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 ...example is the trimeric human nuclear exosome targeting (NEXT) complex, which is composed of hMTR4, the Zn-finger protein ZCCHC8, and the RNA-binding factor RBM7. NEXT primarily targets early and unprocessed transcripts, which demands a rationale for how the nuclear exosome recognizes processed RNAs. Here, we describe the poly(A) tail exosome targeting (PAXT) connection, which comprises the ZFC3H1 Zn-knuckle protein as a central link between hMTR4 and the nuclear poly(A)-binding protein PABPN1. Individual depletion of ZFC3H1 and PABPN1 results in the accumulation of common transcripts that are generally both longer and more extensively 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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•ZFC3H1 links MTR4 with PABPN1 in the poly(A) tail exosome targeting (PAXT) connection•ZFC3H1 and RBM7/ZCCHC8 (NEXT) interact with MTR4 in a mutually exclusive manner•PAXT and NEXT direct distinct RNA species for nuclear exosome degradation•PAXT targets tend to be longer and more extensively polyadenylated than NEXT targets
Meola et al. describe the poly(A) tail exosome targeting (PAXT) connection, which is biochemically and functionally distinct from the previously characterized NEXT complex. PAXT is involved in the degradation of polyadenylated nuclear RNAs. The discovery of PAXT expands our knowledge of the exosome’s role in controlling nuclear transcript levels.
The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase ...Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model.
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► Identification of the human Nuclear Exosome Targeting complex ► NEXT targets promoter upstream transcripts for exosomal degradation ► A putative human TRAMP complex is involved in rRNA degradation in nucleoli ► hMTR4 is a central activator of the human nuclear RNA exosome
Background The main aim of specific immunotherapy is sustained effect due to changes in the immune system that can be demonstrated only in long-term trials. Objective To investigate sustained ...efficacy and disease modification in a 5-year double-blind, placebo-controlled trial, including 2 years of blinded follow-up after completion of a 3-year period of treatment, with the SQ-standardized grass allergy immunotherapy tablet, Grazax ( Phleum pratense 75,000 SQ-T/2,800 BAU, ALK, Denmark) or placebo. Methods A randomized, double-blind, placebo-controlled, multinational, phase III trial included adults with a history of moderate-to-severe grass pollen–induced allergic rhinoconjunctivitis, with or without asthma, inadequately controlled by symptomatic medications. Two hundred thirty-eight participants completed the trial. End points included rhinoconjunctivitis symptom and medication scores, combined scores, asthma symptom and medication scores, quality of life, days with severe symptoms, immunologic end points, and safety parameters. Results The mean rhinoconjunctivitis daily symptom score was reduced by 25% to 36% ( P ≤ .004) in the grass allergy immunotherapy tablet group compared with the placebo group over the 5 grass pollen seasons covered by the trial. The rhinoconjunctivitis DMS was reduced by 20% to 45% ( P ≤ .022 for seasons 1-4; P = .114 for season 5), and the weighted rhinoconjunctivitis combined score was reduced by 27% to 41% ( P ≤ .003) in favor of active treatment. The percentage of days with severe symptoms during the peak grass pollen exposure was in all seasons lower in the active group than in the placebo group, with relative differences of 49% to 63% ( P ≤ .0001). Efficacy was supported by long-lasting significant effects on the allergen-specific antibody response. No safety issues were identified. Conclusion The results confirm disease modification by SQ-standardized grass allergy immunotherapy tablet in addition to effective symptomatic treatment of allergic rhinoconjunctivitis.
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.
Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of ...AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.
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•Nutrient depletion causes a reduction of cytosolic acetyl-coenzyme A•Specific depletion of cytosolic acetyl-coenzyme A induces autophagy•Exogenous supply of acetyl-coenzyme A prevents starvation-induced autophagy•Cytosolic acetyl-coenzyme A represses autophagy via the acetyltransferase EP300
Mariño et al. demonstrate that nutrient starvation causes rapid depletion of acetyl-coenzyme A (AcCoA), reduction in the acetylation of cytoplasmic proteins, and induction of autophagy. This is at least partially dependent on the acetyltransferase EP300. Thus AcCoA and EP300 link cellular nutritional status with the regulation of autophagy.
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 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.
Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to ...hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.
Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components ...and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT‐A and IFT‐B that are transported by molecular motors. The IFT‐B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high‐resolution structures are only available for smaller IFT‐B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15‐subunit IFT‐B complex. The model was validated using cross‐linking/mass‐spectrometry data on reconstituted IFT‐B complexes, X‐ray scattering in solution, diffraction from crystals as well as site‐directed mutagenesis and protein‐binding assays. The IFT‐B structure reveals an elongated and highly flexible complex consistent with cryo‐electron tomographic reconstructions of IFT trains. The IFT‐B complex organizes into IFT‐B1 and IFT‐B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT‐B1 complex. Our data present a structural framework to understand IFT‐B complex assembly, function, and ciliopathy variants.
Synopsis
Construction and maintenance of cilia is mediated by the the bidirectional trafficking of 22 different proteins organized into IFT‐A and IFT‐B complexes, with only structures of smaller IFT‐B assemblies solved. Here, a combination of Alphafold structural modeling and biochemical analysis offers a structural model of the 15‐subunit IFT‐B complex.
Validated structural model for 15‐subunit IFT‐B reveals an elongated and highly flexible complex consistent with cryo‐electron tomographic reconstructions of IFT trains.
The IFT‐B complex divides into IFT‐B1 and IFT‐B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively.
Structural modeling and biochemical data are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of two different conformations of the IFT‐B1 complex.
A combination of alphafold structural modeling and biochemical experiments reveals the structure of the large IFT‐B intraflagellar transport complex and offers insight into IFT‐B complex assembly and cilia formation.
To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins ...after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.
We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 ...interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetylCoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity of a bacterial ortholog of AceCS2, called ACS, is controlled via deacetylation by a bacterial sirtuin protein, our observation highlights the conservation of a metabolic regulatory pathway from bacteria to humans.
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