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Rapid factor depletion highlights intricacies of nucleoplasmic RNA degradation

ABSTRACT: 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. Generally, proteome changes are sparse and largely dominated by co-depletion of other exosome and adaptor subunits, reflecting possible subcomplex compositions. While parallel high-resolution 3 end sequencing of newly synthesized RNA confirms previously established factor specificities, it concomitantly demonstrates an inflation of long-term depletion datasets by secondary effects. Most strikingly, a general intron degradation phenotype, observed in long-term NEXT depletion samples, is undetectable upon short-term depletion, which instead emphasizes NEXT targeting of snoRNA-hosting introns. Further analysis of these introns uncovers an unusual mode of core exosome-independent RNA decay. Our study highlights the accumulation of RNAs as an indirect result of long-term decay factor depletion, which we speculate is, at least partly, due to the exhaustion of alternative RNA decay pathways.

ORGANISM(S): Homo sapiens

PROVIDER: GSE179106 | GEO | 2022/01/20

REPOSITORIES: GEO

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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

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Distinct and redundant roles of nuclear RNA exosome targeting complexes

Project description:Degradation of transcripts in mammalian nuclei is primarily facilitated by the RNA exosome. To obtain substrate specificity, the exosome is aided by adaptors; in the nucleoplasm, the Nuclear EXosome Targeting (NEXT) complex and the PolyA (pA) eXsome Targeting (PAXT) connection. However, how exact targeting is achieved remains enigmatic. Employing high-resolution 3’end sequencing of both steady state and newly produced pA+ and pA- RNA, we demonstrate that NEXT substrates arise from heterogenous and predominantly pA- 3’ends often covering kb-wide genomic regions. In contrast, PAXT targets harbor well-defined pA+ 3’ends defined by canonical pA site usage. Irrespective this clear division, NEXT and PAXT act redundantly in two ways: i) Regional redundancy: The majority of exosome-targeted transcription units produce both NEXT- and PAXT-sensitive RNA isoforms; and ii) Isoform redundancy: The PAXT connection ensures the fail-safe decay of post-transcriptionally polyadenylated NEXT targets. In conjunction, this provides for the efficient nuclear removal of superfluous RNA.

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

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Identification of a nuclear exosome decay pathway for processed transcripts

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

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2020-01-09 | GSE131255 | GEO

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

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

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. [Original project description]

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