Biology subjects

Martin, S. E.

Publications and source records attributed to Martin, S. E..

4 recordsLinked to original sources

bioRxiv2023

Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway in human cells. Here, we identified RNF146 as a ubiquitin ligase (E3) of TEADs, which negatively regulates their stability in cells through proteasome-mediated degradation. We show that RNF146-mediated TEAD ubiquitination is dependent on the TEAD PARylation state. We further validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Despite the RNF146 and proteasome-mediated degradation mechanisms, TEADs are stable proteins with a long half-life in cells. We demonstrate that degradation of TEADs can be greatly enhanced pharmacologically with heterobifunctional chemical inducers of protein degradation (CIDEs). These TEAD-CIDEs can effectively suppress activation of YAP/TAZ target genes in a dose-dependent manner and exhibit significant anti-proliferative effects in YAP/TAZ-dependent tumor cells, thus phenocopying the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates that the ubiquitin-proteasome system plays an important role in regulating TEAD functions and provides a proof-of-concept demonstration that pharmacologically induced TEAD ubiquitination could be leveraged to target YAP/TAZ-driven cancers.

cancer biology↗

bioRxiv2021

Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs

N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reactions cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the birds-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/460679v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@15845aeorg.highwire.dtl.DTLVardef@46a95aorg.highwire.dtl.DTLVardef@118633dorg.highwire.dtl.DTLVardef@1b52f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication.

Polyadenylated nuclear (PAN) RNA is a non-coding transcript involved in Kaposis sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have shown that PAN RNA has a dynamic secondary structure and protein binding profiles that can be influenced by the epitranscriptomic modifications. N6-methyladenosine (m6A) is an abundant signature found in viral and virus-encoded RNAs. Here, we combined an antibody-independent next-generation mapping with direct RNA sequencing to elucidate the m6A landscape of PAN RNA during the KSHV latent and lytic stages of infection. Using a newly developed method, termed Selenium-modified deoxythymidine triphosphate reverse transcription and Ligation Assisted PCR analysis of m6A (SLAP), we gained insight into the fraction of modification at identified sites. Using comprehensive proteomic approaches, we identified writers, erasers, and readers that regulate the m6A status of PAN. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing outlined structural alterations invoked by m6A in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay between the cellular epitranscriptomic machinery and a specific viral RNA.

molecular biology↗

bioRxiv2018

Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens

Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed yet. In this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity, and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We found that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner, and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks.

bioinformatics↗