Biology subjects

Arsenijevic, Y.

Publications and source records attributed to Arsenijevic, Y..

5 recordsLinked to original sources

bioRxiv2026

Transcript-Specific Site-Directed RNA Editing Reveals Principles for Splice-aware guide RNA Design

Site-directed RNA editing (SDRE) utilizing the adenosine deaminase acting on RNA (ADAR) enzymes is commonly facilitated by guide RNAs (gRNAs) optimized to enhance on-target editing and minimize bystander effects. However, the impact of gRNA binding and ADAR-mediated SDRE on canonical pre-mRNA splicing remains poorly understood. Here, we developed an in vitro transcript-specific editing strategy that enables selective targeting and direct comparison of SDRE in pre-mRNA and mature mRNA. Using splice-relevant variants associated with inherited retinal diseases, we investigated the effects of SDRE on exonic, near-canonical intronic, and deep intronic splice variants. We identified gRNA-induced splice perturbation at exonic and intronic targets and observed that higher editing levels could be associated with increased splice disruption. Conversely, SDRE of two exonic splice variants and a deep intronic variant resulted in increased production of correctly spliced transcripts, demonstrating the potential of SDRE for splice modulation. Finally, by dissecting the effects of ADAR expression and gRNA design on editing and splicing outcomes, we established a system for identifying design principles that reduce splice interference and enhance the generation of correctly spliced, edited transcripts. These findings highlight the importance of considering transcript context and splicing consequences in the development of SDRE-based therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741163v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@55e0cforg.highwire.dtl.DTLVardef@1a5c29org.highwire.dtl.DTLVardef@98aec7org.highwire.dtl.DTLVardef@1fab73b_HPS_FORMAT_FIGEXP M_FIG Created in BioRender. Schneider, N. (2026) https://BioRender.com/59i0tiy C_FIG

molecular biology↗

bioRxiv2024

Dual CRALBP isoforms unveiled: iPSC-derived retinal modelling and AAV2/5-RLBP1 gene transfer raise considerations for effective therapy

Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous disorders characterised by progressive vision loss. Over 270 causative genes have been identified and variants within the same gene can give rise to clinically distinct disorders. Human induced pluripotent stem cells (iPSCs) have revolutionised disease modelling, by allowing pathophysiological and therapeutic studies in the patient and tissue context. The IRD gene RLBP1 encodes CRALBP, an actor of the rod and cone visual cycles in the retinal pigment epithelium (RPE) and Muller cells, respectively. Variants in RLBP1 lead to three clinical subtypes: Bothnia dystrophy, Retinitis punctata albescens and Newfoundland rod-cone dystrophy. We modelled RLBP1-IRD subtypes by patient-specific iPSC-derived RPE and identified pertinent therapeutic read-outs. We developed an AAV2/5-mediated gene replacement strategy and provided a proof-of-concept in the ex vivo human models that was validated in an in vivo Rlbp1-/- murine model. Most importantly, we identified a previously unsuspected smaller CRALBP isoform that is naturally and differentially expressed in both human and murine retina. The new isoform arises from an alternative methionine initiation site and plays a role in the visual cycle. This work provides novel insights into CRALBP expression and RLBP1-associated pathophysiology and raises important considerations for successful gene supplementation therapy.

molecular biology↗

bioRxiv2024

Glutamylation imbalance leads to photoreceptor degeneration

The stereotypic structure of microtubules, assembled from conserved /{beta}-tubulin dimers is subject to a complex diversity of Post-translational Modifications (PTMs). PTMs are predicted to fine-tune microtubule properties and interactions with other proteins, thus allowing microtubules to perform specific functions. Cilia accumulate several types of tubulin PTMs, such as polyglutamylation, polyglycylation, detyrosination and acetylation, whose functions are not yet fully understood. Recently, mutations of AGBL5, coding for the deglutamylating enzyme CCP5, have been associated to retinitis pigmentosa, suggesting that perturbation of polyglutamylation leads to the degeneration of photoreceptor cells. However, the molecular mechanisms underlying this degeneration remain unknown. Here, using super-resolution Ultrastructure Expansion Microscopy in mouse and human photoreceptor cells, we found that most tubulin PTMs are accumulated at the level of the connecting cilium, a structure linking the outer and inner segments of photoreceptor cells. Using mouse models with increased glutamylation (Ccp5-/- and Ccp1-/-), or loss of tubulin acetylation (Atat1-/-), we demonstrated that aberrant glutamylation, but not loss of acetylation, resulted in perturbed molecular architecture of the outer segment, with the loss of the bulge region and destabilization of the distal axoneme. Concurrently, we observed a substantial impairment in tubulin glycylation and intraflagellar transport. Altogether our results indicate that glutamylation plays a crucial role in the maintenance of the molecular architecture of the outer segment and point to tubulin PTM imbalance as possible culprit in retinal degeneration.

cell biology↗

bioRxiv2023

Fine-tuning FAM161A gene augmentation therapy to restore retinal function

In 15 years, inherited retinal diseases have seen gene therapy as a springboard to hope. Many preclinical investigations focused on vectors with maximal gene expression capabilities. But despite an efficient gene transfer, little physiological improvement was noted for certain ciliopathies. FAM161A is an essential protein for the structure of photoreceptor connecting cilium (CC). In the absence of FAM161A, cilia disorganize resulting in outersegment collapses and vision impairment. Within the human retina, FAM161A produces two isoforms: the long with exon 4, and the short, lacking it. To restore CC in Fam161a-deficient mice, we compared AAV vectors with different promoter activities, doses, and human isoforms injected subretinally in 14-days Fam161atm1b/tm1b mice, shortly after the onset of cilium disorganization. All vectors improved cell survival, but only combining both isoforms using the weak FCBR1-F0.4 promoter allowed precise FAM161A expression in the CC and enhanced retinal function. Our study on FAM161A gene replacement for RP28, a rod-cone-related disease, underscores the critical need for precise therapeutic gene regulation, appropriate vector dosing and delivery of both isoforms. Fine tuning of therapeutic gene expression, tailored to disease traits, is crucial for restoring retinal function. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

neuroscience↗

bioRxiv2021

The connecting cilium inner scaffold provides a structural foundation to maintain photoreceptor integrity

Retinal degeneration is a leading cause of human blindness due to progressive loss of ciliated photoreceptors cells. While this degradation can be associated with cohesion defects of the microtubule-based connecting cilium (CC) structure, the underlying mechanism is not understood. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC-inner scaffold (CC-IS) containing POC5, CENTRIN and FAM161A. Monitoring CC-IS assembly during photoreceptor development in mouse reveals that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Consistently, Fam161a mutations lead to a specific CC-IS loss and trigger microtubule doublets spreading, prior to outer segment collapse and photoreceptor degeneration, providing a molecular mechanism for retinitis pigmentosa disease. One Sentence SummaryThe connecting cilium inner scaffold acts as a structural zipper granting photoreceptor integrity.

cell biology↗