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JPST000693 An extended PAXT connection is required for turnover of nuclear polyadenylated RNA in human cells [Reanalysis: JPST000693]

ABSTRACT: Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26/RBM27 and the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of either identified PAXT component results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity. [Original project description]

ORGANISM(S): Hek293

SUBMITTER: Yasushi Ishihama

PROVIDER: RPXD034322 | JPOST Repository | Sat Mar 25 00:00:00 GMT 2023

REPOSITORIES: jPOST

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An extended PAXT connection is required for turnover of nuclear polyadenylated RNA in human cells

Project description:Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26/RBM27 and the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of either identified PAXT component results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.

2019-10-31 | PXD016070 | JPOST Repository

The Human ZC3H3 and RBM26/27 Proteins Are Critical for PAXT-mediated Nuclear RNA Decay

Project description:Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.

2020-01-09 | GSE131255 | GEO

Total poly(A)+ RNA sequencing

Project description:Nuclear 3’ to 5’ nuclease RNA exosome plays a key role in quality control and processing of multiple protein-coding and non-coding transcripts made by RNA polymerase II. A mechanistic understanding of exosome function remains a challenge given the large number of RNA species and intervening RNA processing factors. Here we analysed changes in the poly(A)+ RNA proteins interactome provoked by mutations in three distinct subunits of the nuclear RNA exosome. Our data demonstrate a functional connection between Rrp6 and Mtl1 in controlling processing and levels of multiple protein-coding and non-coding transcripts. Furthermore, we show that exosome mutants accumulate components of U1 and U2 snRNPs and show depletion of NTC components from RNA suggesting that the stage prior to the activation of the spliceosome represents a critical quality control step. We have also identified potential new RNA binding factors involved in exosome regulation, including a zinc-finger protein called Mub1 that controls levels of selected transcripts encoding for proteins implicated in stress response. Collectively, our data have provided a global view of RNA metabolism alterations in exosome deficient cells and revealed RNA binding proteins that may act as novel exosome cofactors.

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Identification of a nuclear exosome decay pathway for processed transcripts

Project description:The RNA exosome is fundamental for the degradation of RNA in eukaryotic nuclei. Substrate targeting is facilitated by its co-factor Mtr4p/hMTR4, which links to RNA-binding protein adaptors. One such activity is the human Nuclear EXosome Targeting (NEXT) complex, composed of hMTR4, the Zn-finger protein ZCCHC8 and the RNA-binding factor RBM7. NEXT primarily targets early and unprocessed transcripts, demanding a rationale for how the nuclear exosome recognizes processed RNAs. Here, we describe the PolyA tail eXosome Targeting (PAXT) connection, comprising the hitherto uncharacterized ZFC3H1 Zn-knuckle protein as a central link between hMTR4 and the nuclear polyA binding protein PABPN1. Individual depletion of ZFC3H1 and PABPN1 results in the accumulation of common transcripts, that are generally both longer and more 3’polyadenylated than NEXT substrates. Importantly, ZFC3H1/PABPN1 and ZCCHC8/RBM7 contact hMTR4 in a mutually exclusive manner, revealing that the exosome targets nuclear transcripts of different maturation status by substituting its hMTR4-associating adaptors.

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RNA-seq reveals that Air1 and Air2 control TRAMP substrate specificity for nuclear RNA surveillance

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2012-11-14 | E-GEOD-42231 | biostudies-arrayexpress

The RNA exosome adaptor protein ZFC3H1 functionally competes with nuclear export activity to retain target transcripts

Project description:Mammalian genomes are promiscuously transcribed, yielding protein-coding and non-coding products. Many transcripts are short-lived due to their nuclear degradation by the ribonucleolytic RNA exosome. Here, we show that abolished nuclear exosome function causes the formation of distinct nuclear foci, containing polyadenylated (pA+) RNA secluded from nucleocytoplasmic export. We asked whether exosome co-factors could serve such nuclear retention. Co-localization studies revealed the enrichment of pA+ RNA foci with ‘pA-tail exosome targeting (PAXT) connection’ components MTR4, ZFC3H1 and PABPN1, but no overlap with known nuclear structures, such as Cajal bodies, speckles, paraspeckles or nucleoli. Interestingly, ZFC3H1 is required for foci formation, and in its absence selected pA+ RNAs, including coding and non-coding transcripts, are exported to the cytoplasm in a process dependent on the mRNA export factor AlyREF. Our results establish ZFC3H1 as a central nuclear RNA retention factor, counteracting nuclear export activity.

2018-05-14 | GSE108197 | GEO

Rapid factor depletion highlights intricacies of nucleoplasmic RNA degradation

Project description:Turnover of nucleoplasmic transcripts by the 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 been based on long-term factor depletion strategies. Here, we rapidly deplete a NEXT, a PAXT and a core-exosome component, uncovering direct consequences of their losses. Global proteome analyses displayed only minor changes, yet exposed the immediate co-depletion of exosome and adaptor subunits, reflecting possible sub-complex compositions. Parallel high-resolution 3’end sequencing of newly synthesized RNA confirmed earlier established long-term depletion phenotypes. Still, rapid NEXT depletion displayed a narrower phenotype, illustrated by its restricted effect on snoRNA-hosting introns, whereas long-term NEXT depletion data had suggested a more general role of the complex in mediating intron turnover. While this validates snoRNA-hosting introns as primary NEXT targets, our data suggest that nuclear 5’-3’ RNA decay is altered after prolonged NEXT depletion, obscuring interpretation. Further analysis of snoRNA-hosting introns also uncovered an unusual mode of core exosome-independent RNA decay. Due to its non-processive nature, we speculate that this reflects a hitherto undisclosed exosome-independent activity of the EXOSC10 ribonuclease.

2021-12-23 | PXD026774 | Pride

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

Project description: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 PolyA eXosome Targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts. Conversely, PAXT/exosome was considered to target longer and adenylated substrates via their poly(A) tails. Here, mutational analysis of the core PAXT component ZFC3H1 uncovers a separate branch of the PAXT pathway, which targets short adenylated RNAs and relies on a direct ARS2-ZFC3H1 interaction. We further demonstrate that similar acidic-rich short linear motifs of ZFC3H1 and ZC3H18 compete for a common ARS2 epitope. Consequently, while promoting NEXT function, ZC3H18 antagonizes PAXT activity. We suggest that this unprecedented organization of RNA decay complexes provides co-activation of NEXT and PAXT at loci with abundant production of short exosome substrates.

2024-01-26 | PXD045842 | Pride

RNA-seq reveals that Air1 and Air2 control TRAMP substrate specificity for nuclear RNA surveillance

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