Biology subjects

Lan, T. C. T.

Publications and source records attributed to Lan, T. C. T..

5 recordsLinked to original sources

bioRxiv2024

Aging disrupts spatiotemporal coordination in the cycling ovary

Throughout the female reproductive lifespan, the ovary completes hundreds of cycles of follicle development, ovulation, and tissue regeneration1-3. These processes rely on the precisely coordinated intricate multicellular interactions across time and space4. How aging disrupts these interactions, leading to an overall decline in reproductive and endocrine functions, remains understudied. To understand the multicellular dynamics that underlie ovarian function and their changes with age, here we use Slide-seq, a near-cellular spatial transcriptomics method, to profile 22 mouse ovaries across the reproductive cycle and chronological age, representing 610,620 near-cellular spots across 69 spatial transcriptomic profiles5,6. We develop a segmentation analysis to identify spatial niches that capture different states of folliculogenesis from static snapshots in situ, allowing us to examine the multicellular dynamics of 358 oocytes, 668 follicles, and 236 corpora lutea. We find that aging disrupts both the spatial organization and temporal coordination of folliculogenesis before the cessation of cycling, which may contribute to the dysregulation of hormone production and signaling. These disruptions are marked by altered immune cell dynamics, inflammatory signaling, and global tissue disorganization that impair the cyclic remodeling required for ovarian function. Our findings reveal how multicellular niches orchestrate ovarian function and demonstrate how age-related breakdown of tissue organization across time and space precedes reproductive decline.

genomics↗

bioRxiv2024

Structure-informed mutagenesis identifies a conserved region critical for mouse insulin receptor 5'UTR IRES function

Cells under stress shift their proteome by repressing cap-dependent translation initiation. RNA elements called internal ribosome entry sites (IRES) can allow key cellular transcripts to remain efficiently translated to support an effective stress response. Well- characterized IRESes depend on RNA structures that reduce the protein requirements for translation initiation, thus circumventing translation inhibition. We have previously determined that the insulin receptor 5 untranslated region (5UTR) possesses a capacity for IRES activity that is conserved from insects to mammals. There are several prominent examples of viral IRES structures solved in solution; however, the RNA secondary structures of cellular IRESes remain mostly elusive, especially in vivo. Here we probe the secondary structure of the Insr 5UTR IRES in tandem with two well-studied viral IRESes from Hepatitis C virus (HCV) and Encephalomyocarditis virus (EMCV) using dimethyl sulfate mutational profiling by sequencing (DMS-MaPseq) in cells and in vitro. We find that the viral IRES structures in cells are consistent with their known in vitro structures and that significant linearization of these well-studied IRESes occurs in the region surrounding their translation start codon in cells. Using the concurrent DMS- MaPseq probing as a constraint, we present a model of the mouse insulin receptor (Insr) 5UTR. With this model as a guide, we employed a mutation strategy which allowed us to identify a conserved segment distal from the translation start codon as critical for Insr IRES function. This knowledge informed the design of a minimal IRES element with equivalent activity to the full-length Insr 5UTR across translation contexts. BackgroundThe RNA structural requirements for cap-independent translation initiation facilitated by the Mus musculus insulin receptor (Insr) 5 untranslated region (5UTR) are unknown. ResultsRNA secondary structure probing of the Insr 5UTR in cells provides a folding model used to identify elements required for cap-independent translation initiation. ConclusionA small region of the Insr 5UTR distal to the translation start codon is necessary for cap-independent translation initiation and is fully sufficient in the proper structural context.

molecular biology↗

bioRxiv2023

Functional profiling of the Toxoplasma genome during acute mouse infection

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.

microbiology↗

bioRxiv2021

Translational activation by an alternative sigma factor in Bacillus subtilis

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, {sigma}B, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, {sigma}A, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from {sigma}B-dependent promoters liberates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between {sigma}B- and {sigma}A-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.

microbiology↗

bioRxiv2020

Structure of the full SARS-CoV-2 RNA genome in infected cells

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on native RNA structures in SARS-CoV-2 and most putative regulatory sequences are functionally uncharacterized. Here we report secondary structure ensembles of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and the algorithm detection of RNA folding ensembles using expectation-maximization clustering (DREEM). Our results reveal previously undescribed alternative RNA conformations across the genome, including structures of the frameshift stimulating element (FSE), a major drug target, that are drastically different from prevailing in vitro population average models. Importantly, we find that this structural ensemble promotes frameshifting rates (~40%) similar to in vivo ribosome profiling studies and much higher than the canonical minimal FSE (~20%). Overall, our result highlight the value of studying RNA folding in its native, dynamic and cellular context. The genomic structures detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

biochemistry↗