A functional connection between the Microprocessor and a variant NEXT complex.

In mammalian cells, primary miRNAs are cleaved at their hairpin structures by the Microprocessor complex, whose core is composed of DROSHA and DGCR8. Here, we show that 5' flanking regions, resulting from Microprocessor cleavage, are targeted by the RNA exosome in mouse embryonic stem cells (mESCs). This is facilitated by a physical link between DGCR8 and the nuclear exosome targeting (NEXT) component ZCCHC8.

DNA-directed termination of mammalian RNA polymerase II.

The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage of the nascent RNA. The RNAPII-associated cleavage product is then degraded by XRN2, dislodging RNAPII from the DNA template. In contrast, prokaryotic RNAP and eukaryotic RNAPIII often terminate directly at T-tracts in the coding DNA strand.

Nuclear sorting of short RNA polymerase II transcripts.

Mammalian genomes produce an abundance of short RNA. This is, to a large extent, due to the genome-wide and spurious activity of RNA polymerase II (RNAPII). However, it is also because the vast majority of initiating RNAPII, regardless of the transcribed DNA unit, terminates within a ∼3-kb early "pausing zone.

Substrate recognition principles for the PP2A-B55 protein phosphatase.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in substrates bind B55 through an evolutionary conserved mechanism.

Protocol for generating customizable and reproducible plots of sequencing coverage data using the seqNdisplayR package.

The widespread usage of next-generation sequencing methods for functional genomics studies requires standardized tools for consistent visualization of the associated data. Here, we present seqNdisplayR, an R package for plotting standard sequencing data coverage within a genomic region of interest in a customizable and reproducible manner. We describe steps for installing software, preparing data files, choosing options, and plotting data.

Substrate recognition principles for the PP2A-B55 protein phosphatase.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that α-helices in substrates bind B55 through an evolutionary conserved mechanism.

Dual agonistic and antagonistic roles of ZC3H18 provide for co-activation of distinct nuclear RNA decay pathways.

The RNA exosome is a versatile ribonuclease. In the nucleoplasm of mammalian cells, it is assisted by its adaptors the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts.

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts.

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4.

Computational identification of signals predictive for nuclear RNA exosome degradation pathway targeting.

The RNA exosome degrades transcripts in the nucleoplasm of mammalian cells. Its substrate specificity is mediated by two adaptors: the 'nuclear exosome targeting (NEXT)' complex and the 'poly(A) exosome targeting (PAXT)' connection. Previous studies have revealed some DNA/RNA elements that differ between the two pathways, but how informative these features are for distinguishing pathway targeting, or whether additional genomic features that are informative for such classifications exist, is unknown.

Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression.

Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels.

Control of non-productive RNA polymerase II transcription via its early termination in metazoans.

Transcription establishes the universal first step of gene expression where RNA is produced by a DNA-dependent RNA polymerase. The most versatile of eukaryotic RNA polymerases, RNA polymerase II (Pol II), transcribes a broad range of DNA including protein-coding and a variety of non-coding transcription units. Although Pol II can be configured as a durable enzyme capable of transcribing hundreds of kilobases, there is reliable evidence of widespread abortive Pol II transcription termination shortly after initiation, which is often followed by rapid degradation of the associated RNA.

Rapid factor depletion highlights intricacies of nucleoplasmic RNA degradation.

Turnover of nucleoplasmic transcripts by the mammalian multi-subunit RNA exosome is mediated by two adaptors: the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely utilized long-term factor depletion strategies, facilitating the appearance of indirect phenotypes. Here, we rapidly deplete NEXT, PAXT and core exosome components, uncovering the direct consequences of their acute losses.

hnRNPH1-MTR4 complex-mediated regulation of stability is critical for expression.

Many long noncoding RNAs (lncRNAs) are localized in the nucleus and play important roles in various biological processes, including cell proliferation, differentiation and antiviral response. Yet, it remains unclear how some nuclear lncRNAs are turned over. Here we show that the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) controls expression levels of , a lncRNA involved in the formation of nuclear paraspeckles.

The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers.

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers.

Global view on the metabolism of RNA poly(A) tails in yeast Saccharomyces cerevisiae.

The polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases.

Three-layered control of mRNA poly(A) tail synthesis in .

Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in , we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components.

3' End sequencing of pA and pA RNAs.

The identity and metabolism of RNAs are often governed by their 5' and 3' ends. Single gene loci produce a variety of transcript isoforms, varying primarily in their RNA 3' end status and consequently facing radically different cellular fates. Knowledge about RNA termini is therefore key to understanding the diverse RNA output from individual transcription units.

ARS2/SRRT: at the nexus of RNA polymerase II transcription, transcript maturation and quality control.

ARS2/SRRT is an essential eukaryotic protein that has emerged as a critical factor in the sorting of functional from non-functional RNA polymerase II (Pol II) transcripts. Through its interaction with the Cap Binding Complex (CBC), it associates with the cap of newly made RNAs and acts as a hub for competitive exchanges of protein factors that ultimately determine the fate of the associated RNA. The central position of the protein within the nuclear gene expression machinery likely explains why its depletion causes a broad range of phenotypes, yet an exact function of the protein remains elusive.

Integrator is a genome-wide attenuator of non-productive transcription.

Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter.

nucleates a transcription inhibitory complex to balance neuronal differentiation.

Circular RNAs are important for many cellular processes but their mechanisms of action remain poorly understood. Here, we map circRNA inventories of mouse embryonic stem cells, neuronal progenitor cells and differentiated neurons and identify hundreds of highly expressed circRNAs. By screening several candidate circRNAs for a potential function in neuronal differentiation, we find that represses expression of key neuronal markers, suggesting that this molecule negatively regulates neuronal differentiation.

Integrator restrains paraspeckles assembly by promoting isoform switching of the lncRNA .

RNA 3' end processing provides a source of transcriptome diversification which affects various (patho)-physiological processes. A prime example is the transcript isoform switch that leads to the read-through expression of the long non-coding RNA , at the expense of the shorter polyadenylated transcript . is required for assembly of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy.