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bioRxiv2018

Dysregulation of circular RNAs in myotonic dystrophy type 1

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponge for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by \"back-splicing\", linking covalently 3- and 5-ends of exons. Thus, circRNA levels might be deregulated in conditions associated to altered RNA-splicing. Indeed, increasing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG-repeats in the DMPK gene, resulting in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of 9 tested, 4 transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03 and ZNF609. The circular fraction values correlated positively with skeletal muscle strength and Receiver-Operating-Characteristics curves showed that these four circRNAs allow to distinguish DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and plasma of DM1 patients, but they were not regulated significantly, indicating a tissue-selectivity of the identified modulations. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients.\n\nIn conclusion, this proof-of-principle study identified circRNA dysregulation in DM1 patients.

molecular biology↗

bioRxiv2018

A Haploid Genetic Screening Method for Proteins Influencing Mammalian Nonsense-Mediated mRNA Decay Activity

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in the destruction of faulty, disease-causing mRNAs, as well as its role in the maintenance of normal, endogenous transcript abundance, systematic unbiased methods for uncovering modifiers of NMD activity in mammalian cells remain scant. Here we present and validate a haploid genetic screening method for identifying proteins and processes that stimulate NMD activity involving a 3'-untranslated region exon-junction complex. This reporterbased screening method can be adapted for interrogating other pathways whose output can be measured by the intracellular production of fluorescent proteins.

molecular biology↗

bioRxiv2018

Multiple decay events target HAC1 mRNA during splicing to regulate the unfolded protein response

In the unfolded protein response (UPR), protein-folding stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, the trans-ER-membrane kinase-endoribonuclease Ire1 excises an intron from the HAC1 mRNA and the exon cleavage products are ligated and translated to a transcription factor that induces hundreds of stress-response genes. HAC1 cleavage by Ire1 is thought to be the rate limiting step of its processing. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates by Trl1 RNA 5'-kinase is required for their degradation by the 5'[->]3' exonuclease Xrn1 to enact opposing effects on the UPR. Kinase-mediated decay (KMD) of cleaved HAC1 3'-exon competes with its ligation to limit productive splicing and suppress the UPR, whereas KMD of the excised intron activates HAC1 translation, likely by relieving an inhibitory base-pairing interaction between the intron and 5'-untranslated region. We also found that ligated but 2'-phosphorylated HAC1 mRNA is endonucleolytically cleaved, yielding a KMD intermediate with both 5'- and 2'-phosphates at its 5'-end that inhibit 5'[->]3' decay, and suggesting that Ire1 initiates the degradation of incompletely processed HAC1s to proofread ligation or attenuate the UPR. These multiple decay events expand the scope of RNA-based regulation in the budding yeast UPR and may have implications for the control of the metazoan UPR by mRNA processing.

molecular biology↗

bioRxiv2018

Maternal and zygotic gene regulatory effects of endogenous RNAi pathways

Endogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch sRNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we find that sRNA abundance, the pattern of origin of sRNA and 3 UTR length are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we discovered that ALG-3- and ALG-4-bound 26G-RNAs are dampening the expression of their own mRNAs, revealing a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.\n\nAuthor SummarySmall RNAs (sRNAs) and their partner Argonaute proteins regulate the expression of target RNAs. When sperm and egg meet upon fertilization, a diverse set of proteins and RNA, including sRNA-Argonaute complexes, is passed on to the developing progeny. Thus, these two players are important to initiate specific gene expression programs in the next generation. The nematode Caenorhabditis elegans expresses several classes of sRNAs. 26G-RNAs are a particular class of sRNAs that are divided into two subpopulations: one expressed in the spermatogenic gonad and another expressed in oocytes and in embryos. In this work, we describe the dynamics whereby oogenic 26G-RNAs setup gene silencing in the next generation. We also show several ways that spermatogenic 26G-RNAs and their partner Argonautes, ALG-3 and ALG-4, use to regulate their targets. Finally, we show that ALG-3 and ALG-4 are fine-tuning their own expression, a rare role of Argonaute proteins. Overall, we provide new insights into how sRNAs and Argonautes are regulating gene expression.

molecular biology↗

bioRxiv2018

The beta propellers of eIF3b and eIF3i relocate together to the ribosomal intersubunit interface during translation initiation

During eukaryotic translational initiation, the 48S ribosomal pre-initiation complex (PIC) scans the 5 untranslated region of mRNA until it encounters a start codon. We present a single particle electron cryomicroscopy (cryo-EM) reconstruction of a yeast 48S PIC in an open scanning-competent state in which eIF3b is observed bound on the 40S subunit interface. eIF3b is re-located with eIF3i from their solvent-interface locations observed in other PIC structures; however, eIF3i is not in contact with the 40S. Re-processing of micrographs of our previous 48S PIC in a closed state using currently available tools reveal a similar re-location of eIF3b and eIF3i from the solvent to subunit interface. Genetic analysis indicates that high fidelity initiation in vivo depends strongly on eIF3b interactions at the subunit interface that either promote the closed conformation of the PIC on start codon selection or facilitate subsequent relocation back to the solvent side of the 40S subunit.

molecular biology↗

bioRxiv2018

Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here we present the cryo-EM structure of the type I-F CRISPR RNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a I180-degree rotation of the C-terminal helical bundle on the \"large\" Cas8f subunit. We show that the dsDNA-induced conformational change in Cas8f exposes a Cas2/3 \"nuclease recruitment helix\" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f \"nuclease recruitment helix\", implying that cas genes may sometimes serve as genetic fodder for the evolution of anti-CRISPRs.

molecular biology↗

bioRxiv2018

RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators

In bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2-3 nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well-defined. RNAP contains a clamp domain that closes around the nucleic-acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine-crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.\n\nHighlightsO_LIDisulfide bond crosslinks probe the role of the RNAP clamp domain in termination\nC_LIO_LIRNA but not DNA can release at terminators when the RNAP clamp is closed\nC_LIO_LIRestricting RNAP clamp movement affects elongation rate more than termination rate\nC_LIO_LIInhibiting TL conformational flexibility impairs both RNA and DNA release\nC_LI

molecular biology↗

bioRxiv2026

Design and Validation of New Primers for Specific and Sensitive Real-time PCR Detection and Quantification of Seven Botulinum Encoding Genes (Serotype A-G) of Clostridium botulinum

Botulinum neurotoxins (BoNTs) comprise a highly diverse group of seven serotypes (from A-G) and over 40 subtypes worldwide. Previous primer- and probe-based nucleic acid amplification tests (NAATs) for detection of BoNT encoding genes are challenged by high levels of nucleotide polymorphism both across and within subtypes. In this study, multiple BoNT gene sequences were aligned to identify highly conserved regions for the design of new primers that enable the detection of all seven serotypes under the same conditions. Specific primer sets were designed and validated using in silico, conventional and real-time PCR with constructed plasmids carrying the target fragments and spiked food matrices. The established procedure achieved highly specific and sensitive detection of BoNT serotypes A-G with sensitivity of 10 copies/reaction and a total turnaround time of approximately 1.5 hours. The procedure also eliminated the carryover PCR product by using uracil-N-glycosylase in combination with dUTP in the assay reaction mix. This study provides an alternative NAAT with higher coverage and compliments the traditional mouse bioassays in enhancing global botulism surveillance capabilities.

molecular biology↗

bioRxiv2026

m1A58 acts as a conformational checkpoint coupling human initiator tRNA maturation to translation initiation

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

molecular biology↗

bioRxiv2026

Gene duplication of SNAPC1 generates transcription factors for snRNAs and sex-specific piRNAs

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs essential for transposon silencing and germline integrity across metazoans. In many species, piRNA expression is sexually dimorphic, yet the molecular mechanisms underlying this sex specificity remain poorly understood. In Caenorhabditis elegans, sexually dimorphic piRNA expression is regulated at the transcriptional level. We previously identified SNPC-1.3, a paralog of the small nuclear RNA (snRNA) activating protein complex (SNAPc/SNPC) subunit SNAPC1, as a male-specific piRNA transcription factor. However, the factors governing female piRNA expression remained elusive. Here, we identify SNPC-1.2, a second SNPC-1 paralog, as a female-specific piRNA transcription factor. SNPC-1.2 interacts with the core piRNA transcriptional machinery, binds female piRNA loci, is required for female piRNA expression, and promotes hermaphrodite fertility. In contrast, a third paralog, SNPC-1.1, retains the ancestral SNAPc function in snRNA transcription and is dispensable for piRNA biogenesis. Together, these findings reveal how gene duplication and functional specialization within the snpc-1 gene family generate specificity factors that direct the core SNAP complex to distinct genomic targets, providing a molecular mechanism for sexually dimorphic piRNA expression while maintaining canonical snRNA transcription.

molecular biology↗

bioRxiv2026

RSV competes with the host for translational machinery without a host shutoff strategy

RNA viruses often enhance ribosome recruitment to their own mRNAs through non-canonical sequence elements or by degrading host mRNA. Respiratory syncytial virus (RSV) produces mRNAs with host-like features, including 5'-cap and poly(A) tail. Therefore, the virus lacks an obvious mechanism to preferentially protect its own mRNAs or recruit ribosomes. Furthermore, it remains unknown how RSV interacts with antiviral defense pathways that would reduce cap-dependent translation. Using spike-in normalized sequencing of total and ribosome-associated RNA, we found that RSV does not appear to evoke any host shutoff mechanisms to limit the expression of host genes. These findings show that RSV manages to make use of available ribosomes by competing effectively with host mRNAs and any translational shutoff mechanism would be detrimental. Consistent with this, we found that following activation of antiviral host pathways that reduce cap-dependent translation, translation of RSV mRNAs is decreased to the same extent as host mRNAs. Furthermore, we found that RSV infection does not trigger the dsRNA-activated kinase PKR (which initiates the ISR) and OAS (activates endonuclease RNase L) pathways. These data support a model in which RSV achieves viral protein production, not though inhibiting the host, but by successfully competing with host mRNAs and avoiding activation of antiviral pathways.

molecular biology↗

bioRxiv2026

Invasive mosquito species Aedes aegypti and Aedes albopictus are competent vectors for Barmah Forest Virus

Barmah Forest Virus (BFV), an arthropod-born virus transmitted by mosquitoes, is of significant public health concern in Australia and regions in the Pacific. Recent climate change and globalization raise the potential for BFV to extend its geographic distribution. Despite the rising importance of BFV, its vector dynamics remain poorly understood, particularly concerning the vector competence of different mosquito species. This study aims to investigate the vector competence of BFV across various mosquito species, beyond those endemic to the Australasian region, especially focusing on global relevant vector species. No transmission was observed for Culex quinquefasciatus and Cx. torrentium as well as Anopheles stephensi. In contrast, both investigated Aedes species, Ae. aegypti as well as Ae. albopictus, exhibited BFV-positive saliva across all four temperature profiles (18{degrees}C, 21{degrees}C, 24{degrees}C or 27{degrees}C) examined. These two invasive mosquito species must therefore be classified as potential vectors for BFV, indicating the potential risk of BFV transmission outside of Australia.

molecular biology↗

bioRxiv2026

Structural basis for catalytic and inhibitory divergence between archaeal and bacterial ammonia monooxygenases

Ammonia oxidation initiates nitrification and is closely linked to microbial N2O production. Ammonia monooxygenase (AMO) catalyzes the first and rate-limiting step of nitrification and is widespread across evolutionarily distinct ammonia-oxidizing archaea (AOA) and bacteria (AOB). The ocean is the largest biome for AOA and AOB, which have distinct ecological niches and markedly different sensitivities to nitrification inhibitors. However, the lack of archaeal AMO structures and inhibitor-bound AMO complexes has hindered mechanistic understanding of the architectural, catalytic, and inhibitory divergence between these two enzyme systems. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of marine archaeal AMO captured in active and inactivated states within its native membrane environment, together with inhibitor-bound structures of estuarine bacterial AMO. Archaeal AMO forms an unexpected cup-shaped homotrimer composed of eight subunits per protomer and exhibits substantial architectural divergence from bacterial AMO. Integrated structural, biochemical, kinetic, and computational analyses reveal distinct periplasmic architectures, copper-center organization, and hydrophobic channels between archaeal and bacterial AMOs for ammonium acquisition, catalysis and inhibitor response. These findings provide a structural and mechanistic framework for understanding how archaeal and bacterial AMOs have diverged to distinct ammonia-oxidizing strategies and inhibitor susceptibilities across environmentally important ammonia oxidizers.

molecular biology↗

bioRxiv2026

Inheritance of a Single Edited CD46 Allele Is Associated with Reduced Ex Vivo Susceptibility to Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) remains an economically important pathogen of cattle despite widespread vaccination. A homozygous CD46-edited Gir heifer (Ginger) was previously shown to have significantly reduced susceptibility to BVDV. The edited allele contains an in-frame six amino acid substitution within the virus-binding domain of the BVDV entry receptor CD46, replacing residues G82QVLAL with A82LPTFS. Here, we investigated whether reduced BVDV susceptibility is maintained when the edited allele is inherited in the heterozygous state. Ginger was artificially inseminated with semen from an unedited Gir bull and produced a healthy heterozygous CD46-edited bull calf (Giraldo). Whole-genome sequencing confirmed the inheritance and structural integrity of Giraldo's edited allele. Compared with Ginger, Giraldo exhibited similarly reduced ex vivo BVDV susceptibility across primary fibroblasts, lymphocytes, and monocytes, despite inheriting a wild-type CD46 allele from the sire. Allele-specific CD46 RNA expression analysis demonstrated expression of both the edited and wild-type CD46 alleles. Thus, the reduced-susceptibility phenotype was not attributable to transcriptional silencing of the wild-type allele. Lentiviral complementation studies in CD46-knockout Madin-Darby bovine kidney (MDBK) cells further demonstrated that this wild-type CD46 allele was competent to support BVDV infection when expressed independently. Together, these findings indicate that the CD46 A82LPTFS allele can confer reduced BVDV susceptibility in the heterozygous state despite expression of a functional wild-type CD46 allele. This result suggests the potential to more rapidly disseminate reduced BVDV susceptibility through conventional breeding using homozygous CD46-edited sires.

molecular biology↗

bioRxiv2026

The function of human PIF1 in G quadruplex formation and replication stress response at ALT telomeres

Cancers maintain their telomeres through two telomere maintenance mechanisms: 85-90% of cancers rely on telomerase (TEL+), while 10-15% of cancers adopt the Alternative Lengthening of Telomeres (ALT) pathway. The Break-Induced Replication (BIR) pathway plays a critical role in maintaining telomere length in the ALT+ cells. In both yeast and human, PIF1, a 5' to 3' helicase, is required for the robust activity of BIR. However, the extent of human PIF1 (hPIF1) involvement in the ALT pathway remains unknown. Here we showed that hPIF1 can be recruited to damaged telomeres in ALT+ cells. In addition, we demonstrated that inhibition of hPIF1 induced DNA damage and G quadruplex (G4) accumulation at ALT telomeres, leading to a moderate reduction of the mean telomere length. Most interestingly, we demonstrated that inhibition of hPIF1 also attenuates checkpoint activation, BLM recruitment, single-stranded DNA (ssDNA) formation, DNA damage, and G4s at telomeres in the FANCM deficient ALT+ cells. Finally, we showed that inactivation of hPIF1 affects the viability of both ALT+ and TEL+ cancers, suggesting that hPIF1 is a potential drug target for cancer therapy.

molecular biology↗

bioRxiv2026

Single-Cell Profiling of Dynamic Epicardial Cell States During Myocardial Infarction

Background: The epicardium is reactivated after myocardial infarction (MI); however, the gene expression profiles of post-MI adult epicardial subpopulations remain incompletely defined. Methods: Single-cell RNA sequencing was performed on lineage-traced Wt1+ epicardial cells from Wt1CreERT2/+; R26tdT/+; PdgfranGFP/+ adult mice after sham surgery or at 7 and 14 days after permanent artery ligation to induce MI. Immunostaining was performed on Wt1-lineage-traced cardiac tissue to validate spatial expression after ischemic injury. Results: Unbiased clustering identified nine transcriptionally distinct epicardial populations, encompassing mesothelial, fibroblast/mesenchymal, transitional, and proliferative phenotypes. Fibroblast-like epicardial cells (Wt1+/Pdgfra+) showed time-dependent expression profiles associated with upregulation of epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) gene programs. At 7 days post-MI, there was notable enrichment of genes related to chemokines and Wnt components. By 14 days post-MI, the expression profile shifted toward immune regulation. In contrast, a Wt1high/Msln+ population showed minimal upregulation of EMT gene programs but enhanced paracrine signaling related to wound healing and semaphorins, suggesting reactivation of reparative and angiogenic functions akin to those of the epicardium during embryonic development. Immunostaining and in situ hybridization fluorescence analyses validated laminar epicardial cell placement after MI, comprising a surface Msln+ sheet, an overlapping Wt1-lineage band, and a subadjacent PDGFR+ and Periostin+ compartment that expands 7-14 days after MI and regresses by day 28 post-ischemia. Conclusions: Our data define epicardial gene programs in which a signaling epithelial cell surface overlays an effector mesenchymal cell stroma to coordinate angiogenesis, leukocyte recruitment, and ECM remodeling. This study presents the first integrated single-cell atlas of epicardial-derived cells across multiple post-ischemic timepoints, offering new insights into their reparative potential and dynamic signaling diversity in the injured adult heart.

molecular biology↗

bioRxiv2026

Phosphorylation of spleen tyrosine kinase Y130 positively regulates intracellular signaling and functional responses in platelets

Syk is a non-receptor type protein-tyrosine kinase (PTK), which is associated with platelets surface receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor II-type (CLEC-2). Syk is also expressed in most hematopoietic lineage cells and other cells, such as fibroblasts and neuronal cells. Syk has two tandem SH2 motifs and a C-terminal kinase domain, which are interrupted by interdomains A and B containing multiple tyrosine residues playing a regulatory role upon phosphorylation. This study aims to evaluate the role of Y130 in Syk signaling in platelets. Syk(Y130F) knock-in (KI) mice we generated using the CRISPR-Cas9 technique represent the first in-vivo model harboring this mutation. Using this system, we compared the platelet signaling and responses in wild-type (WT) and Syk(Y130F) littermates. Platelets from homozygous Syk(Y130F) mice showed a decrease in functional responses after activation with CRP, a GPVI agonist, and CLEC-2 crosslinking compared to WT littermates with no significant differences in responses to PAR-4 or purinergic receptor agonists. Key signaling events triggered via both GPVI and CLEC-2, including phosphorylation LAT and PLC-2, were also reduced in Syk(Y130F) platelets at low agonist concentrations. Consistent with these findings, the time to occlusion in the FeCl3 injury model and bleeding time in the tail bleeding assay were significantly enhanced in Syk(Y130F) mice compared to WT littermates. Thus, phosphorylation of Syk Y130 enhances GPVI- and CLEC-2-mediated signaling and functional responses in platelets affecting thrombosis and hemostasis.

molecular biology↗

bioRxiv2026

Half-match recombination drives bridge RNA-guided excision and off-target insertion

IS110-family bridge recombinases are a recently identified class of compact, RNA-guided editors in which a bridge RNA (bRNA) directs the recombination of a donor DNA into a target site. In the current model, the bRNA engages fully complementary donor and target sequences within a single synaptic complex to drive double-stranded recombination, implying that the transposon is cut from its donor site rather than copied, yet neither the strandedness of the excised intermediate nor the requirement for full complementarity has been tested directly. Here we reconstituted IS621 recombination in a cell-free transcription-translation system, building representative arrangements of the excision and insertion reactions and characterizing the outcomes. We find that IS621 predominantly excises a single strand, releasing a single-stranded circle and leaving the donor site intact, consistent with copy-and-paste transposition. By introducing mismatches into the bRNA target sequences, we further find that excision proceeds independently of target-site complementarity, relying strictly on donor-arm recognition; we term this "half-match" recombination, because a substrate matching only half of the bRNA is sufficient. We also find half-match activity during insertion, both in vitro and in a published genome-editing experiment, where it accounts for approximately half of non-target insertion reads. Half-match recombination provides both a mechanistic explanation for off-target insertion and a framework for the rational design of high-fidelity bridge recombinases.

molecular biology↗