RNA最新文献 - Book学术

ykkC-I Riboswitch Aptamer Domain Requires an Interplay Between Metal Triad and Cationic Ligand: Insights into Metal Ion-Induced Allostery from Molecular Dynamics Simulations. 核开关适体结构域需要金属三元体和阳离子配体之间的相互作用：从分子动力学模拟的金属离子诱导变构的见解。

IF 5 3区生物学

RNA Pub Date : 2025-10-07 DOI: 10.1261/rna.080619.125

Indu Negi, Stacey Wetmore

{"title":"ykkC-I Riboswitch Aptamer Domain Requires an Interplay Between Metal Triad and Cationic Ligand: Insights into Metal Ion-Induced Allostery from Molecular Dynamics Simulations.","authors":"Indu Negi, Stacey Wetmore","doi":"10.1261/rna.080619.125","DOIUrl":"https://doi.org/10.1261/rna.080619.125","url":null,"abstract":"Riboswitches are non-coding mRNA regions that regulate gene expression by sensing small molecules. While most riboswitches turn off gene expression, guanidine-I family riboswitches enhance gene expression. Although crystal structures provide insights into the structural basis for guanidinium ion (Gdm+) sensing by the guanidine-I riboswitch aptamer (GRA) domain, the mechanistic interplay between ligand and metal ion binding, RNA conformational changes, and regulatory function remains poorly understood. Using molecular dynamics simulations, we explore the combined effects of the positively charged Gdm+, Mg2+, and K+ observed in close proximity in the experimental crystal structure on the GRA structural dynamics. Our simulations reveal that the binding pocket frequently transitions between ligand bound-like and unbound-like states in the absence of divalent ions, while Mg2+ stabilizes a bound-like RNA conformation. Furthermore, both Mg2+ and Gdm+ facilitate K+ positioning near the binding pocket. As a result, Mg2+, Gdm+, and K+ synergistically increase the structural rigidity of the GRA domain, particularly the P2-P3 junction and the 3' end near the terminator stem. This enhances localized interactions that pull the P1a and P3 domains together to make the transcriptional control region available for expression. Our proposed mechanism is fully consistent with experimental structural and biochemical (including isothermal titration calorimetry and structure-guided mutagenesis) data and rationalizes how the unique triad of ions work together to influence the conformational dynamics of the aptamer domain and riboswitch function. This information can guide future synthetic riboswitch design and the identification of novel therapeutic targets beyond static structural information.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145245011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

UPF1 shuttles between nucleus and cytoplasm independently of its RNA binding and ATPase activities. UPF1在细胞核和细胞质之间穿梭，独立于RNA结合和atp酶活性。

IF 5 3区生物学

RNA Pub Date : 2025-10-07 DOI: 10.1261/rna.080476.125

Sofia Nasif, Andrea Brigitte Eberle, Karin Schranz, Remo Hadorn, Sutapa Chakrabarti, Oliver Mühlemann

{"title":"UPF1 shuttles between nucleus and cytoplasm independently of its RNA binding and ATPase activities.","authors":"Sofia Nasif, Andrea Brigitte Eberle, Karin Schranz, Remo Hadorn, Sutapa Chakrabarti, Oliver Mühlemann","doi":"10.1261/rna.080476.125","DOIUrl":"https://doi.org/10.1261/rna.080476.125","url":null,"abstract":"The ATP-dependent RNA helicase Up-frameshift 1 (UPF1) is an essential protein in mammalian cells and a key factor in nonsense-mediated mRNA decay (NMD), a translation-dependent mRNA surveillance process. UPF1 is mainly cytoplasmic at steady state but accumulates in the nucleus after inhibiting CRM1-mediated nuclear export by Leptomycin B (LMB), indicating that UPF1 shuttles between the nucleus and the cytoplasm. Consistent with its dual localization, there is evidence for nuclear functions of UPF1, for instance in DNA replication, DNA damage response, and telomere maintenance. However, whether any of UPF1's biochemical activities are required for its nuclear-cytoplasmic shuttling remains unclear. To investigate this, we examined two UPF1 mutants: the well-described ATPase-deficient UPF1-DE (D636A/E637A) and a newly generated RNA-binding mutant UPF1-NKR (N524A/K547A/R843A). Biochemical assays confirmed that the UPF1-NKR mutant cannot bind RNA or hydrolyze ATP in vitro but retains interaction with UPF2, UPF3B, and SMG6. Overexpression of UPF1-NKR exerted a dominant-negative effect on endogenous UPF1 and inhibited NMD. Subcellular localization studies revealed that UPF1-DE accumulates in cytoplasmic granules (P-bodies), even in the presence of LMB, whereas UPF1-NKR shuttles normally. This indicates that UPF1's shuttling does not require its RNA-binding or ATPase activities. Notably, the UPF1-DE.NKR double mutant restored nuclear-cytoplasmic shuttling and prevented accumulation in P-bodies, suggesting that the shuttling defect of UPF1-DE arises from its tight binding to RNA. Overall, our findings demonstrate that UPF1's shuttling is independent of its ATPase and RNA-binding activities, with RNA binding itself being a key determinant of its cytoplasmic retention.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145244999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Structural and mechanistic insights into Dis3L2-mediated degradation of structured RNA. dis3l2介导的结构化RNA降解的结构和机制见解。

IF 5 3区生物学

RNA Pub Date : 2025-10-01 DOI: 10.1261/rna.080685.125

Rute G Matos, Ankur Garg, Susana M Costa, Patricia Pereira, Cecilia M Arraiano, Leemor Joshua-Tor, Sandra C Viegas

{"title":"Structural and mechanistic insights into Dis3L2-mediated degradation of structured RNA.","authors":"Rute G Matos, Ankur Garg, Susana M Costa, Patricia Pereira, Cecilia M Arraiano, Leemor Joshua-Tor, Sandra C Viegas","doi":"10.1261/rna.080685.125","DOIUrl":"https://doi.org/10.1261/rna.080685.125","url":null,"abstract":"The RNase II/RNB family of exoribonucleases is present in all domains of life and includes three main eukaryotic members, the Dis3-like proteins (Dis3, Dis3L1, Dis3L2). At the cellular level, Dis3L2 is distinguished by the unique preference for uridylated RNA substrates and the highest efficiency in degrading double-stranded RNA. Defects in these enzymes have been linked to some types of cancers and overgrowth disorders in humans. In this work, we used the Dis3L2 protein from the model organism Schizosaccharomyces pombe (SpDis3L2) to better understand the mechanism of action of Dis3-like exoribonucleases, and to elucidate how single amino acid substitutions in these proteins can affect the biochemical properties of the enzymes, potentially contributing to the molecular basis of the related human diseases. We determined the crystal structure of SpDis3L2 bound to a U13 RNA, in which the protein displays a typical vase-like conformation, accommodating 6 nucleotides of the RNA 3'-end. Furthermore, we constructed two SpDis3L2 protein variants, harbouring single amino acid substitutions mimicking the ones already found in human patients, to test their catalytic activity in vitro. We highlight the A756R SpDis3L2 variant, which loses the ability to degrade double-stranded RNA substrates and accumulates intermediate degradation products when degrading single-stranded RNA substrates. As such, A756 seems to be a key residue responsible for the normal cellular function of Dis3L2, specifically regarding its important role in the degradation of structured RNA substrates.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145207553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The role of Nonsense-Mediated mRNA Decay in restricting long noncoding RNAs expression has been conserved in RNAi-capable budding yeast. 无义介导的mRNA衰变在限制长链非编码rna表达中的作用在rnai能力的芽殖酵母中已被保守。

IF 5 3区生物学

RNA Pub Date : 2025-09-30 DOI: 10.1261/rna.080458.125

Maxime Wery, Ugo Szachnowski, Constance Creux, Quentin Fouilleul, Marina Lefrere, Antonin Morillon

{"title":"The role of Nonsense-Mediated mRNA Decay in restricting long noncoding RNAs expression has been conserved in RNAi-capable budding yeast.","authors":"Maxime Wery, Ugo Szachnowski, Constance Creux, Quentin Fouilleul, Marina Lefrere, Antonin Morillon","doi":"10.1261/rna.080458.125","DOIUrl":"https://doi.org/10.1261/rna.080458.125","url":null,"abstract":"In most Eukaryotes, sense/antisense RNA duplexes can be processed into small interfering RNAs by the ribonuclease III Dicer, a key component of the RNA interference (RNAi) machinery, which has been lost by the budding yeast Saccharomyces cerevisiae Previous studies in this species revealed the pervasive formation of double-stranded (ds)RNA involving antisense Xrn1-sensitive long noncoding (lnc)RNAs, which interferes with their degradation through translation-dependent Nonsense-Mediated mRNA decay (NMD). However, apart from S. cerevisiae, little is known about the post-transcriptional metabolism of lncRNAs, in particular the functional impact of RNAi. Herein, we profiled NMD targets in Naumovozyma castellii, a budding yeast endowed with cytoplasmic RNAi. We identified 592 lncRNAs accumulating in a mutant of the NMD core factor Upf1. Most of them also accumulate in other NMD mutants and upon translation elongation inhibition, indicating a translation-dependent degradation mechanism. Consistently, Ribo-Seq analyses confirmed ribosomes binding for a fraction of them. Within the coding transcriptome, we found that the Dicer-coding mRNA is also regulated by NMD. The resulting upregulation of DCR1 in NMD-deficient cells correlates with an increased production of small RNAs from dsRNA-forming NMD-sensitive lncRNAs and mRNAs. Finally, we observed that Dicer inactivation in Upf1-lacking cells attenuates the accumulation of dsRNA-forming NMD targets. Together, our data highlight the conserved roles of NMD and translation in the post-transcriptional metabolism of lncRNAs, and provide insight into the functional impact of endogenous RNAi on the transcriptome.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145201010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Splice site diversity and abundance of non-canonical introns in diplonemids (Diplonemea, Euglenozoa). 双子体剪接位点的多样性和非规范内含子的丰度（双子体，裸藻）。

IF 5 3区生物学

RNA Pub Date : 2025-09-30 DOI: 10.1261/rna.080641.125

Prasoon Kumar Thakur, Anzhelika Butenko, Filip Karásek, Michaela Svobodov, Drahomíra Faktorová, Hana Pavliskova, Vladimir Varga, Ales Horak, Julius Lukes, David Stanek

{"title":"Splice site diversity and abundance of non-canonical introns in diplonemids (Diplonemea, Euglenozoa).","authors":"Prasoon Kumar Thakur, Anzhelika Butenko, Filip Karásek, Michaela Svobodov, Drahomíra Faktorová, Hana Pavliskova, Vladimir Varga, Ales Horak, Julius Lukes, David Stanek","doi":"10.1261/rna.080641.125","DOIUrl":"https://doi.org/10.1261/rna.080641.125","url":null,"abstract":"Non-coding introns are a unifying feature of protein-coding genes in virtually all extant eukaryotes, with most lineages following the canonical intron structure. However, euglenozoans, unicellular flagellates that include free-living euglenids, human pathogenic kinetoplastids, and highly diverse and abundant marine diplonemids, are a notable exception. Euglenozoan genomes range from extremely intron-poor kinetoplastids to euglenid genomes containing both canonical and non-canonical introns. Here, we present a comprehensive analysis of splice sites and spliceosomal components in six species of understudied diplonemids. All diplonemids examined contain a nearly complete set of spliceosomal snRNP components indicating the presence of a functional U2-type spliceosome. However, the majority of introns in the hemistasiid diplonemids Artemidia motanka and Namystynia karyoxenos are non-canonical and lack conserved GT-AG terminal dinucleotides typical for U2-type introns. These non-canonical introns are capable of extensive base pairing, which brings intron ends into close proximity. Thus, while the splicing apparatus is conserved in diplonemids, the splice sites are highly variable among individual species.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200992","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

New mechanistic insights into Prespliceosome formation-Roles of DEAD-box proteins Prp5 and Sub2. pre - plicosome形成机制的新认识——DEAD-box蛋白Prp5和Sub2的作用

IF 5 3区生物学

RNA Pub Date : 2025-09-30 DOI: 10.1261/rna.080720.125

Ching-Yang Kao, Wei-Yu Tsai, Yu-Lun Su, Che-Sheng Chung, Soo-Chen Cheng

引用次数: 0

Depurination of sarcin/ricin loop 25S rRNA is signaled through the small ribosomal subunit during translation. 在翻译过程中，肌素/蓖麻毒素环25S rRNA的脱嘌呤是通过小核糖体亚基发出信号的。

IF 5 3区生物学

RNA Pub Date : 2025-09-23 DOI: 10.1261/rna.080559.125

Tanya Prashar, Katalin A Hudak

{"title":"Depurination of sarcin/ricin loop 25S rRNA is signaled through the small ribosomal subunit during translation.","authors":"Tanya Prashar, Katalin A Hudak","doi":"10.1261/rna.080559.125","DOIUrl":"https://doi.org/10.1261/rna.080559.125","url":null,"abstract":"In addition to their function in protein synthesis, translating ribosomes serve as sensors that communicate the presence of aberrant messenger RNAs (mRNAs); however, how they recognize damage to their ribosomal RNA (rRNA) remains poorly understood. The conserved sarcin/ricin loop (SRL) of the 25S rRNA is a component of the GTPase centre essential for ribosome movement during translation. In this study, we expressed an RNA N-glycosylase called pokeweed antiviral protein (PAP) in yeast Saccharomyces cerevisiae to specifically damage rRNA by hydrolysis of a purine base from the SRL. 25S rRNA depurination inhibited translation elongation, as shown by reduced incorporation of a methionine analog and binding of eukaryotic elongation factor 2 (eEF2) to ribosomes. PAP expression altered sucrose gradient profiles, increasing free subunits and 80S peaks and reducing polysomes without causing ribosome collisions. We discovered depurinated rRNA associated with 80S monosomes and polysomes, suggesting that cells would detect damage to rRNA during active translation. These ribosomes were ubiquitinated by E3 ligases Mag2 and Hel2, elements of the 18S non-functional rRNA decay (NRD) pathway involved in recognizing slow-moving ribosomes. Furthermore, mass spectrometry analysis revealed ubiquitination of ribosomal protein uS3, characteristic of 18S NRD. Even though the SRL is a component of the large ribosomal subunit, its depurination is signaled by ubiquitin ligases that recognize damage to the small subunit. We suggest that slow translation elongation is the factor that communicates SRL depurination to E3 ubiquitin ligases, which extends our understanding of how rRNA integrity is surveilled in yeast.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145131930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Interdomain assembly between the fungal tRNA ligase adenylyltransferase and kinase domain. 真菌tRNA连接酶腺苷基转移酶与激酶结构域之间的结构域间组装。

IF 5 3区生物学

RNA Pub Date : 2025-09-22 DOI: 10.1261/rna.080592.125

Sandra Köhler, Jirka Peschek

{"title":"Interdomain assembly between the fungal tRNA ligase adenylyltransferase and kinase domain.","authors":"Sandra Köhler, Jirka Peschek","doi":"10.1261/rna.080592.125","DOIUrl":"https://doi.org/10.1261/rna.080592.125","url":null,"abstract":"Trl1-type ligases play an essential role in fungi and plants during the non-conventional tRNA splicing as well as the unfolded protein response. The tripartite enzyme consists of an N-terminal adenylyltransferase domain (LIG), a central polynucleotide kinase domain (KIN) and a C-terminal cyclic phosphodiesterase domain (CPD). The Trl1-mediated reaction can be divided into two steps: (1) RNA end modification by the KIN and CPD domains, and (2) the adenylyltransferase reaction catalyzed by the LIG domain resulting in the phosphodiester bond formation. Due to its absence in humans, Trl1 is often discussed as potential target for antifungal therapy. To date structural information on the full-length Trl1 are missing. Several crystal structures of the individual LIG and KIN as well as a KIN-CPD construct have been solved, thereby elucidating the fold of the individual domains, their cofactor and substrate binding. Here, we provide the missing crystal structure of the two-domain LIG-KIN construct from the thermophilic fungus Chaetomium thermophilum revealing the interdomain assembly and interface. Based on our structure and complementing AlphaFold3 predictions, we further propose a model with implications for interdomain RNA substrate transfer.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145126007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Integrated NMR and MD structure and dynamics of the stem loop II motif (s2m) from the Omicron variant of SARS-CoV-2. SARS-CoV-2 Omicron变体茎环II基序（s2m）的NMR和MD集成结构和动力学

IF 5 3区生物学

RNA Pub Date : 2025-09-19 DOI: 10.1261/rna.080576.125

Tobias Matzel, Joseph Makowski, Adam H Kensinger, Andreas Oxenfarth, Maria A Wirtz Martin, Jeffrey Evanseck, Harald Schwalbe

{"title":"Integrated NMR and MD structure and dynamics of the stem loop II motif (s2m) from the Omicron variant of SARS-CoV-2.","authors":"Tobias Matzel, Joseph Makowski, Adam H Kensinger, Andreas Oxenfarth, Maria A Wirtz Martin, Jeffrey Evanseck, Harald Schwalbe","doi":"10.1261/rna.080576.125","DOIUrl":"https://doi.org/10.1261/rna.080576.125","url":null,"abstract":"The stem-loop-II motif (s2m) is a conserved viral RNA element located in the 3'UTR of different viruses including SARS-CoV-2. High resolution 3D structural data for s2m are only available for the fundamentally different SCoV-1 version and difficult to access for SARS-CoV-2 due to the highly dynamic nature of the s2m RNA element. With the omicron variant, a large deletion occurred for s2m resulting in a relatively short hairpin with an apical pentaloop. We determined the NMR solution structure of s2m_omicron using a variety of torsion-angle sensitive NMR parameters in addition to NOE distance restraints. Surprisingly, relatively high {1H},13C heteronuclear NOE values, averaged ribose 3JHH-coupling constants (H1'H2'; H3'H4') and dipole(H1'-C1'), dipole(H6/8-C6/8)-CCRs hinted towards significant dynamics for the small pentaloop making structure calculations solely relying on NMR data insufficient. To address this problem, we performed ten 1 microsecond MD-simulations from the NMR structure bundle as a starting point and applied Bayesian Maximum Entropy (BME) reweighting to refine the ensemble with the 3J-coupling constant data. Our results from the combined methodology provide a detailed view of the conformational dynamics of the omicron variant of s2m characterized by different stacking patterns, ribose repuckering and overall heterogeneity of the torsion angles for the loop nucleotides. Strikingly, despite the deletion of the initial nonaloop, as present in the Wuhan and Delta variants of s2m, our combined methodology reveals substantial dynamics and reorganization of a conserved UAC triplet at the tip of the pentaloop, adding physical insight that may be leveraged for the ultimate determination of the still unknown function of the RNA element.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145092495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Desthiobiotin (DTB)-modified and TAMRA-modified 2''OMeNAD⁺ are RNA 2'-phosphotransferase (Tpt1) poisons that enable affinity-tagging and fluorescence-tagging of internal RNA 2'-phosphate groups. 去硫代生物素（DTB）修饰和tamra修饰的2 " OMeNAD+是RNA 2'-磷酸转移酶（Tpt1）毒药，能够对内部RNA 2'-磷酸基团进行亲和标记和荧光标记。

IF 5 3区生物学

RNA Pub Date : 2025-09-18 DOI: 10.1261/rna.080707.125

Renata Kasprzyk, Shreya Ghosh, Stewart Shuman

{"title":"Desthiobiotin (DTB)-modified and TAMRA-modified 2''OMeNAD+ are RNA 2'-phosphotransferase (Tpt1) poisons that enable affinity-tagging and fluorescence-tagging of internal RNA 2'-phosphate groups.","authors":"Renata Kasprzyk, Shreya Ghosh, Stewart Shuman","doi":"10.1261/rna.080707.125","DOIUrl":"https://doi.org/10.1261/rna.080707.125","url":null,"abstract":"RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1'' to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. We showed previously that 2''OMeNAD+, a synthetic NAD+ analog that cannot support step 2 transesterification, is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate as an abortive product. Here we report the chemical synthesis of two novel 2''OMeNAD+ compounds, containing desthiobiotin (DTB) linked to adenine C2 or N6 via a diaminohexane linker. Whereas both analogs poison the RslTpt1 reaction after step 1, the 2''OMeNAD-2-DTB derivative supports a higher yield of RNA-2'-phospho-(DTB-ADP-2''OMe-ribose) product, which can be recovered by adsorption to streptavidin beads and elution with biotin. Our results recommend 2''OMeNAD-2-DTB as a novel affinity-tag probe of RNA 2'-phosphate modification. We synthesized a fluorescent derivative, 2''OMeNAD-2-TAMRA, and find that it, too, is an effective step 1 substrate for RslTpt1 that allows fluorescent labeling of an RNA 2'-phosphate.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":5.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0