Nuclear Exosome Targeting Complex Research Articles

The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex

The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA invitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.

A restrictor complex of ZC3H4, WDR82, and ARS2 integrates with PNUTS to control unproductive transcription

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.

What’s NEXT for the exosome?

Two recent studies by Gerlach etal. (2022) and Puno and Lima (2022) provide new structural and functional insight into the assembly of the nuclear exosome targeting complex (NEXT) and how it may target specific classes of RNA for degradation.

Structure and regulation of the nuclear exosome targeting complex guides RNA substrates to the exosome

SummaryIn mammalian cells, spurious transcription results in a vast repertoire of unproductive non-coding RNAs, whose deleterious accumulation is prevented by rapid decay. The nuclear exosome targeting (NEXT) complex plays a central role in directing non-functional transcripts to exosome-mediated degradation, but the structural and molecular mechanisms remain enigmatic. Here, we elucidated the architecture of the human NEXT complex, showing that it exists as a dimer of MTR4-ZCCHC8-RBM7 heterotrimers. Dimerization preconfigures the major MTR4-binding region of ZCCHC8 and arranges the two MTR4 helicases opposite to each other, with each protomer able to function on many types of RNAs. In the inactive state of the complex, the 3′ end of an RNA substrate is enclosed in the MTR4 helicase channel by a ZCCHC8 C-terminal gatekeeping domain. The architecture of a NEXT-exosome assembly points to the molecular and regulatory mechanisms with which the NEXT complex guides RNA substrates to the exosome.

Atypical monocytes conspire with nuclear exosomes to promote organ fibrosis

Li discusses the discovery of novel mechanisms and pathways involving atypical monocytes and nuclear paraspeckles in organ fibrosis. Li discusses the discovery of novel mechanisms and pathways involving atypical monocytes and nuclear paraspeckles in organ fibrosis. CITATIONS Fukushima K, Satoh T, Sugihara F, et al. Dysregulated expression of the nuclear exosome targeting complex component Rbm7 in non-hematopoietic cells licenses the development of fibrosis. Immunity. 2020;52:542–556. Satoh T, Nakagawa K, Sugihara F, et al. Identification of atypical monocyte and committed progenitor involved in fibrosis. Nature. 2017;541:96–101. Fibrosis is a significant topic in transplantation. It is the final presentation of a plethora of chronic diseases, including chronic rejection, and we do not have an effective therapy. The Akira group in Japan reported the discovery of a unique monocytic cell type in tissue fibrosis called segregated-nucleus–containing atypical monocytes (SatMs; Satoh et al). The group recently described exciting new molecular mechanisms involving nuclear exosomes and the death of parenchymal cells in the recruitment of SatMs to fibrotic organs (Fukushima et al). These findings are of great interest to the field of organ transplantation, especially in tackling some of the key problems in chronic rejection. The authors used a chemically induced lung fibrosis model in mice, in which the intratracheal injection of bleomycin led to excessive lung inflammation in the first week due to airway epithelial injury, followed by the development of prominent lung fibrosis. In this model, the authors showed that the inflammatory and fibrotic responses are two separate events involving distinct mechanisms and cell types and that SatMs exclusively mediate the fibrotic response. The authors provided compelling evidence that SatMs represent a unique cell type different from any other myeloid cell types. Phenotypically, SatMs are identified as Ceacam1+/Msr1+/Mac1+/Ly6C–/F4/80– cells, and, functionally, they are superb activators of tissue fibroblasts (producers of matrix proteins). They possess a bilobed and segmented nucleus and have ample cytoplasmic granules, showing a transcriptional profile that straddles that of monocytes and granulocytes. They depend entirely on the transcription factor CCAAT enhancer binding protein beta (C/EBPβ) for development, as genetic knockout of C/EBPβ (i.e., Cebpb–/– mice) selectively abolished SatMs. Importantly, Cebpb–/– mice are highly resistant to the induction of lung fibrosis, although the inflammatory response in the lungs is not perturbed, demonstrating that SatMs are necessary and sufficient in promoting tissue fibrosis in vivo. In pursuing the mechanisms that account for the selective recruitment of SatMs to fibrotic lesions, the authors meticulously designed and carefully executed a series of elegant experiments using state-of-the-art sequencing and molecular tools. These led to the discovery of an RNA-binding protein called RNA-binding motif protein 7 (or Rbm7), as well as a detailed revelation of how Rbm7 works in vivo in promoting fibrosis. The authors showed that Rbm7 is expressed in nonhematopoietic cells in the lungs, especially in airway epithelial cells. Moreover, Rbm7 expression is inducible, especially in response to injury inflicted by bleomycin, and is confined to areas of fibrosis and associated with SatM recruitment. Additionally, genetic knockout of Rbm7, similar to C/EBPβ knockout, prevented the induction of fibrosis by bleomycin. The mechanistic aspect of the pursuit was really creative and novel. The authors presented extensive data showing that, in response to injury, the airway epithelial cells assemble nuclear exomes, also called paraspeckles. The long noncoding (lnc)RNA (i.e., nuclear paraspeckle assembly transcript [NEAT]1/2) serves as a scaffold to bring together DNA repair molecules (e.g., breast cancer [BRCA]1) to form nuclear paraspeckles. As a consequence of cellular stress responses, such paraspeckles are critical in repairing DNA damage and preventing epithelial cell death. Interestingly, once induced, Rbm7 preferentially binds the lncRNA, resulting in the degradation of lncRNA and the subsequent dismantling of the paraspeckles. This renders the airway epithelial cells extremely sensitive to DNA damage and cell death, and the death of epithelial cells then releases the chemokine CXCL12, which acts as a potent chemoattractant to SatMs. Thus, these experiments identify Rbm7 as a critical regulator of tissue fibrosis. The authors extended their findings to include human fibrotic samples (fibrotic kidney, liver and lungs), showing that similar mechanisms may also be operative in tissue fibrosis in humans. Clearly, these studies may open the door to the development of new strategies and therapeutics specifically targeting organ fibrosis, which is an area of tremendous importance in transplantation. Xian C. Li, MD, PhD, is a professor and director at the Immunobiology and Transplant Science Center and Department of Surgery at Houston Methodist Hospital. He is also section editor of “Literature Watch.”

Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Rbm7 in Structural Cells: A NEAT Way to Control Fibrosis

The initial molecular events and the cell type(s) responsible for the development of fibrosis are unclear. Fukushima, Satoh, etal. find that increased expression of the nuclear exosome targeting complex component Rbm7 in lung epithelial cells promotes the degradation of the long non-coding RNA NEAT1, impairs DNA repair, and triggers apoptosis. Dying epithelial cells release chemokines that recruit atypical monocytes, which drive tissue fibrosis.

Nuclear Exosome Targeting Complex Core Factor Zcchc8 Regulates the Degradation of LINE1 RNA in Early Embryos and Embryonic Stem Cells

The nuclear exosome targeting (NEXT) complex is responsible for specific nuclear RNA degradation in mammalian cells. However, its function in development remains unknown. Here, we find that the depletion of a central factor of the NEXT complex, Zcchc8, in mouse results in developmental defects, a shortened lifespan, and infertility. We find that Zcchc8-deficient embryonic stem cells (ESCs) exhibit proliferation abnormalities and reduced developmental potencies. Importantly, the transcripts of retrotransposon element LINE1 are found to be targeted by Zcchc8 and degraded by a Zcchc8-mediated mechanism. We further find that sustained expression of higher levels of LINE1 RNA is detected in maternal Zcchc8-depleted oocytes and embryos. Zcchc8-depleted oocytes show higher chromatin accessibility and developmental defects in both meiotic maturation and embryogenesis after fertilization. Collectively, our study defines Zcchc8-mediated RNA degradation as an important post-transcription regulation of LINE1 transcripts in early embryos and ESCs, which play vital roles in the pluripotency and early development.

Structural basis for MTR4–ZCCHC8 interactions that stimulate the MTR4 helicase in the nuclear exosome-targeting complex

The nuclear exosome-targeting (NEXT) complex functions as an RNA exosome cofactor and is involved in surveillance and turnover of aberrant transcripts and noncoding RNAs. NEXT is a ternary complex composed of the RNA-binding protein RBM7, the scaffold zinc-knuckle protein ZCCHC8, and the helicase MTR4. While RNA interactions with RBM7 are known, it remains unclear how NEXT subunits collaborate to recognize and prepare substrates for degradation. Here, we show that MTR4 helicase activity is enhanced when associated with RBM7 and ZCCHC8. While uridine-rich substrates interact with RBM7 and are preferred, optimal activity is observed when substrates include a polyadenylated 3' end. We identify a bipartite interaction of ZCCHC8 with MTR4 and uncover a role for the conserved C-terminal domain of ZCCHC8 in stimulating MTR4 helicase and ATPase activities. A crystal structure reveals that the ZCCHC8 C-terminal domain binds the helicase core in a manner that is distinct from that observed for Saccharomyces cerevisiae exosome cofactors Trf4p and Air2p. Our results are consistent with a model whereby effective targeting of substrates by NEXT entails recognition of elements within the substrate and activation of MTR4 helicase activity.

Structural studies of the human Nuclear EXosome Targeting complex

Structural studies of the human Nuclear EXosome Targeting complex

The fission yeast MTREC complex targets CUTs and unspliced pre-mRNAs to the nuclear exosome.

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. However, the mechanism by which they are recognized and targeted to the exosome is not fully understood. Here we report that the MTREC complex, which has recently been shown to promote degradation of meiotic mRNAs and regulatory ncRNAs, is also the major nuclear exosome targeting complex for CUTs and unspliced pre-mRNAs in Schizosaccharomyces pombe. The MTREC complex specifically binds to CUTs, meiotic mRNAs and unspliced pre-mRNA transcripts and targets these RNAs for degradation by the nuclear exosome, while the TRAMP complex has only a minor role in this process. The MTREC complex physically interacts with the nuclear exosome and with various RNA-binding and RNA-processing complexes, coupling RNA processing to the RNA degradation machinery. Our study reveals the central role of the evolutionarily conserved MTREC complex in RNA quality control, and in the recognition and elimination of CUTs.

The Human Nuclear Exosome Targeting Complex Is Loaded onto Newly Synthesized RNA to Direct Early Ribonucleolysis

The RNA exosome complex constitutes the major nuclear eukaryotic 3'-5' exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT) complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and 3'-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3' ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3'-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3' ends.

P38MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex.

The nuclear exosome targeting complex (NEXT) directs a major 3′–5′ exonuclease, the RNA exosome, for degradation of nuclear noncoding (nc) RNAs. We identified the RNA-binding component of the NEXT complex, RBM7, as a substrate of p38MAPK/MK2-mediated phosphorylation at residue S136. As a result of this phosphorylation, RBM7 displays a strongly decreased RNA-binding capacity, while inhibition of p38MAPK or mutation of S136A in RBM7 increases its RNA association. Interestingly, promoter-upstream transcripts (PROMPTs), such as proRBM39, proEXT1, proDNAJB4, accumulated upon stress stimulation in a p38MAPK/MK2-dependent manner, a process inhibited by overexpression of RBM7S136A. While there are no stress-dependent changes in RNA-polymerase II (RNAPII) occupation of PROMPT regions representing unchanged transcription, stability of PROMPTs is increased. Hence, we propose that phosphorylation of RBM7 by the p38MAPK/MK2 axis increases nuclear ncRNA stability by blocking their RBM7-binding and subsequent RNA exosome targeting to allow stress-dependent modulations of the noncoding transcriptome.

A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response.

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response.