Lack of affinity signature for germinal center cells that have initiated plasma cell differentiation.

Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. High-affinity cells are proposed to preferentially select into this compartment, potentiating the immune response. We used single-cell RNA-seq to track the germinal center (GC) development of Ighg2A10 B cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP). Following immunization with Plasmodium sporozoites, we identified 3 populations of cells in the GC light zone (LZ). One LZ population expressed a gene signature associated with the initiation of PC differentiation and readily formed PCs in vitro. The estimated affinity of these pre-PC B cells was indistinguishable from that of LZ cells that remained in the GC. This remained true when high- or low-avidity recombinant PfCSP proteins were used as immunogens. These findings suggest that the initiation of PC development occurs via an affinity-independent process.

Reversible, tunable epigenetic silencing of TCF1 generates flexibility in the T cell memory decision.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway.

iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signaling; they function primarily by recognizing transmembrane domains of their clients. Here, we report a mechanistically distinct nuclear function of iRhoms, showing that both human and mouse iRhom2 are non-canonical substrates of signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the transcriptome, in part by binding C-terminal binding proteins (CtBPs). The biological significance of nuclear iRhom2 is indicated by elevated levels in skin biopsies of patients with psoriasis, tylosis with oesophageal cancer (TOC), and non-epidermolytic palmoplantar keratoderma (NEPPK); increased iRhom2 cleavage in a keratinocyte model of psoriasis; and nuclear iRhom2 promoting proliferation of keratinocytes. Overall, this work identifies an unexpected SPC-dependent ER-to-nucleus signaling pathway and demonstrates that iRhoms can mediate nuclear signaling.

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.

Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3' modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3' ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions.

Impact of genome build on RNA-seq interpretation and diagnostics.

Transcriptomics is a powerful tool for unraveling the molecular effects of genetic variants and disease diagnosis. Prior studies have demonstrated that choice of genome build impacts variant interpretation and diagnostic yield for genomic analyses. To identify the extent genome build also impacts transcriptomics analyses, we studied the effect of the hg19, hg38, and CHM13 genome builds on expression quantification and outlier detection in 386 rare disease and familial control samples from both the Undiagnosed Diseases Network and Genomics Research to Elucidate the Genetics of Rare Disease Consortium. Across six routinely collected biospecimens, 61% of quantified genes were not influenced by genome build. However, we identified 1,492 genes with build-dependent quantification, 3,377 genes with build-exclusive expression, and 9,077 genes with annotation-specific expression across six routinely collected biospecimens, including 566 clinically relevant and 512 known OMIM genes. Further, we demonstrate that between builds for a given gene, a larger difference in quantification is well correlated with a larger change in expression outlier calling. Combined, we provide a database of genes impacted by build choice and recommend that transcriptomics-guided analyses and diagnoses are cross referenced with these data for robustness.

The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

The clinical utility and diagnostic implementation of human subject cell transdifferentiation followed by RNA sequencing.

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.

Ornaments for efficient allele-specific expression estimation with bias correction.

Allele-specific expression plays a crucial role in unraveling various biological mechanisms, including genomic imprinting and gene expression controlled by cis-regulatory variants. However, existing methods for quantification from RNA-sequencing (RNA-seq) reads do not adequately and efficiently remove various allele-specific read mapping biases, such as reference bias arising from reads containing the alternative allele that do not map to the reference transcriptome or ambiguous mapping bias caused by reads containing the reference allele that map differently from reads containing the alternative allele. We present Ornaments, a computational tool for rapid and accurate estimation of allele-specific transcript expression at unphased heterozygous loci from RNA-seq reads while correcting for allele-specific read mapping biases. Ornaments removes reference bias by mapping reads to a personalized transcriptome and ambiguous mapping bias by probabilistically assigning reads to multiple transcripts and variant loci they map to. Ornaments is a lightweight extension of kallisto, a popular tool for fast RNA-seq quantification, that improves the efficiency and accuracy of WASP, a popular tool for bias correction in allele-specific read mapping. In experiments with simulated and human lymphoblastoid cell-line RNA-seq reads with the genomes of the 1000 Genomes Project, we demonstrate that Ornaments improves the accuracy of WASP and kallisto, is nearly as efficient as kallisto, and is an order of magnitude faster than WASP per sample, with the additional cost of constructing a personalized index for multiple samples. Additionally, we show that Ornaments finds imprinted transcripts with higher sensitivity than WASP, which detects imprinted signals only at gene level.

The cells of the sensory epithelium, and not the stria vascularis, are the main cochlear cells related to the genetic pathogenesis of age-related hearing loss.

Age-related hearing loss (ARHL) is a major health concern among the elderly population. It is hoped that increasing our understanding of its underlying pathophysiological processes will lead to the development of novel therapies. Recent genome-wide association studies (GWASs) discovered a few dozen genetic variants in association with elevated risk for ARHL. Integrated analysis of GWAS results and transcriptomics data is a powerful approach for elucidating specific cell types that are involved in disease pathogenesis. Intriguingly, recent studies that applied such bioinformatics approaches to ARHL resulted in disagreeing findings as for the key cell types that are most strongly linked to the genetic pathogenesis of ARHL. These conflicting studies pointed either to cochlear sensory epithelial or to stria vascularis cells as the cell types most prominently involved in the genetic basis of ARHL. Seeking to resolve this discrepancy, we integrated the analysis of four ARHL GWAS datasets with four independent inner-ear single-cell RNA-sequencing datasets. Our analysis clearly points to the cochlear sensory epithelial cells as the key cells for the genetic predisposition to ARHL. We also explain the limitation of the bioinformatics analysis performed by previous studies that led to missing the enrichment for ARHL GWAS signal in sensory epithelial cells. Collectively, we show that cochlear epithelial cells, not stria vascularis cells, are the main inner-ear cells related to the genetic pathogenesis of ARHL.

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease.

A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N = ∼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs in TPM2, in which siRNA-mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.

Investigating the basis of lineage decisions and developmental trajectories in the dorsal spinal cord through pseudotime analyses.

Dorsal interneurons (dIs) in the spinal cord encode the perception of touch, pain, heat, itchiness and proprioception. Previous studies using genetic strategies in animal models have revealed important insights into dI development, but the molecular details of how dIs arise as distinct populations of neurons remain incomplete. We have developed a resource to investigate dI fate specification by combining a single-cell RNA-Seq atlas of mouse embryonic stem cell-derived dIs with pseudotime analyses. To validate this in silico resource as a useful tool, we used it to first identify genes that are candidates for directing the transition states that lead to distinct dI lineage trajectories, and then validated them using in situ hybridization analyses in the developing mouse spinal cord in vivo. We have also identified an endpoint of the dI5 lineage trajectory and found that dIs become more transcriptionally homogeneous during terminal differentiation. This study introduces a valuable tool for further discovery about the timing of gene expression during dI differentiation and demonstrates its utility in clarifying dI lineage relationships.

Integrative multi-omics increase resolution of the sea urchin posterior gut gene regulatory network at single cell level.

Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing (-seq) to obtain the necessary information for drafting Strongylocentrotus purpuratus posterior gut GRN. Here we present an update to the GRN using i) a single cell RNA-seq derived cell atlas highlighting the 2 day post fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at single cell level, ii) a set of putative cis-regulatory modules and transcription factor (TF) binding sites obtained from chromatin accessibility ATAC-seq data, and iii) interactions directionality obtained from differential bulk RNA-seq following knockdown of the TF Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1 positive cells in the 2 dpf gastrula embryo. Overall, our data suggests the complex connectivity of the posterior gut GRN and increases the resolution of gene regulatory cascades operating within it.

Complementary environmental analysis and functional characterization of lower glycolysis-gluconeogenesis in the diatom plastid.

Organic carbon fixed in chloroplasts through the Calvin-Benson-Bassham Cycle can be diverted towards different metabolic fates, including cyoplasmic and mitochondrial respiration, gluconeogenesis, and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known to exist in non-photosynthetic tissue. Here, we characterized a lower plastid glycolysis-gluconeogenesis pathway enabling the direct interconversion of glyceraldehyde-3-phosphate and phospho-enol-pyruvate in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis-gluconeogenesis, Enolase and bis-phospho-glycerate mutase (PGAM), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative 'omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that the cpEnolase and cpPGAM genes are expressed at elevated levels in high latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway.

Psychosocial experiences are associated with human brain mitochondrial biology.

Psychosocial experiences affect brain health and aging trajectories, but the molecular pathways underlying these associations remain unclear. Normal brain function relies on energy transformation by mitochondria oxidative phosphorylation (OxPhos). Two main lines of evidence position mitochondria both as targets and drivers of psychosocial experiences. On the one hand, chronic stress exposure and mood states may alter multiple aspects of mitochondrial biology; on the other hand, functional variations in mitochondrial OxPhos capacity may alter social behavior, stress reactivity, and mood. But are psychosocial exposures and subjective experiences linked to mitochondrial biology in the human brain? By combining longitudinal antemortem assessments of psychosocial factors with postmortem brain (dorsolateral prefrontal cortex) proteomics in older adults, we find that higher well-being is linked to greater abundance of the mitochondrial OxPhos machinery, whereas higher negative mood is linked to lower OxPhos protein content. Combined, positive and negative psychosocial factors explained 18 to 25% of the variance in the abundance of OxPhos complex I, the primary biochemical entry point that energizes brain mitochondria. Moreover, interrogating mitochondrial psychobiological associations in specific neuronal and nonneuronal brain cells with single-nucleus RNA sequencing (RNA-seq) revealed strong cell-type-specific associations for positive psychosocial experiences and mitochondria in glia but opposite associations in neurons. As a result, these "mind-mitochondria" associations were masked in bulk RNA-seq, highlighting the likely underestimation of true psychobiological effect sizes in bulk brain tissues. Thus, self-reported psychosocial experiences are linked to human brain mitochondrial phenotypes.

Matrix stiffness-dependent regulation of immunomodulatory genes in human MSCs is associated with the lncRNA CYTOR.

Cell-matrix interactions in 3D environments significantly differ from those in 2D cultures. As such, mechanisms of mechanotransduction in 2D cultures are not necessarily applicable to cell-encapsulating hydrogels that resemble features of tissue architecture. Accordingly, the characterization of molecular pathways in 3D matrices is expected to uncover insights into how cells respond to their mechanical environment in physiological contexts, and potentially also inform hydrogel-based strategies in cell therapies. In this study, a bone marrow-mimetic hydrogel was employed to systematically investigate the stiffness-responsive transcriptome of mesenchymal stromal cells. High matrix rigidity impeded integrin-collagen adhesion, resulting in changes in cell morphology characterized by a contractile network of actin proximal to the cell membrane. This resulted in a suppression of extracellular matrix-regulatory genes involved in the remodeling of collagen fibrils, as well as the upregulation of secreted immunomodulatory factors. Moreover, an investigation of long noncoding RNAs revealed that the cytoskeleton regulator RNA (CYTOR) contributes to these 3D stiffness-driven changes in gene expression. Knockdown of CYTOR using antisense oligonucleotides enhanced the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding those achievable by modulating matrix stiffness alone. Taken together, our findings further our understanding of mechanisms of mechanotransduction that are distinct from canonical mechanotransductive pathways observed in 2D cultures.

The role of mitochondria in sex- and age-specific gene expression in a species without sex chromosomes.

Mitochondria perform an array of functions, many of which involve interactions with gene products encoded by the nucleus. These mitochondrial functions, particularly those involving energy production, can be expected to differ between sexes and across ages. Here, we measured mitochondrial effects on sex- and age-specific gene expression in parental and reciprocal F1 hybrids between allopatric populations of Tigriopus californicus with over 20% mitochondrial DNA divergence. Because the species lacks sex chromosomes, sex-biased mitochondrial effects are not confounded by the effects of sex chromosomes. Results revealed pervasive sex differences in mitochondrial effects, including effects on energetics and aging involving nuclear interactions throughout the genome. Using single-individual RNA sequencing, sex differences were found to explain more than 80% of the variance in gene expression. Males had higher expression of mitochondrial genes and mitochondrially targeted proteins (MTPs) involved in oxidative phosphorylation (OXPHOS), while females had elevated expression of non-OXPHOS MTPs, indicating strongly sex-dimorphic energy metabolism at the whole organism level. Comparison of reciprocal F1 hybrids allowed insights into the nature of mito-nuclear interactions, showing both mitochondrial effects on nuclear expression, and nuclear effects on mitochondrial expression. While based on a small set of crosses, sex-specific increases in mitochondrial expression with age were associated with longer life. Network analyses identified nuclear components of strong mito-nuclear interactions and found them to be sexually dimorphic. These results highlight the profound impact of mitochondria and mito-nuclear interactions on sex- and age-specific gene expression.

Aging and comprehensive molecular profiling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aging-related and heterogeneous hematopoietic malignancy. In this study, a total of 1,474 newly diagnosed AML patients with RNA sequencing data were enrolled, and targeted or whole exome sequencing data were obtained in 94% cases. The correlation of aging-related factors including age and clonal hematopoiesis (CH), gender, and genomic/transcriptomic profiles (gene fusions, genetic mutations, and gene expression networks or pathways) was systematically analyzed. Overall, AML patients aged 60 y and older showed an apparently dismal prognosis. Alongside age, the frequency of gene fusions defined in the World Health Organization classification decreased, while the positive rate of gene mutations, especially CH-related ones, increased. Additionally, the number of genetic mutations was higher in gene fusion-negative (GF-) patients than those with GF. Based on the status of CH- and myelodysplastic syndromes (MDS)-related mutations, three mutant subgroups were identified among the GF- AML cohort, namely, CH-AML, CH-MDS-AML, and other GF- AML. Notably, CH-MDS-AML demonstrated a predominance of elderly and male cases, cytopenia, and significantly adverse clinical outcomes. Besides, gene expression networks including HOXA/B, platelet factors, and inflammatory responses were most striking features associated with aging and poor prognosis in AML. Our work has thus unraveled the intricate regulatory circuitry of interactions among different age, gender, and molecular groups of AML.

Improved RNA stability estimation through Bayesian modeling reveals most Salmonella transcripts have subminute half-lives.

RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in posttranscriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 min, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with cross-linking and immunoprecipitation followed by RNA sequencing (CLIP-seq) revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3' untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for ProQ in the oxidative stress response. Our findings provide insights into posttranscriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

Improvement of a mouse infection model to capture Pseudomonas aeruginosa chronic physiology in cystic fibrosis.

Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.

Dissection and integration of bursty transcriptional dynamics for complex systems.

RNA velocity estimation is a potentially powerful tool to reveal the directionality of transcriptional changes in single-cell RNA-sequencing data, but it lacks accuracy, absent advanced metabolic labeling techniques. We developed an approach, TopicVelo, that disentangles simultaneous, yet distinct, dynamics by using a probabilistic topic model, a highly interpretable form of latent space factorization, to infer cells and genes associated with individual processes, thereby capturing cellular pluripotency or multifaceted functionality. Focusing on process-associated cells and genes enables accurate estimation of process-specific velocities via a master equation for a transcriptional burst model accounting for intrinsic stochasticity. The method obtains a global transition matrix by leveraging cell topic weights to integrate process-specific signals. In challenging systems, this method accurately recovers complex transitions and terminal states, while our use of first-passage time analysis provides insights into transient transitions. These results expand the limits of RNA velocity, empowering future studies of cell fate and functional responses.