Biology subjects

Schertzer, M. D.

Publications and source records attributed to Schertzer, M. D..

8 recordsLinked to original sources

bioRxiv2026

Targeted proteomics informed by long-read RNA-sequencing reveals protein isoform expression associated with bone mineral density

The expanding catalog of sQTLs from projects like GTEx has established that genetic risk for complex traits is frequently mediated through RNA splicing. Yet it is generally unknown whether disease-associated splice events produce stable, distinct protein isoforms and whether they are functionally consequent. Even the deepest shotgun proteomics efforts only capture a minority of annotated splice events; isoform-specific peptides are systematically under-sampled by nature of being lower abundance and intrinsically harder to detect. Here, we implement a genetics-guided targeted mass spectrometry (MS) strategy to detect splice-derived protein isoforms prioritized by sQTL colocalization. Using internal standard parallel reaction monitoring (IS-PRM), we targeted 288 peptides corresponding to 166 putative BMD-associated isoforms in differentiating osteoprogenitor cells and patient-derived primary mesenchymal stem cells (MSCs). Targeted acquisition increased recovery of isoform-informative peptides relative to conventional DDA, rescued splice events that were undetected or sparsely sampled under global acquisition, and confirmed protein-level expression of genetically prioritized isoforms in clinical material. Together, this work establishes that targeted MS is a useful approach to support validation of protein expression in studies of the genetics of splicing.

systems biology↗

bioRxiv2026

LRP2: A proteogenomics pipeline for long-read informed protein isoform analysis and discovery

Most human genes produce multiple RNA isoforms, yet it remains unclear which isoforms are translated into stable, functional proteins. Long-read RNA sequencing resolves full-length transcript structures and, when paired with mass spectrometry, can provide empirical evidence of isoform translation. Despite this opportunity, comprehensive workflows integrating isoform discovery, open reading frame prediction, peptide identification, and protein inference remain limited, leaving users to handle these steps piecemeal. Here, we present LRP2, a modular, end-to-end long-read proteogenomics pipeline built in Nextflow. LRP2 scales transcript discovery to hundreds of samples via PacBio's latest Isocall tool, removes technical artifacts with SQANTI QC, generates and classifies predicted proteomes via CPAT and SQANTI Protein, performs multi-group differential expression and usage analysis via edgeR, DRIMSeq, and a long-read adaptation of LeafCutter, and integrates protein-level evidence from DDA and DIA MS data through FragPipe. For cross-dataset comparison of novel isoforms, LRP2 employs deterministic splice-junction, coordinate-based isoform identifiers. Used as an integrated pipeline, LRP2 enables the detection of novel peptides and improves the protein isoform inference to confirm protein isoform translation.

bioinformatics↗

bioRxiv2025

Perplexity as a Metric for Isoform Diversity in the Human Transcriptome

Characterizing the extensive isoform diversity revealed by long-read RNA-sequencing remains challenging. After removal of technical artifacts, existing pipelines apply arbitrary expression thresholds that filter out bona fide transcript structures, misrepresenting diversity and hindering reproducibility. Instead of discarding isoforms, we propose a fundamentally distinct approach to quantifying isoform diversity using perplexity-the effective number of isoforms for a gene, derived from Shannon entropy-wherein every isoform, including low-abundance ones, contributes proportionally to a genes diversity. Analyzing 124 ENCODE4 PacBio datasets spanning 55 human cell types, we show that perplexity provides interpretable and reproducible isoform diversity measurements across genes, regulatory levels, and tissues.

genomics↗

bioRxiv2025

A computational framework for mapping isoform landscape and regulatory mechanisms from spatial transcriptomics data

Transcript diversity including splicing and alternative 3 end usage is crucial for cellular identity and adaptation, yet its spatial coordination remains poorly understood. Here, we present SPLISOSM (SpatiaL ISOform Statistical Modeling), a computational framework for detecting isoform-resolution patterns from spatial transcriptomics data. SPLISOSM leverages multivariate testing to account for spot- and isoform-level dependencies, demonstrating robust and theoretically grounded performance on sparse data. In the mouse brain, we identify over 1,000 spatially variable transcript diversity events, primarily in synaptic signaling pathways linked to neuropsychiatric disorders, and uncover both known and novel regulatory relationships with region-specific RNA binding proteins. We further show that these patterns are evolutionarily conserved between mouse and human prefrontal cortex. Analysis of human glioblastoma highlights pervasive transcript diversity in antigen presentation and adhesion genes associated with specific microenvironmental conditions. Together, we present a comprehensive spatial splicing analysis in the brain under normal and neoplastic conditions.

bioinformatics↗

bioRxiv2023

Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

Alternative splicing is an essential mechanism for diversifying proteins, in which mature RNA isoforms produce proteins with potentially distinct functions. Two major challenges in characterizing the cellular function of isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design. To address these gaps, we developed and methodically tested a strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all splicing and transcriptional events, including exon skipping and inclusion, alternative 5 and 3 splice sites, and alternative starts and ends.

genomics↗

bioRxiv2022

Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn

During mouse embryogenesis, expression of the lncRNA Airn induces gene silencing and recruits Polycomb Repressive Complexes (PRCs) to varying extents over a 15 megabase domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between Airn lncRNA and target chromatin correlated with underlying intensity of PRC-directed chromatin modifications. Deletion of CpG islands that form contacts with Airn altered long-distance silencing and PRC activity in a manner that correlated with changes in chromatin architecture. We conclude that Airn is a potent cis-acting lncRNA whose primary functions of transcriptional repression and PRC recruitment are controlled by an equilibratory network of DNA regulatory elements that modulate its frequency of contact with target chromatin.

genomics↗

bioRxiv2020

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for the production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of multiple downstream polyadenylation sequences. Readthrough required Repeat A and the ~750 nucleotides downstream but did not require SPEN. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a transgene comprising Xists first 5.5 kilobases robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

molecular biology↗

bioRxiv2018

A piggyBac-based toolkit for inducible genome editing in mammalian cells

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically-dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is co-transfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. Using Mus musculus-derived embryonic and trophoblast stem cells, we show that CRISPR-Bac can be used to knockdown proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

molecular biology↗