Check out the latest publication from the Woodson lab!
Rapid turnover of glmS mRNA in Bacillus subtilis by 5′-3′ exoribonuclease RNase J is essential for feedback regulation of glucosamine-6-phosphate (GlcN6P) synthase expression, upon self-cleavage of a GlcN6P-activated ribozyme in the glmS 5′ UTR. We used biochemical assays and single molecule fluorescence microscopy to show that initiation of RNase J decay is inefficient and requires approximately 15 5′ unpaired nucleotides to form a processive exonuclease complex that is insensitive to downstream RNA structure. When stably folded, the cleaved glmS ribozyme blocks RNase J initiation. However, co-transcriptional ribozyme cleavage and physiological Mg2+ levels increase decay by weakening the ribozyme structure. At 22 °C, the processive velocity of RNase J, 23 ± 8 nt/s, is equal to or faster than transcription, indicating that RNase J has the potential to catch elongating polymerases. The results show how the folding stability of 5′ mRNA structure contributes to RNase J recognition and the control of mRNA half-life.
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Check out the latest publication from the Liu lab!
The coronavirus proofreading exoribonuclease (ExoN) is essential for genome fidelity and immune evasion of the viruses. Despite its critical roles in the viral life cycle, it is unclear how ExoNs across different coronaviruses diverge in their structures and catalytic properties, which may lead to differences in viral genome mutation rates and, consequently, viral fitness, immune evasion, and resistance to antiviral drugs. Here, we present comparative structural and biochemical analyses of ExoNs between two most representative human coronaviruses, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the merbecovirus subgenus and SARS-CoV-2 from the sarbecovirus subgenus. Our results reveal a markedly lower catalytic activity of ExoN from MERS-CoV than that from SARS-CoV-2. The molecular basis of such a divergence across the two coronaviruses is unveiled by the cryo-EM structures of MERS-CoV ExoN in complex with RNA substrates bearing different 3′-end base pairs or mismatch, which represent the first set of ExoN structures from a coronavirus outside the sarbecovirus subgenus. Our findings also identify two highly conserved structural determinants that dictate efficient excision of different nucleotides at the 3′ terminus of RNA substrates by coronavirus ExoNs, a property that is pivotal for their roles in both viral RNA proofreading and immune evasion.
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Check out the latest publication from the Fukunaga lab!
The self-renewal and differentiation of germline stem cells (GSCs) are tightly regulated during oogenesis. The Drosophilafemale germline provides a powerful model to study these regulatory mechanisms. We previously identified Sakura (also known as Bourbon/CG14545) as a crucial factor for maintenance and differentiation of GSCs and oogenesis, and demonstrated that Sakura binds to Ovarian Tumor (Otu), another essential regulator of these processes. Here, we identify MYCBP (c-Myc binding protein) as an additional essential component of this regulatory network. We show that MYCBP physically associates with itself, Sakura, and Otu, forming binary and ternary complexes including a MYCBP•Sakura•Otu complex. MYCBP is highly expressed in the ovary, and mycbp null mutant females exhibit rudimentary ovaries with germline-less and tumorous ovarioles, fail to produce eggs, and are completely sterile. Germline-specific depletion of mycbp disrupts Dpp/BMP signaling, causing aberrant expression of bag-of-marbles (bam) and leading to defective differentiation and GSC loss. In addition, mycbp is required for female-specific splicing of sex-lethal (sxl), a master regulator of sex identity determination. These phenotypes closely resemble those observed in sakura and otu mutants. Together, our findings reveal that MYCBP functions in concert with Sakura and Otu to coordinate self-renewal and differentiation of GSCs and oogenesis in Drosophila.
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Check out the latest preprint from the Xiao lab!
Chromosomal topology and transcription are tightly coupled, yet the quantitative impact of topological constraints on transcription, supercoiling, and the potential coupling between neighboring genes in vivo remains unclear. In this work, we constructed synthetic chromosomal domains in Escherichia coli that contained two genes inside a topology-controllable domain and a third gene outside. Using three-color single-molecule fluorescence in situ hybridization (smFISH), we measured transcription output from the three genes in individual cells under conditions in which gene orientation, domain formation state, and global chromosomal supercoiling density were varied. We found that topological domain formation repressed transcription, diminished gene orientation-dependent differences in transcription, and modulated the supercoiling sensitivity of genes located both within and near the domain. Relaxing global negative supercoiling through gyrase inhibition broadly repressed transcription; increasing global negative supercoiling level through topoisomerase I inhibition repressed highly expressed genes, while activating lowly expressed ones. Besides single-gene effects, we also observed an intrinsic coupling between neighboring genes with a non-monotonic dependence on the underlying supercoiling state, which shifted with domain topology and gene syntax. Our results establish chromosome topology as a major regulator of both transcription levels and the coupling between adjacent genes.
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Check out the latest publication from the Liu lab!
SARS-CoV-2’s remarkable resistance to nucleotide analog antivirals such as remdesivir, which thwarts RNA synthesis by inhibiting viral polymerase (RdRp), challenges available therapies. We reveal that remdesivir incorporation destabilizes RdRp–RNA complex while enhancing RNA binding to the proofreading exoribonuclease (ExoN), facilitating remdesivir excision. Conserved ExoN determinants for remdesivir recognition and excision underpin ExoN-mediated resistance across all coronaviruses. These findings inform the design of next-generation antivirals and combination therapies capable of overcoming ExoN-mediated resistance.
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Check out the latest publication from the Leung lab!
Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification mediated by ADP-ribosyltransferases, known as PARPs, which attach ADP-ribose units onto proteins, forming negatively charged multimeric chains. This modification relaxes chromatin at DNA damage sites, facilitating repair machinery access. Additionally, PAR polymers serve as docking platforms for effector proteins, termed PAR “readers”, commonly involved in DNA repair. The recruitment of these proteins is mediated through conserved protein domains, including RNA recognition motifs (RRMs). Using an array of hundreds of recombinant RNA-binding domains, we systematically examined RRM interactions with PAR chains of varying lengths. Despite their chemical similarity to RNA, only a small subset of RRMs binds PAR. We identified the RRMs of poly(A)-binding protein (PABPN1) and nucleolin (NCL) as readers of short- and long-chain PAR, respectively. PABPN1 binds short chains via a single RRM unit, while NCL engages long chains using three of its four RRMs. Both proteins are recruited to DNA damage sites marked by PARP activity in a laser micro-irradiation assay, and their RRMs exhibit competitive binding to PAR and RNA. These findings highlight the capacity of specific RRMs to recognize structurally similar ribonucleotide and ADP-ribose polymers, expanding our understanding of RRM versatility and the functional interplay between PARylation and RNA binding.
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Check out the latest publication from the Cochella lab!
The transition from unicellular to multicellular life required the acquisition of coordinated and regulated cellular behaviors, including adhesion and migration. In metazoans, this involves adhesion proteins, signaling systems, and an elaborate extracellular matrix (ECM) that contributes to adhesion and signaling interactions. Innovations that enabled complex multicellularity occurred through new genes in these pathways, novel functions for existing genes, and regulatory changes. Gene regulation by microRNAs (miRNAs) expanded with multicellularity. A single miRNA, miR-100, arose in the last common eumetazoan ancestor and is widely conserved across animals. We reveal the molecular function of its C. elegans homolog, the miR-51 family. This family acts in a dose-dependent manner to control morphogenesis by regulating several genes involved in cell signaling, adhesion, and migration, including ECM modifiers—specifically heparan sulfate sulfotransferases (HSTs). Some of these targets are also predicted to be conserved targets across vertebrates. Our work suggests that this miRNA provided an innovation in the regulation of cellular interactions early in metazoan evolution.
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Check out the latest publication from the Trcek lab!
Drosophila germ granules are enriched with mRNAs critical for development. Within them, mRNAs cluster through intermolecular interactions that may involve base pairing. Here we apply in silico, in vitro and in vivo approaches to examine the type and prevalence of these interactions. We show that RNA clustering can occur without extended sequence complementarity (stretches of six or more continuous complementary bases) and that mRNAs display similar level of foldedness within germ granules as outside. Our simulations predict that clustering is driven by scattered, surface-exposed bases, enabling intermolecular base pairing. Notably, engineered germ granule mRNAs containing exposed GC-rich complementary sequences within stem loops located in the 3! untranslated region promote intermolecular interactions. However, these mRNAs are also expressed at lower levels, leading to developmental defects. Although germ granule mRNAs contain numerous GC-rich complementary sequences, RNA folding renders them inaccessible for intermolecular base pairing. We propose that RNA folding restricts intermolecular base pairing to maintain proper mRNA function within germ granules.
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Check out the latest publication from the Wu lab!
Ribosome-associated protein quality control (RQC) is a surveillance system that identifies and processes aberrant mRNAs with collided ribosomes. ZNF598 plays a key role by ubiquitinating the 40S subunit of collided ribosomes. However, how ZNF598 distinguishes stalled from transient ribosome collisions remains unclear. To address this, we developed a method to visualize the binding of a single protein to a specific mRNA while simultaneously determining its translation status. By endogenously tagging ZNF598 with HaloTag, we observed its strong interaction with RQC reporter mRNAs. We discovered that multiple ZNF598s engage with a single RQC mRNA, suggesting that ZNF598 recognizes more than just the leading collided ribosome in a queue. Overexpressing ZNF598 increased the ribosomal clearance rate, indicating that it is a rate-limiting factor for RQC. Interestingly, a subset of supposedly “normal” mRNAs may be damaged and targeted by ZNF598, underscoring the importance of RQC to maintain the proteome quality even in unstressed conditions. Under global UV-induced RNA damage, ZNF598 recruitment to the reporter RQC mRNA diminished, highlighting its role as a limiting factor in managing widespread ribosome collisions.
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Check out the latest publication from the Coller lab!
All eukaryotic mRNAs bear a 7-methylguanosine cap on their 5′ end. The 5′ cap enables mRNA translation by binding directly to eIF4E; which further recruits other factors and the 40S ribosome. Additionally, the 5′ cap maintains transcript stability; removal of the cap by the enzyme Dcp2 is necessary to degrade the mRNA. An a priori conclusion, therefore, has been that cap binding by eIF4E and DCP2 are antithetical to each other as both need access to the same substrate, i.e., the 5′ cap. In this study, we purified native full-length human eIF4E and Dcp2 and utilize biophysical and biochemical approaches to examine the in vitro interplay between Dcp2 and eIF4E. We confirm that Dcp2 is sufficient to remove the 5′ cap. Moreover, we demonstrate that Dcp2 binds RNA with nanomolar affinity. We discovered that, unexpectedly, eIF4E does not interfere with Dcp2’s decapping function, contradicting previous mechanistic models. Moreover, eIF4E binding appears to increase the affinity of Dcp2 for RNA. Although limited to in vitro conditions, our findings warrant a reevaluation of the proposed relationship between these mRNA cap-binding proteins.
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Check out the latest publication from the Woodson lab!
Small noncoding RNAs (sRNAs) regulate gene expression in Escherichia coli bacteria in response to diverse stimuli in the environment and the host. Most sRNAs regulate mRNA expression by directly base pairing with complementary sites in the target mRNA with the help of the chaperone protein Hfq. sRNAs and Hfq must rapidly search hundreds of candidate mRNAs for matched (cognate) targets while discriminating against noncognate targets. Here, we use single-molecule fluorescence microscopy to directly observe how cognate and noncognate mRNAs bind immobilized sRNA-Hfq. The results show that initially unstable sRNA-Hfq-mRNA complexes either dissociate within seconds by ejecting one of the two RNAs, depending on their interactions with Hfq, or are stabilized by sRNA-mRNA base pairing. Cognate mRNAs are more likely to form long-lived sRNA-Hfq-mRNA complexes, even in the presence of competing RNA. Active competition for the mutual Hfq chaperone introduces a kinetic barrier to RNA colocalization that is resolved by base pairing, driving the accumulation of cognate sRNA-mRNA interactions while eliminating noncognate interactions.
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Check out the latest publication from the Fukunaga lab!
During oogenesis, self-renewal and differentiation of germline stem cells (GSCs) must be tightly regulated. The Drosophila female germline serves as an excellent model for studying these regulatory mechanisms. Here, we report that a previously uncharacterized gene CG14545, which we named sakura, is essential for oogenesis and female fertility in Drosophila. Sakura is predominantly expressed in the ovaries, particularly in the germline cells, including GSCs. sakura null mutant female flies display rudimentary ovaries with germline-less and tumorous phenotypes, fail to produce eggs, and are completely sterile. The germline-specific depletion of sakura impairs Dpp/BMP signaling, leading to aberrant bag-of-marbles (bam) expression, resulting in faulty differentiation and loss of GSCs. sakura is also necessary for normal levels of piwi-interacting RNAs (piRNAs) levels and for female-specific splicing of sex-lethal (sxl), a master regulator of sex identity determination. We identified Ovarian Tumor (Otu) as a protein binding partner of Sakura and found that loss of otu phenocopies loss of sakura in ovaries. Thus, we identify Sakura as a crucial factor for GSC renewal and differentiation and oogenesis, and propose that Sakura and Otu function together in these processes.
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Check out the latest publication from the Kim lab!
The Argonaute CSR-1 is essential for germline development in C. elegans. Loss of CSR-1 leads to the down-regulation of thousands of germline-expressed genes, supporting a model in which CSR-1 “licenses” gene expression via a poorly understood mechanism. In contrast, a small subset of genes is up-regulated in csr-1 mutants, including morc-1, which encodes a conserved GHKL-type ATPase. We show that morc-1 is overexpressed in csr-1 mutants and accumulates over CSR-1 licensed targets, coinciding with aberrant gain of H3K9me3, reduced H3K36me3, and transcriptional repression. Notably, loss of morc-1 fully rescues these chromatin defects and partially restores gene expression and fertility in csr-1 mutants. Conversely, ectopic overexpression of MORC-1 in the wild-type germ line is sufficient to repress CSR-1 licensed targets and severely compromise fertility. These findings support a model in which CSR-1 prevents MORC-1 overexpression and consequent misregulation of CSR-1 licensed genes.
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Check out the latest publication from the Xiao lab!
In Escherichia coli, FtsN is thought to coordinate septal peptidoglycan (sPG) synthesis and degradation. Its E domain interacts with the sPG synthesis complex, FtsWIQLB, and its SPOR domain interacts with denuded glycan (dnG), intermediates of sPG degradation. Here we used single-molecule tracking of FtsN and FtsW to investigate how FtsN coordinates the two opposing processes. We found that the SPOR domain binds to dnG cooperatively. This binding sequesters FtsWIQLB on dnG, which we call the dnG-track, and prevents dnG degradation. SPOR domain’s release from dnGs exposes dnGs to degradation, moves FtsN to the sPG synthesis track and activates FtsWIQLB. In addition, FtsN self-interacts through the SPOR domain, promoting the multimerization of FtsWIQLB on both tracks. This self-interaction may create a sensitive switch, regulating FtsN’s partitioning between dnG- and sPG-tracks to coordinate sPG degradation and synthesis while also controlling the balance between sequestered and active populations of the sPG synthesis complex. Our data reveal a third track that plays an important role in sPG synthesis and degradation across space and time, complementing the previously discovered sPG-track and FtsZ-track in E. coli for robust septal cell wall constriction.
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Check out the latest publication from the Fukunaga lab!
Pre-mRNA introns are removed by two distinct spliceosomes: the major (U2-type) spliceosome, which splices over 99.5% of introns, and the minor (U12-type) spliceosome, responsible for a rare class of introns known as minor introns. While the major spliceosome contains U1, U2, U4, U5, and U6 small nuclear RNAs (snRNAs) along with numerous associated proteins, the minor spliceosome comprises U11, U12, U4atac, U5, and U6atac snRNAs and includes specialized proteins. The function and regulation of the minor spliceosome are critical. Mutations in its specific component, RNA-binding protein RNPC3/65K, are linked to human diseases such as primary ovarian insufficiency. In this study, we identify RNA-binding protein Miso (CG44249), which shares 31% and 27% amino acid sequence identity with human RNPC3 and RBM41, respectively, as a key factor in minor splicing and oogenesis in Drosophila Miso associates with U11 and U12 snRNAs in ovaries. miso mutant females exhibit smaller ovaries, reduced germline stem cell numbers, disrupted oogenesis, reduced fecundity, and lower fertility. In miso mutant ovaries, significant minor intron retention is observed, accompanied by a reduction in spliced RNAs and protein products. Our findings establish Miso as a critical factor for minor intron splicing and underscore its essential role in Drosophila oogenesis.
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Check out the latest publication from the Sun lab!
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the repeat expansion in C9ORF72. Dipeptide repeat (DPR) proteins translated from both sense and antisense repeats, especially arginine-rich DPRs (R-DPRs), contribute to neurodegeneration. Through CRISPR interference (CRISPRi) screening in human-derived neurons, we identified receptor-type tyrosine-protein phosphatase S (PTPσ) as a strong modifier of poly-GR-mediated toxicity. We showed that reducing PTPσ promotes the survival of both poly-GR- and poly-PR-expressing neurons by elevating phosphatidylinositol 3-phosphate (PI3P), accompanied by restored early endosomes and lysosomes. Remarkably, PTPσ knockdown or inhibition substantially rescues the PI3P-endolysosomal defects and improves the survival of C9ORF72-ALS/FTD patient-derived neurons. Furthermore, the PTPσ inhibitor diminishes GR toxicity and rescues pathological and behavioral phenotypes in mice. Overall, these findings emphasize the critical role of PI3P-mediated endolysosomal deficits induced by R-DPRs in disease pathogenesis and reveal the therapeutic potential of targeting PTPσ in C9ORF72-ALS/FTD.
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Check out the latest publication from the Buskirk group!
Although many antibiotics inhibit bacterial ribosomes, the loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in E. coli. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryoelectron microscopy (cryo-EM) structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA such that, by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.
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Check out the latest publication from the Zhou lab!
Background
Lipid droplets (LDs) are dynamic cytoplasmic lipid-storing organelles that play a pivotal role in maintaining cellular energy balance, lipid homeostasis, and metabolic signaling. Dysregulation of lipid metabolism, particularly excessive lipogenesis, contributes to the abnormal accumulation of LDs in the nervous system, which is associated with several neurodegenerative diseases. Circular RNAs (circRNAs) are a new class of non-coding and regulatory RNAs that are widely expressed in eukaryotes. However, only a subset has been functionally characterized. Here, we identified and functionally characterized a new circular RNA circbabo(5,6,7,8S) that regulates lipogenesis and neuronal integrity in Drosophila melanogaster.
Results
circbabo(5,6,7,8S) is derived from the babo locus which encodes the type I receptor for transforming growth factor β(TGF-β). Depletion of circbabo(5,6,7,8S) in flies causes elevated lipid droplet accumulation, progressive photoreceptor cell loss and shortened lifespan, phenotypes that are rescued by restoring circbabo(5,6,7,8S) expression. In addition, RNA-seq and epistasis analyses reveal that these abnormalities are caused by aberrant activation of the SREBP signaling pathway. Furthermore, circbabo(5,6,7,8S)-depleted tissues display enhanced activation of the TGF-β signaling pathway and compromised mitochondrial function, resulting in upregulation of reactive oxygen species (ROS). Moreover, we provide evidence that circbabo(5,6,7,8S) encodes the protein circbabo(5,6,7,8S)-p, which inhibits TGF-β signaling by interfering with the assembly of babo/put receptor heterodimer complex. Lastly, we show that dysregulation of the ROS/JNK/SREBP signaling cascade is responsible for the LD accumulation, neurodegeneration, and shortened lifespan phenotypes elicited by circbabo(5,6,7,8S) depletion.
Conclusions
Our study demonstrates the physiological role of the protein-coding circRNA circbabo(5,6,7,8S) in regulating lipid metabolism and neuronal integrity.
Check out the latest publication from the Woodson lab!
Ribosome synthesis in bacteria is coupled with transcription of the pre-ribosomal RNA (pre-rRNA), which must fold and assemble with 20 or more ribosomal proteins. In vitro, the Escherichia coli pre-16S rRNA misfolds during transcription, delaying stable binding of ribosomal protein uS4 that nucleates assembly of the 16S 5′ domain. Using single-molecule fluorescence microscopy, we show that the DEAD-box protein CsdA (DeaD) strongly accelerates uS4 binding by facilitating proper folding of the nascent rRNA. Unstable RNA structures are unfolded by CsdA, whereas stable RNA structures resist unwinding. We show that CsdA unfolding becomes less frequent as more ribosomal proteins add to the complex. The results demonstrate that disassembly of unstable, nascent RNA-protein complexes by chaperones fuels the search for native structure. We propose that general chaperones create a gradient of disassembly that steepens the hierarchy of proper protein addition until late assembly intermediates escape unwinding and commit to 30S maturation.
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Check out the latest publication from the Seydoux lab!
Condensates that accumulate small RNA biogenesis factors (nuage) are common in germ cells and often associate with nuclei. In the Caenorhabditis elegans germline, P granules overlay large clusters of nuclear pores and this organization has been proposed to facilitate surveillance of nascent transcripts by Argonaute proteins enriched in P granules. We report that co-clustering of nuclear pores and P granules depends on FG repeat-containing nucleoporins and FG repeats in the Vasa class helicase GLH-1. Worms with mutations that prevent this co-clustering are fertile under standard growth conditions and exhibit misregulation of only a minority of genes, including replication-dependent histones. Our observations suggest that association with nuclear pores, although non-essential for genome surveillance, may serve to tune mRNA flow through P granules and other nuage condensates.
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Check out the latest publication from the Leung lab!
Non-covalent interactions of poly(ADP-ribose) (PAR) facilitate condensate formation, yet the impact of these interactions on condensate properties remains unclear. Here, we demonstrate that PAR-mediated interactions through PARP13, specifically the PARP13.2 isoform, are essential for modulating the dynamics of stress granules—a class of cytoplasmic condensates that form upon stress, including types frequently observed in cancers. Single amino acid mutations in PARP13, which reduce its PAR-binding activity, lead to the formation of smaller yet more numerous stress granules than observed in the wild-type. This fragmented stress granule phenotype is also apparent in PARP13 variants with cancer-associated single-nucleotide polymorphisms (SNPs) that disrupt PAR binding. Notably, this fragmented phenotype is conserved across a variety of stresses that trigger stress granule formation via diverse pathways. Furthermore, this PAR-binding mutant diminishes condensate dynamics and impedes fusion. Overall, our study uncovers the important role of PAR-protein interactions in stress granule dynamics and maturation, mediated through PARP13.
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Check out the latest publication from the Coller lab!
Polyadenosine (poly(A)) tails are nearly ubiquitous in human messenger RNA (mRNA) governing mRNA stability and translation. Crucially, the poly(A) tail regulates cytoplasmic gene expression by undergoing controlled removal upon exposure to the cytoplasm. Upon removal, mRNA ceases protein production and may subsequently be degraded or silenced. We have generated a therapeutic modality that tethers a poly(A) tail mimetic on the 3′ end of specifically targeted mRNAs, thereby enhancing their expression beyond their normal utility. This technology, which we term mRNA boosters, lends itself to uses on haploinsufficiency disorders, where reduced gene expression manifests in a disease state. By polyadenylating short RNA sequences antisense to the 3′ untranslated region (UTR) of specific mRNAs, we demonstrate that we can selectively and significantly enhance mRNA expression both in vitro and in vivo. We showcase the effectiveness of this technology on genes linked to autism spectrum disorders such as SYNGAP1, M E CP2, PURA, and CTNNB1, illustrating increased expression in both human cell cultures and animal models. These findings indicate that small poly(A) tail mimetics can substantially enhance mRNA expression, providing the potential to efficaciously treat haploinsufficiency disorders.
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