Li, N.-S. | bioRxiv Science

bioRxiv2023

Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity

Monitoring the dynamic changes of cellular tRNA pools is challenging, due to the extensive post-transcriptional modifications of individual species. The most critical component in tRNAseq is a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show that the recently developed group-II intron RT Induro has the processivity and efficiency necessary to profile tRNA dynamics. Using our Induro-tRNAseq, simpler and more comprehensive than the best methods to date, we show that Induro progressively increases readthrough of tRNA over time and that the mechanism of increase is selective removal of RT stops, without altering the misincorporation frequency. We provide a parallel dataset of the misincorporation profile of Induro relative to the related TGIRT RT to facilitate the prediction of non-annotated modifications. We report an unexpected modification profile among human proline isoacceptors, absent from mouse and lower eukaryotes, that indicates new biology of decoding proline codons.

genomics↗

bioRxiv2021

Stereo-specific Lasofoxifene Derivatives Reveal the Interplay between Estrogen Receptor Alpha Stability and Antagonistic Activity in ESR1 Mutant Breast Cancer Cells

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend ER cellular lifetime, accumulation, and are antagonists in the breast and agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders (SERDs) reduce ER cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ER cellular lifetime and accumulation influences antagonistic activity, we synthesized a series of methylpyrollidine lasofoxifene derivatives that maintained the drugs antagonistic activities while uniquely tuning ER cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ER cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ER ligand binding domain in complex with the methylated lasofoxifene derivatives, SERMs, and SERDs show that molecules that favor a highly buried helix 12 conformation achieve the greatest transcriptional suppression activities. Together these results show that chemical reduction of ER cellular lifetime does not necessarily correlate with transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our approach shows how minor chemical additions modulate receptor cellular lifetime while maintaining other activities to achieve desired SERM or SERD profiles. SIGNIFICANCEThis study shows that antiestrogens that enforce a wild-type-like antagonist conformation demonstrate improved therapeutic activities in hormone-resistant breast cancer cells harboring Y537S and D538G ESR1.

cancer biology↗