An RNA topoisomerase complex interacts with Fragile X syndrome protein to promote neurodevelopment and maintain normal life-span

National Institute On Aging

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Abstract

Topoisomerases are magicians of the DNA world, working their wizardry to solve topological problems of DNA during replication, repair, and transcription. Many DNA metabolizing enzymes (polymerases, helicases, nucleases, and ligases) have counterparts in the RNA world. One exception is topoisomerase, which seems to be absent from the RNA world. During our research on DNA topoisomerases that participate in DNA repair, we discovered that topoisomerase 3beta (Top3b) has many features of an RNA topoisomerase. First, Top3b associates with the Fragile X syndrome protein, FMRP, which is known to bind mRNA and to regulate mRNA translation and transport. Second, Top3b resembles FMRP in associating with polyribosomes, which are units for mRNA translation. Third, Top3b colocalizes with FMRP in RNA stress granules, which are cytoplasmic compartments for stalled mRNA and translation machinery. Fourth, Top3b binds mRNA in cells as shown by a crosslinked-RNA immunoprecipitation assay (HITS-CLIP). Fourth, Top3b mutants in Drosophila display abnormal neuromuscular junctions similar to those in FMR1 mutants. Fifth, Top3b mutations in Drosophila modify the rough eye phenotype induced by FMRP over-expression. Sixth and most importantly, Top3b can directly catalyze topoisomerase reactions on RNA substrates. In addition, a point mutation that inactivates its DNA topoisomerase activity also disrupts its RNA topoisomerase activity, indicating that the same catalytic residue may be used for reactions on both DNA and RNA substrates. Furthermore, the paralog of Top3b, Top3a, completely lacks RNA topoisomerase activity, suggesting that the observed RNA topoisomerase activity is specific for Top3b. We were able to create Drosophila Top3b/Fmr1 double mutant, and found that the abnormal neuromuscular junction phenotype observed in each single mutant is suppressed in the double mutant. This further illustrates that the two proteins genetically interact in antagonistic manner. Moreover, the data suggest that the inhibitors of the RNA topoisomerase may be used as drugs to alleviate conditions of the Fragile X patients. Several human studies have linked Top3b mutation with schizophrenia, intellectual disability and autism. Consistent with these findings, we found that multiple Top3b bound mRNAs are encoded by schizophrenia and autism-related genes. We further showed that one schizophrenia-related gene, ptk2/FAK, displayed reduced expression in neuromuscular junctions of the Drosophila Top3b mutant, Fmr1 mutant, and their double mutant, suggesting that Top3b and Fmr1 work in the same pathway to promote ptk2 expression in synapse. We also observed abnormal synapse formation in both Drosophila and mouse that are inactivated of Top3b. In summary, we have identified Top3b as the first dual-activity topoisomerase in eukaryotes that can act on not only DNA, but also RNA. We showed that Top3b works with FMRP to promote neurodevelopment and mental health. A manuscript describing this work has been published in Nature Neuroscience (Xu et al. Nature Neuroscience, 2013), and is featured by highlights in Nature, Nature Neuroscience, Nature Review Neurology, and other journals and organizations. One important issue is how prevalent is RNA topoisomerase activity in various species. We have tested topoisomerases from a variety of species, and found that RNA topoisomerase activity is present in all three domains of life, bacteria, archaea, and eukarya. The data support the notion that the RNA topoisomerases are important so that they are conserved through evolution. This work was published in 2016 (Ahmad et al., NAR 2016) We and others have previously shown that Top3b in human associate with polyribosomes, suggesting that it participates in mRNA translation. We have found that Top3b from other animal species also associate with polyribosomes, whereas those from bacteria and yeast do not. Moreover, this association requires TDRD3, a partner of Top3b which only exists in animals. Our data thus suggest that only in animals, the dual-activity topoisomerase Top3b works in mRNA translation as part of a complex. Human cells have 5 topoisomerases in nucleus and cytoplasm. We found that only Top3b strongly binds mRNAs. Moreover, we found that this binding activity strongly depends on the RNA-binding domain of Top3b. These data are consistent with the notion that Top3b acts as an RNA topoisomerase and works in mRNA metabolism. We have examined two de novo single nucleotide variants of Top3b discovered in schizophrenia and autism patients, and found that an autism patient-derived point mutant lost the RNA topoisomerase activity and mRNA binding activity. In addition, both mutants have defective ability to interact with FMRP The data provide additional evidence for involvement of Top3b in mental disorders. We produced transgenic flies expressing different mutants of Top3b. We found that the RNA binding activity and the topoisomerase activity are both required for formation of normal Drosophila synapse. Moreover, the autism patient derived point mutant is defective in promoting synapse formation. The data support the notion that the RNA topoisomerase activity of Top3b is needed for normal neurodevelopment. This work was published in NAR (Ahmad et al., 2017) We have shown that Top3-knockout mice exhibit behavioral phenotypes related to psychiatric disorders and cognitive impairment, including increased anxiety and fear, impaired social interactions, and defective spatial learning and memory. In addition, these mice display deficits in adult hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many that are critical for neuronal functions and mental health. Our data suggest that Top3 is essential for normal brain function in multiple domains, and defective neuronal activity-dependent transcription may be a mechanism by which Top3 mutations cause cognitive impairment and psychiatric disorders. This work is published in Nature Communications (Joo et al., 2020). We found that TDRD3 forms a stable complex with Top3b in not only human, but also Drosophila. We show that in Drosophila, Top3b biochemically and genetically interacts with the RNAi-induced silencing complex (RISC) containing AGO2, p68 RNA helicase, and FMRP. Top3b and RISC mutants are both defective in heterochromatin formation and transcriptional silencing; and this defect is suppressed in the double mutants between Top3b and AGO2, p68, and RNAi biogenesis enzyme, Dicer-2, suggesting coordinated interactions between Top3b and siRNA machinery. Moreover, both Top3b and AGO2 single mutant flies exhibit reduced heterochromatin markers in pericentric and telomeric heterochromatin; and the reduction in pericentric heterochromatin is also suppressed in their double mutant. Furthermore, expression of several genes and transposable elements within telomeric heterochromatin is increased in the Top3b mutant. Finally, we found that Top3b depends on its RNA binding domain and its catalytic activity to promote heterochromatin formation and transcriptional silencing. Our data suggest that Top3b works with siRNA machinery to promote heterochromatin formation and transcriptional silencing. This work is in Nature Communications (Lee et al., Nat. Commun. 2018). We have established CRISPR-CAS9 system in the lab, and were able to knockout TDRD3 in Drosophila. We found that TDRD3 flies also display abnormal development of DA neurons, suggesting that the entire Top3b-TDRD3 complex is required for normal neurodevelopment. Human TDRD3 genomic variants have been associated with schizophrenia, verbal short-term memory and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. We generated a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, olfaction, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive and psychiatric disorders. This work was published in 2024 (Zhu et al., Progress in NeuroBio. 2024). Topoisomerases typically function in the nucleus to relieve topological stress in DNA. We recently show that Top3b-TDRD3 largely localizes in the cytoplasm and interacts biochemically and genetically with PIWI-interacting RNA (piRNA) processing enzymes to promote piRNA biogenesis, post-transcriptional gene silencing (PTGS) of transposons, and Drosophila germ cell development. Top3b requires its topoisomerase activity to promote PTGS of a transposon reporter and preferentially silences long and highly expressed transposons, suggesting that RNAs with these features may produce more topological stress for topoisomerases to solve. The double mutants between Top3b and piRNA processing enzymes exhibit stronger disruption of the signatures and levels of germline piRNAs, more de-silenced transposons, and larger defects in germ cells than either single mutant. Our data suggest that Top3b can act in a new RNA-based process--piRNA biogenesis and PTGS of transposons-and this function is required for Top3b to promote normal germ cell function. This work was published in Cell Reports 2025 (Lee et al., 2025). TOP3B-TDRD3 complex has been reported to associate with the exon-junction complex (EJC) and other mRNA splice regulatory factors (SRFs). However, no mRNAs have been shown with altered alternative splicing (AS) patterns in its absence. We used long-read RNA sequencing to show that Top3b inactivation in mouse brains alters 10-30% of AS events. We confirmed alterations of 4 types of AS in 6 representative genes by sequencing their AS products. The frequently altered AS events include activation or repression of cryptic splice sites, and alternative 3’ splice sites, which overlap with those of cells depleted of EJC. Further analysis revealed that Top3b-TDRD3 interacts with SRSF1, a component of the EJC-SR network acting at multiple steps in mRNA processing. Top3b-TDRD3 resembles SRSF1 in suppressing exon 9 skipping of Caspase-2 gene, and in suppressing cryptic splice sites. Notably, several genes displaying altered AS in Top3b KO mice have been linked to neurological disorders. Our data suggest that Top3b may interact with EJC-SR network to maintain normal splicing and safeguard transcriptome integrity, and disruption of this function may contribute to the neurological disorders observed in individuals carrying Top3b mutations. A manuscript describing this work has been submitted for publication.