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A dual-activity topoisomerase complex regulates mRNA translation and turnover.

ABSTRACT: Topoisomerase 3β (TOP3B) and TDRD3 form a dual-activity topoisomerase complex that interacts with FMRP and can change the topology of both DNA and RNA. Here, we investigated the post-transcriptional influence of TOP3B and associated proteins on mRNA translation and turnover. First, we discovered that in human HCT116 colon cancer cells, knock-out (KO) of TOP3B had similar effects on mRNA turnover and translation as did TDRD3-KO, while FMRP-KO resulted in rather distinct effects, indicating that TOP3B had stronger coordination with TDRD3 than FMRP in mRNA regulation. Second, we identified TOP3B-bound mRNAs in HCT116 cells; we found that while TOP3B did not directly influence the stability or translation of most TOP3B target mRNAs, it stabilized a subset of target mRNAs but had a more complex effect on translation-enhancing for some mRNAs whereas reducing for others. Interestingly, a point mutation that specifically disrupted TOP3B catalytic activity only partially recapitulated the effects of TOP3B-KO on mRNA stability and translation, suggesting that the impact of TOP3B on target mRNAs is partly linked to its ability to change topology of mRNAs. Collectively, our data suggest that TOP3B-TDRD3 can regulate mRNA translation and turnover by mechanisms that are dependent and independent of topoisomerase activity.

SUBMITTER: Su S

PROVIDER: S-EPMC9262614 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A dual-activity topoisomerase complex regulates mRNA translation and turnover

Project description:Topoisomerase 3β (TOP3B) and TDRD3 form a dual-activity topoisomerase complex that interacts with FMRP and can change the topology of both DNA and RNA. Here, we investigated the post-transcriptional influence of TOP3B and associated proteins on mRNA translation and turnover. First, we discovered that in human HCT116 colon cancer cells, knock-out (KO) of TOP3B had similar effects on mRNA turnover and translation as did TDRD3-KO, while FMRP-KO resulted in rather distinct effects, indicating that TOP3B had stronger coordination with TDRD3 than FMRP in mRNA regulation. Second, we identified TOP3B-bound mRNAs in HCT116 cells; we found that while TOP3B did not directly influence the stability or translation of most TOP3B target mRNAs, it stabilized a subset of target mRNAs but had a more complex effect on translation--enhancing for some mRNAs whereas reducing for others. Interestingly, a point mutation that specifically disrupted TOP3B catalytic activity only partially recapitulated the effects of TOP3B-KO on mRNA stability and translation, suggesting that the impact of TOP3B on target mRNAs is only in part linked to its ability to change topology of mRNAs. Collectively, our data suggest that TOP3B-TDRD3 can regulate mRNA translation and turnover by mechanisms that are dependent and independent of topoisomerase activity.

2022-06-08 | GSE188574 | GEO

A dual-activity topoisomerase complex regulates mRNA translation and turnover

Project description:A dual-activity topoisomerase complex regulates mRNA translation and turnover

| PRJNA779461 | ENA

A dual-activity topoisomerase complex promotes both transcriptional activation and repression in response to starvation

Project description:Topoisomerases are required to release topological stress generated by RNA polymerase II (RNAPII) during transcription. Here we show that in response to starvation, the complex of topoisomerase 3b (TOP3B) and TDRD3 can promote transcriptional activation or repression. Human HCT116 cells individually inactivated for TOP3B, TDRD3, or TOP3B topoisomerase activity, exhibit similarly disrupted transcription for both starvation-activated genes (SAGs) and starvation-repressed genes (SRGs). Responding to starvation, both TOP3B-TDRD3 and the elongating form of RNAPII exhibit concomitantly increased binding to TOP3B-dependent SAGs, at binding sites that overlap. Strikingly, TOP3B inactivation decreases the binding of elongating RNAPII to TOP3B-dependent SAGs while increased it to SRGs. Furthermore, TOP3B-ablated cells display reduced transcription of several autophagy-associated genes and autophagy per se. Our data suggest that TOP3B-TDRD3 can promote both transcriptional activation and repression by regulating RNAPII distribution. In addition, the findings that it can facilitate autophagy may account for the shortened lifespan of Top3b-KO mice.

2023-02-01 | GSE214322 | GEO

RNase L Reprograms Translation by Widespread mRNA Turnover Escaped by Antiviral mRNAs.

Project description:In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2? phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-?. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.

| S-EPMC6754297 | biostudies-literature

Non-equivalent Roles of AGO1 and AGO2 in mRNA Turnover and Translation of Cyclin D1 mRNA.

Project description:Mammalian Argonaute proteins (AGO1-4), in combination with microRNAs (miRs), bind to target mRNAs to initiate degradation and/or translation repression, but the relationships between these two effects is unclear. Although the AGO isoforms ofDrosophilaand plants perform different functions, mammalian AGO isoforms are considered to be functionally degenerate in terms of miR loading and downstream silencing effects. However, we found that, in quiescent (G0) rat myoblasts transiting to the G1 phase, cyclin D1 (Ccnd1) mRNA was associated with two functionally distinct AGO-miR complexes. While AGO1-miR-1 down-regulated the mRNA level, AGO2-let-7 delayed the timing of translation. Knockdown (KD) of AGO2, or antisense-mediated depletion of Let-7, caused Ccnd1 translation to occur earlier, but had no significant effect on mRNA abundance. Conversely, down-regulation of either AGO1 or miR-1, resulted in elevated Ccnd1 mRNA levels at early times, but failed to affect the timing of translation. These results show that the two miR-mediated silencing effects, viz. mRNA decay and translation repression, are independent processes induced by individual AGO isoforms in association with specific miRs.

| S-EPMC4807293 | biostudies-literature

A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

Project description:Chromatin modification is an important epigenetic mechanism underlying neuroplasticity. Histone methylation and acetylation have both been shown to modulate gene expression, but the machinery responsible for mediating these changes in neurons has remained elusive. Here we identify a chromatin-modifying complex containing the histone demethylase PHF8 and the acetyltransferase TIP60 as a key regulator of the activity-induced expression of Arc, an important mediator of synaptic plasticity. Clinically, mutations in PHF8 cause X-linked mental retardation while TIP60 has been implicated in the pathogenesis of Alzheimer's disease. Within minutes of increased synaptic activity, this dual function complex is rapidly recruited to the Arc promoter, where it specifically counteracts the transcriptionally repressive histone mark H3K9me2 to facilitate the formation of the transcriptionally permissive H3K9acS10P, thereby favoring transcriptional activation. Consequently, gain-of-function of the PHF8-TIP60 complex in primary rat hippocampal neurons has a positive effect on early activity-induced Arc gene expression, whereas interfering with the function of this complex abrogates it. A global proteomics screen revealed that the majority of common interactors of PHF8 and TIP60 were involved in mRNA processing, including PSF, an important molecule involved in neuronal gene regulation. Finally, we proceeded to show, using super-resolution microscopy, that PHF8 and TIP60 interact at the single molecule level with PSF, thereby situating this chromatin modifying complex at the crossroads of transcriptional activation. These findings point toward a mechanism by which an epigenetic pathway can regulate neuronal activity-dependent gene transcription, which has implications in the development of novel therapeutics for disorders of learning and memory.

| S-EPMC4586916 | biostudies-literature

Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

Project description:Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

| S-EPMC3325215 | biostudies-literature

Phytochrome regulates translation of mRNA in the cytosol.

Project description:An array of photoreceptors including cryptochromes, phototropin, and phytochromes regulates various light responses in plants. Among these photoreceptors, phytochromes perceive red and far-red light by switching between two interconvertible spectral forms (Pr and Pfr). The Pfr form promotes light responses partly by destabilizing negatively acting, phytochrome-interacting basic helix-loop-helix transcription factors (PIFs), thus modulating transcription in the nucleus. The Pfr form is also present in the cytosol. However, the role of phytochromes in the cytosol is not well understood. Here we show that the Pfr form interacts with the cytosolic protein PENTA1 (PNT1) and inhibits the translation of protochlorophyllide reductase (PORA) mRNA. PNT1 possesses five C3H-type zinc finger domains and displays similarity to various RNA binding proteins including Tristetraprolin, which regulates stabilities of mRNAs such as TNF-? mRNA in humans. Consistent with its function as an RNA binding protein, PNT1 directly binds to mRNA of a key chlorophyll biosynthetic gene, protochlorophyllide reductase in vivo and inhibits the translation of PORA mRNA in the presence of phytochromes. The present results demonstrate that phytochromes transmit light signals to regulate not only transcription in the nucleus through PIFs, but also translation in the cytosol through PNT1.

| S-EPMC3268310 | biostudies-literature

Dual-acting riboswitch control of translation initiation and mRNA decay.

Project description:Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.

| S-EPMC3528543 | biostudies-other

Antibacterial activity of novel dual bacterial DNA type II topoisomerase inhibitors.

Project description:In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.

| S-EPMC7029851 | biostudies-literature

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