Biology subjects

Roy, K. R.

Publications and source records attributed to Roy, K. R..

6 recordsLinked to original sources

bioRxiv2026

The m6A motif is an anti-3' splice site

N6-methyladenosine (m6A) is the most abundant internal mRNA modification and can inhibit splicing when present on a 3' splice site (3'SS). Whether methylation of splicing signals contributes broadly to splicing regulation is unknown. We analyzed in vitro and in vivo m6A maps from 31 studies to investigate m6A deposition at splicing signals. We show that 3'SS-like substitutions block in vitro methylation by the main m6A writer and that intronic m6A can be broadly detected in vivo but spares canonical 3'SS adenosines, as opposed to compatible adenosines at other splicing signals, including the 3'SS of AT-AC introns. Human variant alleles compatible with the writer motif are modified at higher rates, and allelic comparisons revealed a preference for A at -4, where most 3'SS carry a pyrimidine. These results show that the specificity of the main m6A writer spares the 3'SS from methylation, which reconciles cotranscriptional methylation with efficient splicing.

molecular biology↗

bioRxiv2025

Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1.

Proteins operate through a few critical residues, yet most proteins remain uncharacterized at the deep molecular resolution, particularly within essential genes, where functional dissection is obstructed by lethality. Here, we establish a platform for mutational scanning of essential genes at their endogenous locus, combining a repressible complementation system with multiplexed CRISPR-based genome editing in budding yeast. Our approach provides a generalizable framework for dissecting essential protein function in vivo, expanding the capacity to map critical residues underlying essential cellular processes. We applied this strategy to NRD1, encoding an essential RNA Polymerase II (RNAPII) termination factor and performed a systematic alanine scanning with near-saturation coverage. We discovered novel and unexpected lethal mutations in the CTD-interacting domain (CID), thus revealing an unanticipated importance for this domain. Overall, our results demonstrate the power of our mutation scanning platform to map critical residues underlying essential cellular processes.

genetics↗

bioRxiv2025

MLL1 complex is a critical regulator of fetal hemoglobin repression

Increasing fetal-type hemoglobin (HbF) expression in adult erythroid cells holds promise in the treatment of sickle cell disease (SCD) and {beta}-thalassemia. We have identified MLL1 complex as a critical regulator of fetal and embryonic hemoglobin repression. Knockdowns of MEN1 and KMT2A, encoding essential components of the complex, caused a significant downregulation of BCL11A expression and a substantial increase in {gamma}- and {varepsilon}-globin mRNA levels in HUDEP-2 cells. Significant binding of MEN1 and KMT2A were readily detected at the promoter and a critical enhancer of BCL11A in HUDEP-2 cells, suggesting that BCL11A is a direct transcriptional target of MLL1 complex. Consistent with these results, MEN1 or KMT2A knockdown in normal human CD34+ hematopoietic stem and progenitor cells (HSPCs) induced to undergo erythroid differentiation also significantly decreased their BCL11A expression and increased their {gamma}- and {varepsilon}-globin expression and the production of F cells in the culture. Treatment of these cells with MENIN inhibitors yielded similar results and promoted erythroid differentiation with minimal effects on their growth. These findings underscore a critical role of MLL1 complex in regulating fetal and embryonic hemoglobin expression and suggest that MENIN inhibitors could offer a promising therapeutic approach for sickle cell disease and {beta}-thalassemia.

developmental biology↗

bioRxiv2024

Dissecting quantitative trait nucleotides by saturation genome editing

Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.

genetics↗

bioRxiv2023

Genome-scale analysis of interactions between genetic perturbations and natural variation

Interactions between genetic perturbations and segregating loci can cause perturbations to show different phenotypic effects across genetically distinct individuals. To study these interactions on a genome scale in many individuals, we used combinatorial DNA barcode sequencing to measure the fitness effects of 7,700 CRISPRi perturbations targeting 1,712 distinct genes in 169 yeast cross progeny (or segregants). We identified 460 genes whose perturbation has different effects across segregants. Several factors caused perturbations to show variable effects, including baseline segregant fitness, the mean effect of a perturbation across segregants, and interacting loci. We mapped 234 interacting loci and found four hub loci that interact with many different perturbations. Perturbations that interact with a given hub exhibit similar epistatic relationships with the hub and show enrichment for cellular processes that may mediate these interactions. These results suggest that an individuals response to perturbations is shaped by a network of perturbation-locus interactions that cannot be measured by approaches that examine perturbations or natural variation alone.

genetics↗

bioRxiv2023

Spliceosomal mutations decouple 3' splice site fidelity from cellular fitness

The fidelity of splice site selection is thought to be critical for proper gene expression and cellular fitness. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is a daunting task considering the low complexity of the 3'SS consensus sequence YAG. Here we show that inactivating the near-essential splicing factor Prp18p results in a global activation of alternative 3'SS, many of which harbor sequences that highly diverge from the YAG consensus, including some highly unusual non-AG 3'SS. We show that the role of Prp18p in 3'SS fidelity is promoted by physical interactions with the essential splicing factors Slu7p and Prp8p and synergized by the proofreading activity of the Prp22p helicase. Strikingly, structure-guided point mutations that disrupt Prp18p-Slu7p and Prp18p-Prp8p interactions mimic the loss of 3'SS fidelity without any impact on cellular growth, suggesting that accumulation of incorrectly spliced transcripts does not have a major deleterious effect on cellular viability. These results show that spliceosomes exhibit remarkably relaxed fidelity in the absence of Prp18p, and that new 3'SS sampling can be achieved genome-wide without a major negative impact on cellular fitness, a feature that could be used during evolution to explore new productive alternative splice sites.

molecular biology↗