoregulation in G1-S. Furthermore, the switching of RNAi gene orientation from convergent to tandem causes loss of their G1-S down-regulation. Surprisingly, yeast mutants with tandemized dcr1, ago1, or clr4 genes display aberrant centromeric heterochromatin, which results in abnormal cell morphology. Our results emphasize the significance of gene orientation for correct RNAi gene expression, and suggest a role for cell cycle-dependent formation of RNAi CG heterochromatin in cellular integrity.

Rct1, a Nuclear RNA Recognition Motif-Containing Cyclophilin, Regulates Phosphorylation of the RNA Polymerase II C-Terminal Domain†

ABSTRACT Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates transcription and coordinates it with pre-mRNA processing. We show here that Rct1, a nuclear multidomain cyclophilin from Schizosaccharomyces pombe, is encoded by an essential gene that interacts with the CTD and regulates its phosphorylation in vivo. Downregulation of Rct1 levels results in increased phosphorylation of the CTD at both Ser2 and Ser5 and in a commensurate decrease in RNAP II transcription. In contrast, overexpression of Rct1 decreases phosphorylation on both sites. The close association of Rct1 with transcriptionally active chromatin suggests a role in regulation of RNAP II transcriptional activity. These data, together with the pleiotropic phenotype upon Rct1 deregulation, suggest that this multidomain cyclophilin is an important player in maintaining the correct phosphorylation code of the CTD and thereby regulating CTD function.

Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage

The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform–Piwi/Argonaute/Zwille–connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks. We further demonstrate that turnover of damage-induced nuclear, double-stranded (ds) RNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes endogenous DNA damage and delays the DDR by impaired recruitment of repair factors MDC1 and 53BP1. Collectively, we place Dicer within the context of the DDR by demonstrating a DNA damage-inducible phosphoswitch that causes localized processing of nuclear dsRNA by p-Dicer to promote DNA repair.

Long Non-coding RNA

Rapid development in genome-wide transcriptional analyses has led to the discovery of a large number of non-coding transcripts, also called long non-coding RNA (lncRNA). LncRNAs harbor biological activities including regulation of protein-coding gene expression at epigenetic, transcriptional and post-transcriptional levels. They also take a part in various physiological and pathological processes, participating in cell development, immunity, disease processes and oncogenesis. Here I discuss and summarize, current knowledge about lncRNA origin, function and involvement in human disease.

Gene Silencing CUTs Both Ways

There is extensive transcription throughout the eukaryotic genome resulting in both antisense transcripts from coding regions and cryptic unstable transcripts (CUTs) from intergenic regions. In this issue, Camblong et al. (2007) demonstrate in the budding yeast that antisense transcripts, if stabilized by exosome impairment, are able to mediate gene silencing via the recruitment of histone deacetylases.

Transcription facilitates sister chromatid cohesion on chromosomal arms.

Cohesin is a multi-subunit protein complex essential for sister chromatid cohesion, gene expression and DNA damage repair. Although structurally well studied, the underlying determinant of cohesion establishment on chromosomal arms remains enigmatic. Here, we show two populations of functionally distinct cohesin on chromosomal arms using a combination of genomics and single-locus specific DNA-FISH analysis. Chromatin bound cohesin at the loading sites co-localizes with Pds5 and Eso1 resulting in stable cohesion. In contrast, cohesin independent of its loader is unable to maintain cohesion and associates with chromatin in a dynamic manner. Cohesive sites coincide with highly expressed genes and transcription inhibition leads to destabilization of cohesin on chromatin. Furthermore, induction of transcription results in de novo recruitment of cohesive cohesin. Our data suggest that transcription facilitates cohesin loading onto chromosomal arms and is a key determinant of cohesive sites in fission yeast.