Biology subjects

Kanadia, R. N.

Publications and source records attributed to Kanadia, R. N..

11 recordsLinked to original sources

bioRxiv2026

WATER reveals heterochrony of molecular programs underlies developmental failure caused by minor spliceosome inhibition

The final limb structure reflects coordinated deployment of molecular programs, defined not only by which genes are expressed but when they are activated and silenced across time. Existing omics analyses obscure the temporal unfolding of these programs and conflate program identity with deployment timing by assuming temporal equivalence between conditions. We developed WATER (Weighted Windowed Assignment of Temporal Expression of RNA), a framework that reconstructs temporal gene expression trajectories independently within each condition, enabling direct comparison of temporal program architecture between wild-type and perturbed systems. Applying WATER to U11-null mouse forelimb development revealed that minor spliceosome inhibition redistributes genes across inappropriate temporal trajectories. Minor spliceosome inhibition causes splicing defects in minor intron-containing genes such as the PRC2 component Eed, leading to reduced H3K27me3 deposition and chromatin-transcription divergence. Single-cell RNA sequencing revealed persistence of progenitor states, impaired chondrogenic progression, and p53-dependent apoptotic checkpoint activation. Orthogonal WATER analysis of Eed-knockout stem cells recapitulated key features of chromatin gating failure, including temporal redistribution of skeletal development programs and progenitor state persistence, confirming that Eed loss alone is sufficient to produce temporal program redistribution independently of other splicing defects. Trp53 ablation in U11-null limbs partially rescued distal limb structures without correcting the underlying splicing defects, establishing that checkpoint activation amplifies rather than initiates the timing disruption. The limb retains much of its molecular toolkit but executes it in the wrong order, demonstrating that developmental failure arises from mistimed deployment of intact molecular programs. Thus, temporal program architecture is a fundamental organizing principle of morphogenesis.

developmental biology↗

bioRxiv2025

Disentangling spatial organization and splicing of rare intron classes in the human genome

Three-dimensional (3D) genome organization influences transcription and RNA processing, yet how the spatial positioning of genes contributes to pre-mRNA splicing has only recently come into focus. Despite these advances, it remains unclear how introns, particularly rare intron classes, are organized within the 3D genome and whether this organization influences their splicing. Here, we mapped the spatial organization of six intron classes including major, minor, minor-like, hybrid, major-like and non-canonical across four human cell lines (K562, H1, HCT116, and HFFc6) using Hi-C, TSA-seq, and DamID-seq data. This revealed minor intron enrichment in active compartments A and speckle-associated domains (SPADs) and depletion from lamina-associated domains (LADs), whereas hybrid and non-canonical introns showed the opposite trend. Integrating TSA-seq with RNA-seq data suggested that splicing efficiency depends on intron identity rather than nuclear positioning. For example, major-like, minor-like, and non-canonical introns in SPADs were less efficiently spliced than major and minor introns despite their proximity to nuclear speckles. These patterns were consistent across cancer (K562, HCT116) and stem cells (H1) but not fibroblasts (HFFc6). Comparison of minor intron splicing in and out of SPADs across cell lines revealed that, relative to fibroblasts, minor introns outside of SPADs in cancer cells were more efficiently spliced. This suggests that increased efficiency of minor intron splicing in cancer cell lines is not necessarily due to 3D positioning. In all, these findings reveal that intron subclasses show distinct nuclear organization, yet for minor introns, identity rather than position governs splicing efficiency.

bioinformatics↗

bioRxiv2022

Integrated multi-omics reveals minor spliceosome inhibition causes molecular stalling and developmental delay of the mouse forelimb.

Developmental insults causing limb progenitor cell cycle defects or death tend to produce micromelic limbs with maintained segmentation. This suggests that the developing limb is plastic yet has a bias towards proximo-distal patterning. Here we use a minor spliceosome-deficient (U11-null) mouse forelimb, which has severe micromelia yet maintains proximo-distal segmentation, to decipher the mechanism(s) underlying this form of developmental robustness. We show that U11 loss triggers transcriptomic stalling upon spatially heterogenous mis-splicing of minor intron-containing genes. Through spatial transcriptomics, we detected a failure of the U11-null forelimb to separate its distal patterning program from its proximal differentiation program, which was supported by single-cell RNAseq-determined developmental delay of U11-null chondroprogenitors. Ultimately, these molecular and cellular deficits culminated in perturbed chondrogenesis, myogenesis, and axonogenesis. Taken together, we suggest that, upon sensing depletion of progenitors, the limb halts its transcriptional networks to pause its cellular trajectory, affording time to restructure its developmental program.

developmental biology↗

bioRxiv2022

Taxonomy of introns, their evolution, and the role of minor introns in stress response

Despite the high conservation of minor introns across eukaryotic supergroups, specific lineages have completely lost minor intron splicing, which has raised questions about their evolution and purpose. Addressing these questions requires identification of the introns that are affected by minor spliceosome inhibition. To this end, we applied principles of Linnaean taxonomy combined with position-weight matrices to produce five intron classes: minor, minor-like, hybrid, major-like and major. We classified introns across the genomes of 263 species of six eukaryotic supergroups, which can be viewed at the Minor Intron Database (MIDB). Transcriptomic analysis revealed that [~]40% of the minor introns are responsive to minor spliceosome inhibition, while an additional 5% of the minor-like and hybrid introns are also affected. We propose that minor-like introns represent an intermediate in the conversion of minor to major introns and uncover the importance of a guanine at the -1 position of the 5 splice site in facilitating this shift in spliceosome dependence. Finally, we find that minor introns are aberrantly spliced in fish and plants upon cold stress, thereby providing a potential explanation for their high degree of conservation in these lineages.

molecular biology↗

bioRxiv2022

Integrity of the minor spliceosome in the developing mouse hypothalamus determines neuronal subtype composition regulating energy balance.

While gene regulatory networks underlying hypothalamic development are being characterized, minor intron splicing remains unexplored. Here, we used Nkx2.1-Cre to ablate Rnu11, encoding the minor spliceosome-specific U11 snRNA, in the progenitors of the ventral diencephalon (VD), to study minor intron splicing in hypothalamic development and control of energy balance in mice. Loss of U11 resulted in aberrant minor intron splicing, mitotic stalling, apoptosis, and altered neurogenesis. Mutant mice exhibited gross dysgenesis of hypothalamic architecture, while single-cell RNA sequencing (scRNAseq) revealed aberrant composition of neuronal subtypes implicated in feeding and energy balance. Mutant weanlings failed to thrive, followed by rapid weight gain, resulting in obesity. Assessment of energy imbalance and pair-feeding demonstrated that hyperphagia in adult mutants initiates weight gain, and is compounded by metabolic dysfunction, ultimately resulting in obesity. Our findings suggest a key role of minor intron splicing in the developmental patterning of hypothalamic neuronal subtypes underlying energy balance.

neuroscience↗

bioRxiv2022

Minor intron containing genes: Achilles' heel of viruses?

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the worlds unpreparedness to deal with the emergence of novel pathogenic viruses, pointing to the urgent need to identify targets for broad-spectrum antiviral strategies. Here, we report that proteins encoded by Minor Intron-containing Genes (MIGs) are significantly enriched in datasets of cellular proteins that are leveraged by SARS-CoV-2 and other viruses. Pointing to a general gateway for viruses to tap cellular machinery, MIG-encoded proteins (MIG-Ps) that react to the disruption of the minor spliceosome are most important points of viral attack, suggesting that MIG-Ps may pan-viral drug targets. While contemporary anti-viral drugs shun MIG-Ps, we surprisingly found that anti-cancer drugs that have been repurposed to combat SARS-CoV-2, indeed target MIG-Ps, suggesting that such genes can potentially be tapped to efficiently fight viruses.

systems biology↗

bioRxiv2021

Oxytocin Signaling Regulates the Homeostatic Response to Cold Stress in Poikilothermic Vertebrates

When exposed to low temperature, homeothermic vertebrates maintain internal body temperature by activating thermogenesis and by altered metabolism, synchronized by neuroendocrine responses. Although such physiological responses also occur in poikilothermic vertebrates, the prevailing notion is that their reactions are passive. Here, we explored molecular hypothalamic and physiological responses to cold stress in the tropical poikilotherm Nile tilapia (Oreochromis niloticus). We show that cold exposed tilapia exhibit complex homeostatic responses, including increased hypothalamic oxytocin, plasma glucose and cortisol concomitant with reduced plasma lactate and metabolic rate. Pharmacological or genetic blockage of oxytocin signaling further affected metabolic rate in two cold-exposed poikilothermic models. This indicates that oxytocin, a known thermoregulator in homeotherms, actively regulates temperature-related homeostasis in poikilotherms. Overall, our findings show that the brain of poikilotherms actively responds to cold temperature by regulating metabolic physiology. Moreover, we identify oxytocin signaling as an adaptive and evolutionarily conserved metabolic regulator of temperature-related homeostasis.

physiology↗

bioRxiv2021

The minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers

Minor splicing is a second splicing system required for the correct expression of [~]700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38. Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3. We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

cancer biology↗

bioRxiv2020

The minor and major spliceosome interact to regulate alternative splicing around minor introns

Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.

molecular biology↗

bioRxiv2020

Minor spliceosome disruption causes limb growth defects without altering patterning

Disruption of the minor spliceosome causes primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite the importance of minor intron splicing in limb size regulation, its role in limb development remains unexplored. Here we show that loss of U11 small nuclear RNA, an essential minor spliceosome component, results in stunted limbs that maintain patterning. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced due to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. Limb progenitor cells experience delayed prometaphase to metaphase transition and prolonged S-phase, resulting in death of rapidly dividing, distally located progenitors. Consequently, crucial limb patterning genes are upregulated and their expression is maintained spatially to achieve basic patterning. Overall, these findings reveal a potential mechanism shared in disease and domestication.

developmental biology↗

bioRxiv2017

Minor spliceosome inactivation in the developing mouse cortex causes self-amplifying radial glial cell death and microcephaly.

Inactivation of the minor spliceosome has been linked to microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). To interrogate how minor intron splicing regulates cortical development, we employed Emx1-Cre to ablate Rnu11, which encodes the minor spliceosome-specific U11 small nuclear RNA (snRNA), in the developing cortex (pallium). Rnu11 cKO mice were born with microcephaly, caused by death of self-amplifying radial glial cells (RGCs). However, both intermediate progenitor cells (IPCs) and neurons were produced in the U11-null pallium. RNAseq of the pallium revealed elevated minor intron retention in the mutant, particularly in genes regulating cell cycle. Moreover, the only downregulated minor intron-containing gene (MIG) was Spc24, which regulates kinetochore assembly. These findings were consistent with the observation of fewer RGCs entering cytokinesis prior to RGC loss, underscoring the requirement of minor splicing for cell cycle progression in RGCs. Overall, we provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.\n\nSummary StatementHere we report the first mammalian model to investigate the role of the minor spliceosome in cortical development and microcephaly.\n\nList of abbreviations usedMOPD1=microcephalic osteodysplastic primordial dwarfism type 1; snRNA=small nuclear RNA; cKO=conditional knockout; NPC=neural progenitor cell; RGC=radial glial cell; IPC=intermediate progenitor cell; MIG=minor intron-containing gene

developmental biology↗