Presentation Order:
Alyssa
Hannah
Shah
Keaton
Time: biweekly, Wednesday 9:30-11:50
Writing 9:30-10:00---5-10 min/person
Research Presentation: 30 minutes talk+discussion
HL
Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit
ST
CFAP20 salvages arrested RNAPII from the path of co-directional replisomes
Cells prevent harmful collisions between transcription and DNA replication, particularly at promoter-proximal regions where RNA polymerase II (RNAPII) frequently pauses and forms R-loops. While most previous work focused on head-on collisions, the mechanism that protects co-directional replication forks from paused RNAPII near promoters remained unclear. Using genome-wide mapping, CRISPR-based genetic screens, and DNA fiber assays, the authors identify CFAP20 as a nuclear factor that suppresses Mediator-dependent R-loops specifically at co-directional promoters. Loss of CFAP20 leads to promoter-specific R-loop accumulation, replication fork acceleration, reduced origin firing, and single-stranded DNA gap formation, which can be rescued by inactivating the Mediator kinase module. The major strength of the study is its demonstration that local transcriptional stress can lead to global replication defects, although the mechanism by which CFAP20 promotes RNAPII elongation remains unclear. Future studies should define the direct molecular interaction between CFAP20 and the transcription machinery and determine whether CFAP20 dysfunction contributes to genome instability in vivo.
AJM
Ligand-specific activation trajectories dictate GPCR signalling in cells
G-protein-coupled receptors (GPCRs) are crucial to cell communication and are considered the most important target for drug development. While biophysical analyses of purified GPCRs have elucidated unique conformational states, there are no studies in living cells that identify such dynamics, nor the impact of these states in signaling intensity and duration. Herein, the authors develop a novel biosensor which utilizes a non-canonical amino acid with a small fluorescent label incorporated into various regions of the extracellular domain of the GPCR M2R that emit measurable differences in fluorescence to ligand binding. Coupled with testing agonists of varying affinity and G-proteins of different activity, the authors revealed two distinct GPCR-G protein complexes of unique extracellular conformation that are dependent on ligand and G-protein binding. In sum, strong agonists favor stabilizing complexes of high activation of G-proteins, whereas weaker affinity agonists stabilize a low-efficacy complex; these complexes occur for the same ligand-receptor pair across time for distinct G-proteins. Overall, the authors demonstrate the utility of their GPCR biosensor in ligand-receptor dynamics to understand unique G-protein signaling cascades, which will aid drug advancements towards greater specificity/less off target effects.
KK
A direct role for a mitochondrial targeting sequence in signalling stress
HL
Synergy between regulatory elements can render cohesin dispensable for distal enhancer function
Long-range enhancers are thought to need cohesin-mediated DNA loops to reach promoters, yet many genes remain active when cohesin is removed. The authors rapidly depleted cohesin in mouse stem cells and used RNA-seq, high-resolution 3D genome mapping, and targeted deletions of promoter-proximal elements to test enhancer dependence. Surprisingly, loss of loops caused only modest transcriptional changes, and many distal enhancers still functioned. Mechanistically, promoter-proximal elements synergize with distal enhancers to drive transcription without physical contact. This shows gene activation depends on the combinatorial logic of regulatory elements, not solely on chromosome folding, explaining why structural disruptions often have limited effects.
AJM
Viral RNA blocks circularization to evade host codon usage control
Human cells have a bias for certain tRNA-codon pairs among synonymous codons, which results in differential translation rates across mRNAs. This fundamental mechanism, termed codon-usage bias, should constrain viral protein replication with discordant codon preference; however, nearly all viral genomes, including several common and pandemic-potential viruses for humans, such as influenza and SARS-CoV-2, have dissimilar codon usage preferences to us. Thus, the authors sought to determine the mechanism of viral codon-usage bias escape via careful plasmid construction to evaluate the impacts of codons, flanking untranslated regions (UTRs), and 3D structures on translation rate. Importantly, the authors revealed codon-usage bias is only relevant for UTR-mediated circularized mRNA. Meanwhile, viral 5’ UTRs enforce a linear structure during translation, which eliminates codon preference and escapes inhibition of protein elongation. This work lends support to several biological theories, including the closed-loop structure of canonical mRNA during translation and codon-usage regulation. Overall, this work has broad impacts on gene editing techniques and therapies. Future work can visualize linearization of viral genomes during translation as well as determine the spectrum of viruses utilizing this means of escape.
ST
AJM
Genetic elements promote retention of extrachromosomal DNA in cancer cells
Extra-chromosomal DNA (ecDNA) is a prevalent oncogene activator in diverse cancers, permitting heritable additive expression of oncogenes across cell generations. How ecDNA is retained has been well studied in the viral replication context, however, its mechanism in the human genome has not been established. Herein, the authors revealed a novel method, termed Retain-Seq, that sequences transduced plasmids with random human genome inserts to identify retention sequences that allow ecDNA retention across several rounds of cell division. The authors identify diverse retention sequences with a common thread of transcription-activating regions, including transcriptional start sites, promoters, and enhancer. Importantly, these regions are enriched in CpG sites. Given CpG methylation is crucial for promoter activity, the authors tested the impact of differential methylation status of ecDNA’s CpGs on retention, finding decreased ecDNA retention and subsequent cell proliferation and viability with increased CpG methylation. Overall, the authors elucidate an important biological means of oncogene activation that could pose a novel target for therapeutic intervention against cancer, and could be further applied to improve targeted personalized gene therapies in chronic disease.
KK
Atomically accurate de novo design of antibodies with RFdiffusion
Antibodies are critical to immune defense and as a novel therapeutic class; however, de novo development of antibodies to target specific epitopes requires costly and time-consuming in vivo screening. To address this challenge, the authors of this study created a computational framework to design antibodies against clinically relevant epitopes. A set of antibody designs generated with a modified version of their RFdiffusion structure prediction model, yielded several candidate antibodies that bound to the target, though not at the desired affinity. The authors then combined their model with an additional step to mutate the candidate designs in silico, eventually reaching the desired affinity. Although the current implementation of this approach requires more optimization and validation than is practical for most potential end users, it represents a promising innovation toward de novo antibody design.
HL
Secretome translation shaped by lysosomes and lunapark-marked ER junctions
The spatial organization that drives the translation of mRNAs encoding secretory and membrane proteins at the endoplasmic reticulum (ER), and their coordination with other organelles such as lysosomes, remains poorly understood. Using real-time single-molecule imaging, Choi et al. define the spatial relationship between secretome mRNA translation, ER junctions, and lysosomal proximity. They demonstrate that secretome mRNAs associate with the ER during active translation but are more diffuse when untranslated, and that translating mRNAs preferentially localize to ER three-way junctions termed “translation hotspots.” These hotspots are enriched for the ER-shaping protein Lunapark and frequently positioned near lysosomes. Functionally, efficient secretome mRNA translation depends on both Lunapark and intact lysosomal activity, with lysosomal metabolic state modulating local translation such that amino-acid starvation enhances, while lysosomal deacidification suppresses, translation near junctions. Together, these findings identify a novel mechanism in which Lunapark-marked ER junctions and lysosomes spatially coordinate the translation of secretory and membrane protein mRNAs, reframing ER architecture as an active regulator of translational control rather than a passive scaffold.
ST
Neoadjuvant immunotherapy in mismatch-repair-proficient colon cancers