In this study, we report the synthesis of 2'-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5-11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2'-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2'-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2'-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.
{"title":"Synthesis of 2'-formamidonucleoside phosphoramidites for suppressing the seed-based off-target effects of siRNAs.","authors":"Kohei Nomura, Seongjin An, Yoshiaki Kobayashi, Jiro Kondo, Ting Shi, Hirotaka Murase, Kosuke Nakamoto, Yasuaki Kimura, Naoko Abe, Kumiko Ui-Tei, Hiroshi Abe","doi":"10.1093/nar/gkae741","DOIUrl":"https://doi.org/10.1093/nar/gkae741","url":null,"abstract":"<p><p>In this study, we report the synthesis of 2'-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5-11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2'-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2'-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2'-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Contact-dependent growth inhibition (CDI) is a bacterial competition mechanism, wherein the C-terminal toxin domain of CdiA protein (CdiA-CT) is transferred from one bacterium to another, impeding the growth of the toxin recipient. In uropathogenic Escherichia coli 536, CdiA-CT (CdiA-CTEC536) is a tRNA anticodon endonuclease that requires a cysteine biogenesis factor, CysK, for its activity. However, the mechanism underlying tRNA recognition and cleavage by CdiA-CTEC536 remains unresolved. Here, we present the cryo-EM structure of the CysK:CdiA-CTEC536:tRNA ternary complex. The interaction between CdiA-CTEC536 and CysK stabilizes the CdiA-CTEC536 structure and facilitates tRNA binding and the formation of the CdiA-CTEC536 catalytic core structure. The bottom-half of the tRNA interacts exclusively with CdiA-CTEC536 and the α-helices of CdiA-CTEC536 engage with the minor and major grooves of the bottom-half of tRNA, positioning the tRNA anticodon loop at the CdiA-CTEC536 catalytic site for tRNA cleavage. Thus, CysK serves as a platform facilitating the recognition and cleavage of substrate tRNAs by CdiA-CTEC536.
{"title":"Mechanism of activation of contact-dependent growth inhibition tRNase toxin by the amino acid biogenesis factor CysK in the bacterial competition system.","authors":"Zhaohang Feng, Yuka Yashiro, Kozo Tomita","doi":"10.1093/nar/gkae735","DOIUrl":"https://doi.org/10.1093/nar/gkae735","url":null,"abstract":"<p><p>Contact-dependent growth inhibition (CDI) is a bacterial competition mechanism, wherein the C-terminal toxin domain of CdiA protein (CdiA-CT) is transferred from one bacterium to another, impeding the growth of the toxin recipient. In uropathogenic Escherichia coli 536, CdiA-CT (CdiA-CTEC536) is a tRNA anticodon endonuclease that requires a cysteine biogenesis factor, CysK, for its activity. However, the mechanism underlying tRNA recognition and cleavage by CdiA-CTEC536 remains unresolved. Here, we present the cryo-EM structure of the CysK:CdiA-CTEC536:tRNA ternary complex. The interaction between CdiA-CTEC536 and CysK stabilizes the CdiA-CTEC536 structure and facilitates tRNA binding and the formation of the CdiA-CTEC536 catalytic core structure. The bottom-half of the tRNA interacts exclusively with CdiA-CTEC536 and the α-helices of CdiA-CTEC536 engage with the minor and major grooves of the bottom-half of tRNA, positioning the tRNA anticodon loop at the CdiA-CTEC536 catalytic site for tRNA cleavage. Thus, CysK serves as a platform facilitating the recognition and cleavage of substrate tRNAs by CdiA-CTEC536.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142126306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jianfeng Chen, Siyuan Su, Adrian Pickar-Oliver, Anna M Chiarella, Quentin Hahn, Dennis Goldfarb, Erica W Cloer, George W Small, Smaran Sivashankar, Dale A Ramsden, Michael B Major, Nathaniel A Hathaway, Charles A Gersbach, Pengda Liu
As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
{"title":"Engineered Cas9 variants bypass Keap1-mediated degradation in human cells and enhance epigenome editing efficiency.","authors":"Jianfeng Chen, Siyuan Su, Adrian Pickar-Oliver, Anna M Chiarella, Quentin Hahn, Dennis Goldfarb, Erica W Cloer, George W Small, Smaran Sivashankar, Dale A Ramsden, Michael B Major, Nathaniel A Hathaway, Charles A Gersbach, Pengda Liu","doi":"10.1093/nar/gkae761","DOIUrl":"https://doi.org/10.1093/nar/gkae761","url":null,"abstract":"<p><p>As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142126304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, which is de-regulated in various cancers, in human Argonaute-2 (hAgo2), the effector protein in RISC. This analysis employs guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced Magic Angle Spinning (MAS) NMR. Homonuclear correlation experiments revealed that the non-A-form helical conformation of miR-34a increases when incorporated into hAgo2 and subsequently bound to SIRT1 mRNA compared to the free miR-34a or the free mRNA:miR duplex. The C8-C1' correlation provided a nucleotide-specific distribution of C2'- and C3'-endo sugar puckering, revealing the capture of diverse dynamic conformations upon freezing. Predominantly C3'-endo puckering was observed for the seed region, while C2'-endo conformation was found in the central region, with a mixture of both conformations elsewhere. These observations provide insights into the molecular dynamics underlying miR-mediated mRNA regulation and demonstrate that experiments conducted under cryogenic conditions, such as at 90 K, can capture and reveal frozen dynamic states, using methods like DNP-enhanced MAS NMR or Cryo-Electron Microscopy.
{"title":"Elucidating microRNA-34a organisation within human Argonaute-2 by dynamic nuclear polarisation-enhanced magic angle spinning NMR.","authors":"Rubin Dasgupta, Walter Becker, Katja Petzold","doi":"10.1093/nar/gkae744","DOIUrl":"https://doi.org/10.1093/nar/gkae744","url":null,"abstract":"<p><p>Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, which is de-regulated in various cancers, in human Argonaute-2 (hAgo2), the effector protein in RISC. This analysis employs guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced Magic Angle Spinning (MAS) NMR. Homonuclear correlation experiments revealed that the non-A-form helical conformation of miR-34a increases when incorporated into hAgo2 and subsequently bound to SIRT1 mRNA compared to the free miR-34a or the free mRNA:miR duplex. The C8-C1' correlation provided a nucleotide-specific distribution of C2'- and C3'-endo sugar puckering, revealing the capture of diverse dynamic conformations upon freezing. Predominantly C3'-endo puckering was observed for the seed region, while C2'-endo conformation was found in the central region, with a mixture of both conformations elsewhere. These observations provide insights into the molecular dynamics underlying miR-mediated mRNA regulation and demonstrate that experiments conducted under cryogenic conditions, such as at 90 K, can capture and reveal frozen dynamic states, using methods like DNP-enhanced MAS NMR or Cryo-Electron Microscopy.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142126220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sandra Martin, Florian Fournes, Giovanna Ambrosini, Christian Iseli, Karolina Bojkowska, Julien Marquis, Nicolas Guex, Justine Collier
The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen.
{"title":"DNA methylation by CcrM contributes to genome maintenance in the Agrobacterium tumefaciens plant pathogen.","authors":"Sandra Martin, Florian Fournes, Giovanna Ambrosini, Christian Iseli, Karolina Bojkowska, Julien Marquis, Nicolas Guex, Justine Collier","doi":"10.1093/nar/gkae757","DOIUrl":"https://doi.org/10.1093/nar/gkae757","url":null,"abstract":"<p><p>The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142126219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Simona Ferraioli, Fatih Sarigol, Celine Prakash, Daria Filipczak, Roland Foisner, Nana Naetar
A-type lamins form a filamentous meshwork beneath the nuclear membrane that anchors large heterochromatic genomic regions at the nuclear periphery. A-type lamins also exist as a dynamic, non-filamentous pool in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). Both proteins associate with largely overlapping euchromatic genomic regions in the nucleoplasm, but the functional significance of this interaction is poorly understood. Here, we report that LAP2α relocates towards regions containing myogenic genes in the early stages of muscle differentiation, possibly facilitating efficient gene regulation, while lamins A and C mostly associate with genomic regions away from these genes. Strikingly, upon depletion of LAP2α, A-type lamins spread across active chromatin and accumulate at regions of active H3K27ac and H3K4me3 histone marks in the vicinity of myogenic genes whose expression is impaired in the absence of LAP2α. Reorganization of A-type lamins on chromatin is accompanied by depletion of the active chromatin mark H3K27ac and a significantly impaired myogenic differentiation. Thus, the interplay of LAP2α and A-type lamins is crucial for proper positioning of intranuclear lamin A/C on chromatin to allow efficient myogenic differentiation.
A 型片层蛋白在核膜下形成丝状网状结构,将大型异染色质基因组区域固定在核外围。A 型片层蛋白在核内部也以动态、非丝状的形式存在,它们与片层相关多肽 2 alpha(LAP2α)相互作用。这两种蛋白都与核质中基本重叠的染色体基因组区域相关联,但这种相互作用的功能意义却鲜为人知。在这里,我们报告了 LAP2α 在肌肉分化的早期阶段向含有肌生成基因的区域迁移,这可能有助于有效的基因调控,而片段蛋白 A 和 C 则主要与远离这些基因的基因组区域结合。令人吃惊的是,当LAP2α缺失时,A型片段蛋白会扩散到活跃的染色质上,并在H3K27ac和H3K4me3组蛋白标记活跃的区域聚集,这些区域位于LAP2α缺失时表达受损的肌生成基因附近。染色质上 A 型片层蛋白的重组伴随着活性染色质标记 H3K27ac 的耗竭和成肌分化的显著受损。因此,LAP2α和A型片层蛋白的相互作用对于核内片层蛋白A/C在染色质上的正确定位至关重要,这样才能实现高效的成肌分化。
{"title":"LAP2alpha facilitates myogenic gene expression by preventing nucleoplasmic lamin A/C from spreading to active chromatin regions.","authors":"Simona Ferraioli, Fatih Sarigol, Celine Prakash, Daria Filipczak, Roland Foisner, Nana Naetar","doi":"10.1093/nar/gkae752","DOIUrl":"https://doi.org/10.1093/nar/gkae752","url":null,"abstract":"<p><p>A-type lamins form a filamentous meshwork beneath the nuclear membrane that anchors large heterochromatic genomic regions at the nuclear periphery. A-type lamins also exist as a dynamic, non-filamentous pool in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). Both proteins associate with largely overlapping euchromatic genomic regions in the nucleoplasm, but the functional significance of this interaction is poorly understood. Here, we report that LAP2α relocates towards regions containing myogenic genes in the early stages of muscle differentiation, possibly facilitating efficient gene regulation, while lamins A and C mostly associate with genomic regions away from these genes. Strikingly, upon depletion of LAP2α, A-type lamins spread across active chromatin and accumulate at regions of active H3K27ac and H3K4me3 histone marks in the vicinity of myogenic genes whose expression is impaired in the absence of LAP2α. Reorganization of A-type lamins on chromatin is accompanied by depletion of the active chromatin mark H3K27ac and a significantly impaired myogenic differentiation. Thus, the interplay of LAP2α and A-type lamins is crucial for proper positioning of intranuclear lamin A/C on chromatin to allow efficient myogenic differentiation.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142126305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ana Quintas, Robert F Harvey, Emilie Horvilleur, Gavin D Garland, Tobias Schmidt, Lajos Kalmar, Veronica Dezi, Alberto Marini, Alexander M Fulton, Tuija A A Pöyry, Cameron H Cole, Martin Turner, Ritwick Sawarkar, Michael A Chapman, Martin Bushell, Anne E Willis
RNA binding proteins drive proliferation and tumorigenesis by regulating the translation and stability of specific subsets of messenger RNAs (mRNAs). We have investigated the role of eukaryotic initiation factor 4B (eIF4B) in this process and identify 10-fold more RNA binding sites for eIF4B in tumour cells from patients with diffuse large B-cell lymphoma compared to control B cells and, using individual-nucleotide resolution UV cross-linking and immunoprecipitation, find that eIF4B binds the entire length of mRNA transcripts. eIF4B stimulates the helicase activity of eIF4A, thereby promoting the unwinding of RNA structure within the 5' untranslated regions of mRNAs. We have found that, in addition to its well-documented role in mRNA translation, eIF4B additionally interacts with proteins associated with RNA turnover, including UPF1 (up-frameshift protein 1), which plays a key role in histone mRNA degradation at the end of S phase. Consistent with these data, we locate an eIF4B binding site upstream of the stem-loop structure in histone mRNAs and show that decreased eIF4B expression alters histone mRNA turnover and delays cell cycle progression through S phase. Collectively, these data provide insight into how eIF4B promotes tumorigenesis.
{"title":"Eukaryotic initiation factor 4B is a multi-functional RNA binding protein that regulates histone mRNAs.","authors":"Ana Quintas, Robert F Harvey, Emilie Horvilleur, Gavin D Garland, Tobias Schmidt, Lajos Kalmar, Veronica Dezi, Alberto Marini, Alexander M Fulton, Tuija A A Pöyry, Cameron H Cole, Martin Turner, Ritwick Sawarkar, Michael A Chapman, Martin Bushell, Anne E Willis","doi":"10.1093/nar/gkae767","DOIUrl":"https://doi.org/10.1093/nar/gkae767","url":null,"abstract":"<p><p>RNA binding proteins drive proliferation and tumorigenesis by regulating the translation and stability of specific subsets of messenger RNAs (mRNAs). We have investigated the role of eukaryotic initiation factor 4B (eIF4B) in this process and identify 10-fold more RNA binding sites for eIF4B in tumour cells from patients with diffuse large B-cell lymphoma compared to control B cells and, using individual-nucleotide resolution UV cross-linking and immunoprecipitation, find that eIF4B binds the entire length of mRNA transcripts. eIF4B stimulates the helicase activity of eIF4A, thereby promoting the unwinding of RNA structure within the 5' untranslated regions of mRNAs. We have found that, in addition to its well-documented role in mRNA translation, eIF4B additionally interacts with proteins associated with RNA turnover, including UPF1 (up-frameshift protein 1), which plays a key role in histone mRNA degradation at the end of S phase. Consistent with these data, we locate an eIF4B binding site upstream of the stem-loop structure in histone mRNAs and show that decreased eIF4B expression alters histone mRNA turnover and delays cell cycle progression through S phase. Collectively, these data provide insight into how eIF4B promotes tumorigenesis.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142120279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The BEN domain is a newly discovered type of DNA-binding domain that exists in a variety of species. There are nine BEN domain-containing proteins in humans, and most have been shown to have chromatin-related functions. NACC1 preferentially binds to CATG motif-containing sequences and functions primarily as a transcriptional coregulator. BANP and BEND3 preferentially bind DNA bearing unmethylated CpG motifs, and they function as CpG island-binding proteins. To date, the DNA recognition mechanism of quite a few of these proteins remains to be determined. In this study, we solved the crystal structures of the BEN domains of NACC1 and BANP in complex with their cognate DNA substrates. We revealed the details of DNA binding by these BEN domain proteins and unexpectedly revealed that oligomerization is required for BANP to select unmethylated CGCG motif-containing DNA substrates. Our study clarifies the controversies surrounding DNA recognition by BANP and demonstrates a new mechanism by which BANP selects unmethylated CpG motifs and functions as a CpG island-binding protein. This understanding will facilitate further exploration of the physiological functions of the BEN domain proteins in the future.
BEN 结构域是一种新发现的 DNA 结合结构域,存在于多种物种中。人类有九种含 BEN 结构域的蛋白质,其中大多数已被证明具有染色质相关功能。NACC1 优先与含 CATG 标记的序列结合,主要作为转录核心调节因子发挥作用。BANP 和 BEND3 优先结合含有未甲基化 CpG 基序的 DNA,它们是 CpG 岛结合蛋白。迄今为止,这些蛋白中仍有不少的 DNA 识别机制尚未确定。在这项研究中,我们解析了 NACC1 和 BANP 的 BEN 结构域与其同源 DNA 底物复合的晶体结构。我们揭示了这些 BEN 结构域蛋白与 DNA 结合的细节,并意外地发现 BANP 在选择含有未甲基化 CGCG 矩阵的 DNA 底物时需要寡聚化。我们的研究澄清了围绕 BANP 识别 DNA 的争议,展示了 BANP 选择未甲基化 CpG 基序并作为 CpG 岛结合蛋白发挥作用的新机制。这一认识将有助于今后进一步探索 BEN 结构域蛋白的生理功能。
{"title":"Structural basis of DNA recognition by BEN domain proteins reveals a role for oligomerization in unmethylated DNA selection by BANP.","authors":"Jiahao Ren, Junmeng Wang, Yanpeng Ren, Yuyang Zhang, Pengshuai Wei, Meng Wang, Yimeng Zhang, Meng Li, Chuyan Yuan, Haipeng Gong, Junyi Jiang, Zhanxin Wang","doi":"10.1093/nar/gkae762","DOIUrl":"https://doi.org/10.1093/nar/gkae762","url":null,"abstract":"<p><p>The BEN domain is a newly discovered type of DNA-binding domain that exists in a variety of species. There are nine BEN domain-containing proteins in humans, and most have been shown to have chromatin-related functions. NACC1 preferentially binds to CATG motif-containing sequences and functions primarily as a transcriptional coregulator. BANP and BEND3 preferentially bind DNA bearing unmethylated CpG motifs, and they function as CpG island-binding proteins. To date, the DNA recognition mechanism of quite a few of these proteins remains to be determined. In this study, we solved the crystal structures of the BEN domains of NACC1 and BANP in complex with their cognate DNA substrates. We revealed the details of DNA binding by these BEN domain proteins and unexpectedly revealed that oligomerization is required for BANP to select unmethylated CGCG motif-containing DNA substrates. Our study clarifies the controversies surrounding DNA recognition by BANP and demonstrates a new mechanism by which BANP selects unmethylated CpG motifs and functions as a CpG island-binding protein. This understanding will facilitate further exploration of the physiological functions of the BEN domain proteins in the future.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142120294","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Proteolysis-targeting chimera (PROTAC) is an emerging therapeutic technology that leverages the ubiquitin-proteasome system to target protein degradation. Due to its event-driven mechanistic characteristics, PROTAC has the potential to regulate traditionally non-druggable targets. Recently, AI-aided drug design has accelerated the development of PROTAC drugs. However, the rational design of PROTACs remains a considerable challenge. Here, we present an updated online database, PROTAC-DB 3.0. In this third version, we have expanded the database to include 6111 PROTACs (87% increase compared to the 2.0 version). Additionally, the database now contains 569 warheads (small molecules targeting the protein), 2753 linkers, and 107 E3 ligands (small molecules recruiting E3 ligases). The number of target-PROTAC-E3 ternary complex structures has also increased to 959. Recognizing the importance of druggability in PROTAC design, we have incorporated pharmacokinetic data to PROTAC-DB 3.0. To enhance user experience, we have added features for sorting based on molecular similarity and literature publication date. PROTAC-DB 3.0 is accessible at http://cadd.zju.edu.cn/protacdb/.
{"title":"PROTAC-DB 3.0: an updated database of PROTACs with extended pharmacokinetic parameters.","authors":"Jingxuan Ge, Shimeng Li, Gaoqi Weng, Huating Wang, Meijing Fang, Huiyong Sun, Yafeng Deng, Chang-Yu Hsieh, Dan Li, Tingjun Hou","doi":"10.1093/nar/gkae768","DOIUrl":"https://doi.org/10.1093/nar/gkae768","url":null,"abstract":"<p><p>Proteolysis-targeting chimera (PROTAC) is an emerging therapeutic technology that leverages the ubiquitin-proteasome system to target protein degradation. Due to its event-driven mechanistic characteristics, PROTAC has the potential to regulate traditionally non-druggable targets. Recently, AI-aided drug design has accelerated the development of PROTAC drugs. However, the rational design of PROTACs remains a considerable challenge. Here, we present an updated online database, PROTAC-DB 3.0. In this third version, we have expanded the database to include 6111 PROTACs (87% increase compared to the 2.0 version). Additionally, the database now contains 569 warheads (small molecules targeting the protein), 2753 linkers, and 107 E3 ligands (small molecules recruiting E3 ligases). The number of target-PROTAC-E3 ternary complex structures has also increased to 959. Recognizing the importance of druggability in PROTAC design, we have incorporated pharmacokinetic data to PROTAC-DB 3.0. To enhance user experience, we have added features for sorting based on molecular similarity and literature publication date. PROTAC-DB 3.0 is accessible at http://cadd.zju.edu.cn/protacdb/.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142120280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kathryn Turnbull, Helge Paternoga, Esther von der Weth, Artyom A Egorov, Agnieszka A Pochopien, Yujie Zhang, Lilit Nersisyan, Tõnu Margus, Marcus J O Johansson, Vicent Pelechano, Daniel N Wilson, Vasili Hauryliuk
The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.
翻译终止的效率取决于终止密码子的性质及其上下文。在真核生物中,A 位点终止密码子的识别和多肽的释放分别由释放因子 eRF1 和 eRF3 介导。翻译终止受其他因子的调节,这些因子或直接与释放因子相互作用,或与 E 位点结合并调节肽基转移酶中心的活性。以前的研究表明,酿酒酵母 ABCF ATPase New1 参与了翻译终止和/或核糖体再循环,但其确切功能仍不清楚。在这里,我们应用了 5PSeq、单颗粒低温电子显微镜和读穿报告实验来深入了解 New1 的生物学功能。 我们发现,缺乏 New1 会导致核糖体在以赖氨酸或精氨酸密码子为前缀的终止密码子处停滞,而且这种停滞并不是由 C 端氨基酸的性质决定的,而是由 P 位点中 tRNA 异位接受者的身份决定的。总之,我们的研究结果表明,当核糖体的 P 位点有特定的 tRNA 同位受体时,翻译终止的效率很低,而 New1 的招募能在这些有问题的终止情况下挽救核糖体。
{"title":"The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site","authors":"Kathryn Turnbull, Helge Paternoga, Esther von der Weth, Artyom A Egorov, Agnieszka A Pochopien, Yujie Zhang, Lilit Nersisyan, Tõnu Margus, Marcus J O Johansson, Vicent Pelechano, Daniel N Wilson, Vasili Hauryliuk","doi":"10.1093/nar/gkae748","DOIUrl":"https://doi.org/10.1093/nar/gkae748","url":null,"abstract":"The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142101026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}