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.

Dicer is a central enzymatic player in RNA interference (RNAi) 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, but that its nuclear levels are tightly regulated. In its nuclear manifestation, Dicer interacts with RNA polymerase II (Pol II) at actively-transcribed gene loci. Loss of Dicer causes the appearance of endogenous dsRNA, leading 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 non-coding RNA transcription units. Failure to do so has catastrophic effects on cell function.

AtCyp59 and its orthologs from different organisms belong to a family of modular proteins consisting of a peptidyl-prolyl cistrans isomerase (PPIase) domain, followed by an RNA recognition motif (RRM), and a C-terminal domain enriched in charged amino acids. AtCyp59 was identified in a yeast two-hybrid screen as an interacting partner of the Arabidopsis SR protein SCL33/ SR33. The interaction with SCL33/SR33 and with a majority of Arabidopsis SR proteins was confirmed by in vitro pull-down assays. Consistent with these interactions, AtCyp59 localizes to the cell nucleus, but it does not significantly colocalize with SR proteins in nuclear speckles. Rather, it shows a punctuate localization pattern resembling transcription sites. Indeed, by using yeast two-hybrid, in vitro pull-down, and immunoprecipitation assays, we found that AtCyp59 interacts with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Ectopic expression of the tagged protein in Arabidopsis cell suspension resulted in highly reduced growth that is most probably due to reduced phosphorylation of the CTD. Together our data suggest a possible function of AtCyp59 in activities connecting transcription and pre-mRNA processing. We discuss our data in the context of a dynamic interplay between transcription and pre-mRNA processing.

Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3 ′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice.

Recent work implicated human Dicer in the DNA damage response. Burger et al. show that DNA damage induces phosphorylation of Dicer and promotes DNA repair in the nucleus.

DNA is constantly exposed to endogenous and exogenous damage. Various types of DNA repair counteract highly toxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilizes small RNA species, which are produced as long non-coding (nc)RNA precursors and promote recognition of DSBs. However, regulatory principles that control production of such transcripts remain largely elusive. Here we show that the Abelson tyrosine kinase c-Abl/ABL1 causes formation of RNA polymerase II (RNAPII) foci, predominantly phosphorylated at carboxy-terminal domain (CTD) residue Tyr1, at DSBs. CTD Tyr1-phosphorylated RNAPII (CTD Y1P) synthetizes strand-specific, damage-responsive transcripts (DARTs), which trigger formation of double-stranded (ds)RNA intermediates via DNA–RNA hybrid intermediates to promote recruitment of p53-binding protein 1 (53BP1) and Mediator of DNA damage checkpoint 1 (MDC1) to endogenous DSBs. Interference with transcription, c-Abl activity, DNA–RNA hybrid formation or dsRNA processing impairs CTD Y1P foci formation, attenuates DART synthesis and delays recruitment of DDR factors and DSB signalling. Collectively, our data provide novel insight in RNA-dependent DDR by coupling DSB-induced c-Abl activity on RNAPII to generate DARTs for consequent DSB recognition.

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.

In mammalian cells, small non-coding RNAs (sncRNAs) negatively regulate gene expression in a pathway known as RNA interference (RNAi). RNAi can be categorized into post-transcriptional gene silencing (PTGS), which involves the cleavage of target messenger RNA (mRNA) or inhibition of translation in the cytoplasm, and transcriptional gene silencing (TGS), which is mediated by the establishment of repressive epigenetic marks at target loci. Transfer RNAs (tRNAs), which are essential for translation, can be processed into small ncRNAs, termed tRNA-derived small RNAs (tsRNAs). The biogenesis of tsRNAs and their role in gene expression regulation has not yet been fully understood. Here, we show that Dicer dependent tsRNAs promote gene silencing through a mechanism distinct from PTGS and TGS. tsRNAs can lead to downregulation of target genes by targeting introns via nascent RNA silencing (NRS) in nuclei. Furthermore, we show that Ago2 slicer activity is required for this mechanism. Synthetic tsRNAs can significantly reduce expression of a target gene at both RNA and protein levels. Target genes regulated by NRS are associated with various diseases, which further underpins its biological significance. Finally, we show that NRS is evolutionarily conserved and has the potential to be explored as a novel synthetic sRNA based therapeutic.