IF 4.3 3区生物学

RNA Pub Date : 2026-10-09 DOI: 10.1261/rna.081162.126

Ilias Skeparnias, Athanasios-Nasir Shaukat, Christos Katsioulas, Dimitrios Anastasakis, Katerina Grafanaki, Vasiliki Chondrou, Argyro Sgourou, Jinwei Zhang, Athanasios Papakyriakou, Constantinos Stathopoulos

{"title":"PNLDC1 deadenylase affects translational rate and cell proliferation in mESCs.","authors":"Ilias Skeparnias, Athanasios-Nasir Shaukat, Christos Katsioulas, Dimitrios Anastasakis, Katerina Grafanaki, Vasiliki Chondrou, Argyro Sgourou, Jinwei Zhang, Athanasios Papakyriakou, Constantinos Stathopoulos","doi":"10.1261/rna.081162.126","DOIUrl":"https://doi.org/10.1261/rna.081162.126","url":null,"abstract":"PNLDC1 is a member of the DEDD deadenylases, known for its specificity in poly(A) tail removal during mRNA turnover. While initially characterized for its function in pre-piRNA trimming critical to spermatogenesis and transposon silencing, its broader deadenylase activity which is especially pronounced during early development and silenced during differentiation, remains poorly understood. In this study, we mapped the epigenetic regulation of Pnldc1 during mESC differentiation, uncovering its promoter methylation pattern associated with transcriptional repression. Pnldc1-knockout mESC models established via CRISPR/Cas9 were shown to retain viability and pluripotency. RNA-seq and subsequent polysome profiling revealed that Pnldc1 deficiency leads to the downregulation of gene networks involved in translation and a lower overall translational rate. Notably, PNLDC1 has been linked to epigenetic changes in various cancers and is associated with oncogenic signaling pathways. Using a homology model based on the related PARN deadenylase, we conducted virtual screening of compound libraries and selected 12 candidates for experimental testing. Experimental validation revealed that two compounds significantly impacted genes involved in stress response and signal transduction, while also suppressing aberrant proliferation. These findings highlight PNLDC1's role in regulating mRNA stability during differentiation and proliferation and as a candidate for targeted therapeutic intervention.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149148122","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

Preferential G3BP1-induced condensation of DNA-RNA mixtures from stress granules over RNA or double-stranded DNA. g3bp1诱导的压力颗粒中DNA-RNA混合物的冷凝优于RNA或双链DNA。

IF 4.3 3区生物学

RNA Pub Date : 2026-10-09 DOI: 10.1261/rna.081135.126

Natalia A Demeshkina, Adrian R Ferré-D'Amaré

{"title":"Preferential G3BP1-induced condensation of DNA-RNA mixtures from stress granules over RNA or double-stranded DNA.","authors":"Natalia A Demeshkina, Adrian R Ferré-D'Amaré","doi":"10.1261/rna.081135.126","DOIUrl":"https://doi.org/10.1261/rna.081135.126","url":null,"abstract":"G3BP1 is a regulator of stress granule assembly capable of driving condensation of RNA in vitro. Recently, we purified stress granule cores from yeast and mammalian cells, and found that in addition to RNA and proteins, they contain large amounts of circular double-stranded DNA (dsDNA). Since G3BP1 has been implicated in DNA-RNA heteroduplex resolution, we compared the ability of G3BP1 to drive condensation of cytoplasmic RNA, circular cytoplasmic dsDNA, and total nucleic acids (comprising both, circular dsDNA and RNA) extracted from purified stress granule cores of HEK293T cells. Imaging under near-physiologic conditions (1.7 mM MgCl2, no crowding agents, 0.1 ng/μL nucleic acids) revealed that G3BP1 most effectively induces condensation of the DNA-RNA mixture from stress granule cores. Comparison of the disruption of these condensates by treatment with RNase A, DNase, or RNase H further shows that they are especially susceptible to the latter two. The sensitivity to DNase and RNase H of G3BP1-induced condensates of the stress granule-derived nucleic acid mixtures underscores the importance of DNA in these cytoplasmic assemblies.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149148599","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

Optimization of Circle-Seq improves transcription error profiling and transcriptome representation. Circle-Seq的优化改善了转录错误分析和转录组表示。

IF 4.3 3区生物学

RNA Pub Date : 2026-10-08 DOI: 10.1261/rna.081090.126

Laura Deus Ramirez, Matthew Brandon Cooke, Jennifer Anne Halliday, Christophe Herman

{"title":"Optimization of Circle-Seq improves transcription error profiling and transcriptome representation.","authors":"Laura Deus Ramirez, Matthew Brandon Cooke, Jennifer Anne Halliday, Christophe Herman","doi":"10.1261/rna.081090.126","DOIUrl":"https://doi.org/10.1261/rna.081090.126","url":null,"abstract":"Errors in transcription generate transient RNA variants that are amplified through translation, with potential consequences for cellular phenotypes and RNA-based therapeutics. Accurate detection of these rare events relies on high-fidelity sequencing approaches such as Circle-Seq, but technical artifacts introduced during RNA preparation can distort error profiles and frequencies. Here, we systematically evaluate the impact of bacterial RNA sample preparation on Circle-Seq outputs. We show that RNA extraction and ribosomal RNA depletion introduce artifactual oxidative damage signatures, particularly C>U and G>A substitutions, that inflate apparent transcription error rates. Optimized mRNA preparation conditions substantially reduce these artifacts. In addition, we identify an RNase III fragmentation bias caused by its preference for structured RNAs, resulting in uneven transcriptome coverage. Chaotropic monovalent ions mitigate this bias and improve coverage uniformity. Finally, our optimized protocol captured, with improved accuracy, the frequency and spectrum of transcription errors in absence of the transcription fidelity factor GreA. Together, these results define the technical limitations of Circle-Seq and provide practical solutions to improve the accuracy and interpretability of transcription error measurements in bacterial systems.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149138302","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

Psi RNA-specific Binding Promotes HIV-1 Gag Conformational Change Critical for Immature Viral Particle Assembly. Psi rna特异性结合促进HIV-1 Gag构象变化对未成熟病毒颗粒组装至关重要。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-29 DOI: 10.1261/rna.081222.126

Yehong Qiu, Puja Banerjee, Kaylee Grabarkewitz, Vicki H Wysocki, Ioulia Rouzina, Greg Voth, Karin Musier-Forsyth

{"title":"Psi RNA-specific Binding Promotes HIV-1 Gag Conformational Change Critical for Immature Viral Particle Assembly.","authors":"Yehong Qiu, Puja Banerjee, Kaylee Grabarkewitz, Vicki H Wysocki, Ioulia Rouzina, Greg Voth, Karin Musier-Forsyth","doi":"10.1261/rna.081222.126","DOIUrl":"10.1261/rna.081222.126","url":null,"abstract":"The immature HIV-1 virion is assembled by the Gag polyprotein using inositol hexakisphosphate (IP6) as an essential assembly co-factor. Gag binds the genomic RNA Psi packaging signal via the nucleocapsid (NC) domain and associates with the plasma membrane via the matrix (MA) domain. Previous studies revealed that Gag exists in both compact (C) and extended (E) conformational states in solution. Only E-Gag formed virus-like particles with the correct size and IP6 shifted the equilibrium of DNA-bound Gag to the E state. The influence of specific RNA elements on this conformational change is unknown. In this work, a dual dye-labeled Gag was prepared for probing the effect of RNA binding on Gag conformation using Förster resonance energy transfer (FRET). In low salt and in the absence of other factors, Gag was primarily in the C state. Psi RNA binding induced a more significant FRET decrease than binding to non-Psi RNAs, consistent with a shift to E-Gag. IP6 alone also promoted the E-Gag state in the absence and presence of RNA. Atomistic molecular dynamics simulations are consistent with and provide detail into the role of NC-Psi RNA binding in the conformational switch of C-Gag to assembly-competent E-Gag. Simulations also showed that this switch is driven by capsid (CA) linker domain orientational flexibility and MA-CA unbinding dynamics. Thus, the highly flexible multi-domain Gag polyprotein leverages both viral and host cell factors to sample and stabilize distinct conformations, thereby orchestrating the viral assembly process.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149081969","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

Enterovirus RNase L inhibiting RNAs are highly conserved with limited phylogenetic distribution. 肠病毒RNase L抑制rna高度保守，系统发育分布有限。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-22 DOI: 10.1261/rna.081191.126

Samantha Zangari, Madeline Sherlock, Jeffrey Kieft

{"title":"Enterovirus RNase L inhibiting RNAs are highly conserved with limited phylogenetic distribution.","authors":"Samantha Zangari, Madeline Sherlock, Jeffrey Kieft","doi":"10.1261/rna.081191.126","DOIUrl":"10.1261/rna.081191.126","url":null,"abstract":"RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the Picornaviridae family, but only a subset of those tested were functional-all are in Enterovirus coxsackiepol. When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026005","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

Verification of human nucleotide sequence reagents and cell line identities in original circRNA articles published in high impact factor journals. 在高影响因子期刊上发表的circRNA原创文章中人类核苷酸序列试剂和细胞系身份的验证。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-22 DOI: 10.1261/rna.081131.126

Pranujan Pathmendra, Francisco J Enguita, Jennifer A Byrne

{"title":"Verification of human nucleotide sequence reagents and cell line identities in original circRNA articles published in high impact factor journals.","authors":"Pranujan Pathmendra, Francisco J Enguita, Jennifer A Byrne","doi":"10.1261/rna.081131.126","DOIUrl":"https://doi.org/10.1261/rna.081131.126","url":null,"abstract":"Numbers of research articles studying circRNAs have increased rapidly since 2017. Previous analyses of human circRNA articles in two high impact factor cancer research journals identified papers with wrongly identified nucleotide sequence reagents and circRNAs whose identities could not be independently verified. In the present study, verification of human nucleotide sequence reagent and cell line identities in retracted circRNA articles published from 2017-2021 in high impact factor journals found wrongly identified nucleotide sequences and/or cell lines in all 13 retracted papers. Similar analyses of human circRNA papers published in high impact factor journals in 2022 found at least one wrongly identified reagent in 51% (60/118) papers, where 52/118 (44%) papers described wrongly identified nucleotide sequence reagents and 22/118 (19%) described wrongly identified cell lines. In addition, some 2022 circRNA papers described circRNA probes of uncertain targeting specificity (34/118, 29%), non-verifiable nucleotide sequence reagents (25/118, 21%), claimed miRNA reagents whose miRNA targets were not indexed by MirGeneDB (13/118, 11%) and/or non-verifiable cell line identifiers (6/118, 5%). Overall, wrongly identified and/or queried reagents were noted in 73% (86/118) 2022 circRNA papers in high IF journals, highlighting the need for critical engagement with the human circRNA literature.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025630","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

Pharmacological inhibitors of the nonsense-mediated RNA decay identified by high-throughput screening. 通过高通量筛选鉴定无义介导的RNA衰变的药理学抑制剂。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-22 DOI: 10.1261/rna.081009.126

Anna Borrelli, Martina De Santis, Rossella De Cegli, Nicolina Cristina Sorrentino, Luis J V Galietta, Arianna Venturini

{"title":"Pharmacological inhibitors of the nonsense-mediated RNA decay identified by high-throughput screening.","authors":"Anna Borrelli, Martina De Santis, Rossella De Cegli, Nicolina Cristina Sorrentino, Luis J V Galietta, Arianna Venturini","doi":"10.1261/rna.081009.126","DOIUrl":"https://doi.org/10.1261/rna.081009.126","url":null,"abstract":"A relevant percentage of genetic diseases (~10%) are caused by premature termination codon (PTC) mutations, including cystic fibrosis (CF). CF patients carrying a nonsense mutation in the CFTR gene are insensitive to CFTR modulators and cannot benefit from the pharmacological therapy with Kaftrio®. The rescue of CFTR with PTCs faces two limiting steps: 1) the nonsense-mediated RNA decay (NMD) mechanism that, by degrading mRNAs carrying PTCs, strongly reduces CFTR transcript levels; 2) PTCs cause the arrest of protein synthesis, with the production of a truncated and nonfunctional CFTR protein. The combination of NMD inhibitors with readthrough agents, which overcome the translation arrest, can maximize rescue of CFTR with PTCs. NMD inhibitors can also be effective in the absence of a readthrough agent for PTCs localized at the carboxy-terminus of the CFTR sequence such as W1282X. In this work, we screened a chemical library with a CFTR functional assay to find novel compounds able to modulate NMD mechanism. At the end of the screening, we identified and validated by secondary assays three possible candidates acting as NMD inhibitors/modulators that could increase CFTR rescue in patients with PTCs, namely CC-115, samotolisib and elimusertib. The study also provides new insights into the changes occurring to cells transcriptome after the inhibition of the physiological NMD pathway, improving our knowledge of possible side effects of NMD inhibitors. This could help in the development of new compounds with more efficacy and less toxicity, that could be useful for the treatment of different genetic disorders caused by PTCs.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026043","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

Transcript-wide m6A methylation defines the efficiency of cap-independent translation initiation. 转录范围内的m6A甲基化决定了帽无关翻译起始的效率。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-17 DOI: 10.1261/rna.081017.126

Egor A Smolin, Andrey I Buyan, Dmitry N Lyabin, Anton A Buzdin, Ivan V Kulakovskiy, Irina A Eliseeva

{"title":"Transcript-wide m6A methylation defines the efficiency of cap-independent translation initiation.","authors":"Egor A Smolin, Andrey I Buyan, Dmitry N Lyabin, Anton A Buzdin, Ivan V Kulakovskiy, Irina A Eliseeva","doi":"10.1261/rna.081017.126","DOIUrl":"https://doi.org/10.1261/rna.081017.126","url":null,"abstract":"N⁶-methyladenosine (m⁶A) plays an important role in translation control and, particularly, in cap-independent initiation. The impact of m⁶A is usually studied in the context of its location in the transcripts, but the global impact of m⁶A along mRNAs remains unclear. Here, we combined ribosome profiling under conditions of mTOR inhibition and m⁶A mapping to identify distinct subsets of mRNAs that differ in their sensitivity to the suppression of cap-dependent initiation. We found that the sensitivity strongly correlated with the total m⁶A methylation of the transcripts. Further, upon decreased mTOR activity, m⁶A methylation facilitated the enhanced association of mRNAs with components of the eIF4F complex. Thus, the efficiency of cap-independent translation initiation is primarily defined not by precise localization but by the total level of m⁶A methylation, and m⁶A has a compensatory role in maintaining translation when the canonical cap-dependent pathway is impaired. Our findings underscore the significance of contemplating global m⁶A methylation status as a pivotal element in translational control, particularly under stress or signaling perturbations.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148991240","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

Disease mutations in the N-terminal extension and α2'-helix of dyskerin do not abolish dimerization but disrupt binding to the telomerase RNA. dyskerin的n端延伸和α2'-螺旋的疾病突变不会取消二聚化，但会破坏与端粒酶RNA的结合。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-16 DOI: 10.1261/rna.081234.126

Jian Qin, Patrick Lambert-Lanteigne, Alexandre Garus, Chantal Autexier

{"title":"Disease mutations in the N-terminal extension and α2'-helix of dyskerin do not abolish dimerization but disrupt binding to the telomerase RNA.","authors":"Jian Qin, Patrick Lambert-Lanteigne, Alexandre Garus, Chantal Autexier","doi":"10.1261/rna.081234.126","DOIUrl":"10.1261/rna.081234.126","url":null,"abstract":"The H/ACA ribonucleoprotein complex component dyskerin is essential for the biogenesis of H/ACA RNAs, including the human telomerase RNA (hTR). The N-terminal extension and α2' helix of dyskerin are hotspots for disease-associated mutations linked to X-linked dyskeratosis congenita (X-DC), a premature aging disorder. Some of these mutations disrupt dyskerin-hTR interactions, leading to hTR destabilization and reduced telomerase activity. Cryo-EM structures of human telomerase have shown that the N-terminal extension and α2' helix participate in dyskerin dimerization. However, biochemical evidence for dyskerin dimerization is still lacking, and it remains unclear whether mutations in these regions impair hTR binding by disrupting dimerization. Here, we provide the first biochemical evidence that dyskerin undergoes dimerization. We further demonstrate that dimerization is RNA independent and not abolished by disease mutations in the N-terminal extension or α2' helix. Instead, these mutations impair hTR binding. Our findings offer new mechanistic insight into how mutations in the dyskerin N-terminal extension and α2' helix contribute to the pathogenesis of X-DC.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1672-1685"},"PeriodicalIF":4.3,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13580451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148649098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

PCanPIE: a group I intron platform for efficient circRNA synthesis at ambient temperatures. PCanPIE：在室温下高效合成环状rna的I族内含子平台。

IF 4.3 3区生物学

RNA Pub Date : 2026-09-16 DOI: 10.1261/rna.081078.126

Ruben Warkentin, Anna Marie Pyle

{"title":"PCanPIE: a group I intron platform for efficient circRNA synthesis at ambient temperatures.","authors":"Ruben Warkentin, Anna Marie Pyle","doi":"10.1261/rna.081078.126","DOIUrl":"10.1261/rna.081078.126","url":null,"abstract":"Ribozyme-based permuted intron-exon (PIE) systems offer a protein-independent route to circRNA production, but existing platforms require elevated temperatures that promote RNA degradation. Here we report the first application of the Candida albicans mitochondrial large subunit (C.a.mtLSU) group I intron as a PIE platform for circRNA synthesis, which we term PCanPIE (Pyle lab Candida PIE). We evaluated three peripheral stems, P5, P6b, and P8, as permutation sites and demonstrated that all three support circularization at ambient temperature and low magnesium (25°C, 6 mM MgCl2), without the 55°C heating step required by existing PIE systems. Kinetic analysis revealed that permutation site does not affect the observed splicing rate constant but does influence PCanPIE folding and therefore influences circularization efficiency. The P6b permutation yielded the highest circularization efficiency, with 95% of the precursor splicing to produce circRNA. Optimization of spacer sequences flanking the circRNA payload eliminated interference from structured native exon sequences and enabled efficient circularization of RNAs up to 1657 nt, including structured, repetitive, and naturally occurring sequences. Together, these results establish PCanPIE as a versatile and ambient-temperature-active addition to the group I intron PIE toolkit.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1603-1611"},"PeriodicalIF":4.3,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13580324/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148549748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

RNA最新文献