Biology subjects

Churchman, L. S.

Publications and source records attributed to Churchman, L. S..

27 records · Page 2Linked to original sources

bioRxiv2021

Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

Oxidative phosphorylation (OXPHOS) complexes consist of nuclear and mitochondrial DNA-encoded subunits. Their biogenesis requires cross-compartment gene regulation to mitigate the accumulation of disproportionate subunits. To determine how human cells coordinate mitochondrial and nuclear gene expression processes, we established an optimized ribosome profiling approach tailored for the unique features of the human mitoribosome. Analysis of ribosome footprints in five cell types revealed that average mitochondrial synthesis rates corresponded precisely to cytosolic rates across OXPHOS complexes. Balanced mitochondrial and cytosolic synthesis did not rely on rapid feedback between the two translation systems. Rather, LRPPRC, a gene associated with Leighs syndrome, is required for the reciprocal translatomes and maintains cellular proteostasis. Based on our findings, we propose that human mitonuclear balance is enabled by matching OXPHOS subunit synthesis rates across cellular compartments, which may represent a vulnerability for cellular proteostasis.

cell biology↗

bioRxiv2020

Essential histone chaperones collaborate to regulate transcription and chromatin integrity

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. We have discovered that the physical interaction between two essential histone chaperones, Spt6 and Spn1/Iws1, is required for transcriptional accuracy and nucleosome organization. To understand this requirement, we have isolated suppressors of an spt6 mutation that disrupts the Spt6-Spn1 interaction. Several suppressors are in a third essential histone chaperone, FACT, while another suppressor is in the transcription elongation factor Spt5/DSIF. The FACT suppressors weaken FACT-nucleosome interactions and bypass the requirement for Spn1, possibly by restoring a necessary balance between Spt6 and FACT on chromatin. In contrast, the Spt5 suppressor modulates Spt6 function in a Spn1-dependent manner. Despite these distinct mechanisms, both suppressors alleviate the nucleosome organization defects caused by disruption of the Spt6-Spn1 interaction. Taken together, we have uncovered a network in which histone chaperones and other elongation factors coordinate transcriptional integrity and chromatin structure.

genetics↗

bioRxiv2020

The spatial distributions of pre-mRNAs suggest post-transcriptional splicing of specific introns within endogenous genes

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription site proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

cell biology↗

bioRxiv2019

GeneWalk identifies relevant gene functions for a biological context using network representation learning

The primary bottleneck in high-throughput genomics experiments is identifying the most important genes and their relevant functions from a list of gene hits. Existing methods such as Gene Ontology (GO) enrichment analysis provide insight at the gene set level. For individual genes, GO annotations are static and biological context can only be added by manual literature searches. Here, we introduce GeneWalk (github.com/churchmanlab/genewalk), a method that identifies individual genes and their relevant functions under a particular experimental condition. After automatic assembly of an experiment-specific gene regulatory network, GeneWalk quantifies the similarity between vector representations of each gene and its GO annotations through representation learning, yielding annotation significance scores that reflect their functional relevance for the experimental context. We demonstrate the use of GeneWalk analysis of RNA-seq and nascent transcriptome (NET-seq) data from human cells and mouse brains, validating the methodology. By performing gene- and condition-specific functional analysis that converts a list of genes into data-driven hypotheses, GeneWalk accelerates the interpretation of high-throughput genetics experiments.

bioinformatics↗

bioRxiv2019

Cytoplasmic mRNA decay factors modulate RNA polymerase II processivity in 5’ and 3’ gene regions in yeast

mRNA levels are determined by the balance between mRNA synthesis and decay. Factors that mediate both processes, including the 5 to 3 exonuclease Xrn1, are responsible for the cross talk between the two processes in a manner that buffers steady-state mRNA levels. However, these proteins roles in transcription remain elusive and controversial. Applying NET-seq to yeast cells, we show that Xrn1 functions mainly as a transcriptional activator and that its disruption manifests via the reduction of RNA polymerase II (Pol II) occupancy downstream of transcription start sites. We combine our data and novel mathematical modeling of transcription to suggest that transcription initiation and elongation of targeted genes is modulated by Xrn1. Furthermore, Pol II occupancy markedly increases near cleavage and polyadenylation sites in xrn1{Delta} cells while its activity decreases, a characteristic feature of backtracked Pol II. We also provide indirect evidence that Xrn1 is involved in transcription termination downstream of polyadenylation sites. Two additional decay factors, Dhh1 and Lsm1, seem to function similarly to Xrn1 in transcription, perhaps as a complex, while the decay factors Ccr4 and Rpb4 also perturb transcription in other ways. Interestingly, DFs are capable of differentiating between SAGA- and TFIID-dominated promoters. These two classes of genes respond differently to XRN1 deletion in mRNA synthesis and differentially utilize mRNA decay pathways, raising the possibility that one distinction between the two types of genes lies in the mechanism(s) that balance these processes.

genomics↗

bioRxiv2019

Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing

Human genes have numerous exons that are differentially spliced within pre-mRNA. Understanding how multiple splicing events are coordinated across nascent transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of CO-transcriptional Processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. nano-COP showed that in both human and Drosophila cells, co-transcriptional splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B globally abolished co-transcriptional splicing in both species. Our findings revealed that splicing order does not strictly follow the order of transcription and is influenced by cis-regulatory elements. In human cells, introns with delayed splicing frequently neighbor alternative exons and are associated with RNA-binding factors. Moreover, neighboring introns in human cells tend to be spliced concurrently, implying that splicing occurs cooperatively. Thus, nano-COP unveils the organizational complexity of metazoan RNA processing.

molecular biology↗

bioRxiv2018

A risk-reward tradeoff of high ribosome production in proliferating cells

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs), which are required to produce thousands of new ribosomes every minute. Although ribosomes are essential in all cells, disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we modeled these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result immediately in acute loss of proteostasis (protein folding homeostasis). Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes. In response, proteostasis genes are activated by an Hsf1-dependent stress response pathway that is required for recovery from r-protein assembly stress. Importantly, we show that exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. Our results highlight ribosome assembly as a linchpin of cellular homeostasis, representing a key proteostasis vulnerability for rapidly proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic conditions that generate orphan r-proteins.

cell biology↗

bioRxiv2018

Spt6 is required for the fidelity of promoter selection

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters, and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation-site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome and reveal the magnitude of the potential for expressing alternative genetic information via intragenic promoters.

genomics↗

bioRxiv2016

FIDDLE: An integrative deep learning framework for functional genomic data inference

Numerous advances in sequencing technologies have revolutionized genomics through generating many types of genomic functional data. Statistical tools have been developed to analyze individual data types, but there lack strategies to integrate disparate datasets under a unified framework. Moreover, most analysis techniques heavily rely on feature selection and data preprocessing which increase the difficulty of addressing biological questions through the integration of multiple datasets. Here, we introduce FIDDLE (Flexible Integration of Data with Deep LEarning) an open source data-agnostic flexible integrative framework that learns a unified representation from multiple data types to infer another data type. As a case study, we use multiple Saccharomyces cerevisiae genomic datasets to predict global transcription start sites (TSS) through the simulation of TSS-seq data. We demonstrate that a type of data can be inferred from other sources of data types without manually specifying the relevant features and preprocessing. We show that models built from multiple genome-wide datasets perform profoundly better than models built from individual datasets. Thus FIDDLE learns the complex synergistic relationship within individual datasets and, importantly, across datasets.

bioinformatics↗