Biology subjects

Rughani, P.

Publications and source records attributed to Rughani, P..

5 recordsLinked to original sources

bioRxiv2025

Transcriptome-wide profiling of alternative splicing regulators with CRISPore-seq

Alternative splicing creates diverse RNA isoforms from individual genes, yet single-cell CRISPR screens are limited to gene-level quantification and cannot detect changes in alternative splicing and transcript isoforms. To overcome this limitation, we develop CRISPore-seq, which couples massively-parallel CRISPR perturbations with joint short- and long-read transcriptomics. CRISPore-seq simultaneously captures genetic perturbations and expression of genes, full-length transcripts and surface proteins in single cells. CRISPore-seq long reads identify 80% more transcript isoforms than short reads. Nearly all long reads map to unique transcript isoforms -- in contrast to existing single-cell perturbation methods, which rarely distinguish specific isoforms. Using CRISPore-seq, we knock-down 15 different RNA-binding proteins (RBPs) and identify thousands of perturbation-driven alternative splicing events (ASEs). We find that exon skipping is the most common ASE observed and that skipped exons are enriched for binding sites of perturbed RBPs. Loss of the Nager syndrome-associated spliceosomal factor SF3B4 triggers skipping of exon 2 in the cell-cycle regulator CCND1, preventing formation of a complex with CDK6 and blocking the G1-S transition. After rescue with a CCND1 isoform containing the skipped exon, both holoenzyme complex formation and cell proliferation are restored. By linking genes to transcriptional phenotypes with isoform-level resolution, CRISPore-seq is a highly scalable tool for understanding the impact of genetic perturbations on the human transcriptome.

genomics↗

bioRxiv2025

Genome evolution in parthenogenetic nematodes shaped by chromosome rearrangements and rare sex

In contrast to their dioecious relatives, members of the parthenogenetic Diploscapter nematode genus harbour their entire genome within a single pair of highly heterozygous chromosomes. To examine how this unusual karyotype relates to the evolution of parthenogenesis, we generated chromosome-level assemblies for two species in this clade: Diploscapter pachys and Diploscapter coronatus. Sequence comparisons revealed that the two genomes are colinear across their entirety, and that multiple ancestral chromosome fusions and extensive genomic rearrangements preceded the divergence of these two species. The presence of shortened telomeres and extended subtelomeric repeats suggests that the fusions arose from defects in telomere function in the lineage. Our analysis also identified an introgression event after divergence of the two species, suggesting that their parthenogenetic lifestyle may have been punctuated by rare sexual reproduction. These findings shed new light on how telomere loss, chromatin architecture, and reproductive strategies interconnect in shaping chromosome evolution.

genomics↗

bioRxiv2023

High resolution long-read telomere sequencing reveals dynamic mechanisms in aging and cancer

Telomeres are the protective nucleoprotein structures at the end of linear eukaryotic chromosomes. Telomeres repetitive nature and length have traditionally challenged the precise assessment of the composition and length of individual human telomeres. Here, we present Telo-seq to resolve bulk, chromosome arm-specific and allele-specific human telomere lengths using Oxford Nanopore Technologies native long-read sequencing. Telo-seq resolves telomere shortening in five population doubling increments and reveals intrasample, chromosome arm-specific, allele-specific telomere length heterogeneity. Telo-seq can reliably discriminate between telomerase- and ALT-positive cancer cell lines. Thus, Telo-seq is a novel tool to study telomere biology during development, aging, and cancer at unprecedented resolution.

cell biology↗

bioRxiv2020

Selection on ancient variations drives the adaptive radiation of Metrosideros across the Hawaiian archipelago

Some of the most spectacular adaptive radiations begin with founder populations on remote islands. How genetically limited founder populations give rise to the striking phenotypic and ecological diversity characteristic of adaptive radiations is a paradox of evolutionary biology. We conducted an evolutionary genomic analysis of genus Metrosideros, a landscape-dominant, incipient adaptive radiation of woody plants that spans a striking range of phenotypes and environments across the Hawaiian Islands. Using nanopore-sequencing, we created a chromosome-level genome assembly for M. polymorpha var. incana and analyzed wholegenome sequences of 131 individuals from 11 taxa sampled across the islands. We found evidence of population structure that grouped taxa by island. Demographic modeling showed concordance between the divergence times of island-specific lineages and the geological formation of individual islands. Gene flow was also detected within and between island taxa, suggesting a complex reticulated evolutionary history. We investigated genomic regions with increased differentiation as these regions may harbor variants involved in local adaptation or reproductive isolation, thus forming the genomic basis of adaptive radiation. We discovered differentiation outliers have arisen from balancing selection on ancient divergent haplotypes that formed before the initial colonization of the archipelago. These regions experienced recurrent divergent selection as lineages colonized and diversified on new islands, and hybridization likely facilitated the transfer of these ancient variants between taxa. Balancing selection on multiple ancient haplotypes-or time-tested variants-may help to explain how lineages with limited gene pools can rapidly diversify to fill myriad ecological niches on remote islands. Significance statementSome of the most spectacular adaptive radiations of plants and animals occur on remote oceanic islands, yet such radiations are preceded by founding events that severely limit genetic variation. How genetically depauperate founder populations give rise to the spectacular phenotypic and ecological diversity characteristic of island adaptive radiations is not known. We generated novel genomic resources for Hawaiian Metrosideros--a hyper-variable incipient adaptive radiation of woody taxa--for insights into the paradox of remote island radiations. We found that Metrosideros colonized each island shortly after formation and diversified within islands through recurrent selection on ancient variations that predate the radiation. Recurring use of ancient variants may explain how genetically depauperate lineages can diversify to fill countless niches on remote islands.

evolutionary biology↗

bioRxiv2019

Nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure

Higher-order chromatin structure arises from the combinatorial physical interactions of many genomic loci. To investigate this aspect of genome architecture we developed Pore-C, which couples chromatin conformation capture with Oxford Nanopore Technologies (ONT) long reads to directly sequence multi-way chromatin contacts without amplification. In GM12878, we demonstrate that the pairwise interaction patterns implicit in Pore-C multi-way contacts are consistent with gold standard Hi-C pairwise contact maps at the compartment, TAD, and loop scales. In addition, Pore-C also detects higher-order chromatin structure at 18.5-fold higher efficiency and greater fidelity than SPRITE, a previously published higher-order chromatin profiling technology. We demonstrate Pore-Cs ability to detect and visualize multi-locus hubs associated with histone locus bodies and active / inactive nuclear compartments in GM12878. In the breast cancer cell line HCC1954, Pore-C contacts enable the reconstruction of complex and aneuploid rearranged alleles spanning multiple megabases and chromosomes. Finally, we apply Pore-C to generate a chromosome scale de novo assembly of the HG002 genome. Our results establish Pore-C as the most simple and scalable assay for the genome-wide assessment of combinatorial chromatin interactions, with additional applications for cancer rearrangement reconstruction and de novo genome assembly.

genomics↗