Biology subjects

Faller, W. J.

Publications and source records attributed to Faller, W. J..

6 recordsLinked to original sources

bioRxiv2024

Mitochondrial damage triggers therapy-induced senescence

Glioblastoma (GBM) is a fatal brain tumor with a critical need for better therapies. It is known that the PI3K, MAPK, and CDK4/6 signaling pathways are hyper-activated in these tumors; however, previous studies have used very high concentration of inhibitors to assess their importance, with mixed results. Here we developed PMCi, a combination approach that targets all three pathways simultaneously, at clinically-relevant doses. PMCi effectively suppresses GBM cell proliferation in vitro and in vivo, and outperforms monotherapies and dual combinations. PMCi acts by inducing cellular senescence, which is mediated solely by the mitochondria, and, unlike other forms of senescence, is independent of nuclear damage. This phenotype is caused by a reactive oxygen species (ROS)\cGAS-STING\senescence-associated secretory phenotype (SASP) signaling cascade, that acts in a paracrine manner to establish and maintain senescence. Our results demonstrate that mitochondrial damage is sufficient to drive senescence, and that this can be leveraged to target GBM cells.

cell biology↗

bioRxiv2024

NAC-mediated ribosome localization regulates cell fate and metabolism in intestinal stem cells

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity, however it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we show that RNA translation is at the root of this interplay. We identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process, and show that it regulates ISC metabolism by relocalizing ribosomes to the mitochondria. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NAC is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of ribosome localization in regulating mitochondrial metabolism and ISC function. TeaserThe location of ribosomes in cells is regulated, and defines the fate of intestinal stem cells.

molecular biology↗

bioRxiv2024

RNA dicing regulates the expression of an oncogenic JAK1 isoform

mRNA transcripts have limited potential for protein synthesis, defined by their open reading frames1. However, recent advances have revealed a far more complex reality, in which the proteome exceeds the perceived limits of the transcriptome2-6 exposing a significant gap in our understanding. Our prior studies have demonstrated that mRNA can undergo further processing, yielding truncated, uncapped mRNAs with translation potential2,7. Yet, the biological importance of this process remains mostly unclear. Here, we demonstrate that cleavage within the coding sequence of JAK1 mRNA produces an uncapped variant downstream to the cleavage site, at the expense of the full-length transcript. This results in the independent translation of the JH1 kinase domain, a process we term RNA dicing. Notably, canonical and diced JAK1 variants have distinct impacts on cell proliferation and tumorigenesis, operating independently, localizing to distinct cellular compartments. In addition, the activation of JAK1 signaling through IFN{gamma} induction promotes the dicing of JAK1, thereby altering the balance of isoforms present. Base editor screens reveal that stop-codon nonsense mutations, which are typically considered loss-of-function, have differing impacts depending on their position relative to the dicing site. In agreement with this, JAK1 nonsense frameshift (JAK1fs) mutations in endometrial tumors inhibit the tumor-suppressive functions of canonical JAK1, while amplifying the oncogenic potential of the diced JH1 kinase domain. Lastly, we demonstrate that Momelotinib8, a JAK1-specific inhibitor, is more effective in cancer cells carrying a "loss-of-function" JAK1fs mutation, highlighting the significant impact of RNA dicing biology as a potent tool for patient stratification. Our findings characterize RNA dicing as a fundamental regulatory machinery that diversifies the potential products of a single mRNA molecule, and allows for significant variation in biological function.

molecular biology↗

bioRxiv2023

An "alert state" ribosome population acts as a master regulator of cytokine mediated processes

Inflammatory cytokines are pivotal to immune responses. Upon cytokine exposure, cells enter an "alert-state" that enhances their visibility to the immune system. Here, we identified an "alert-state" subpopulation of ribosomes (ASRs) defined by the presence of the P-stalk. We show that ASRs are formed in response to cytokines linked to tumor immunity, and are involved in the preferential translation of mRNAs vital for the cytokine response. Mechanistically, ASRs are required for the efficient translation of transmembrane domains of receptor molecules involved in cytokine-mediated processes. Importantly, loss of the ASR prevents CD8+ T cell recognition and killing, and inhibitory cytokines like TGF{beta} hinder ASR formation, suggesting that the ASR is a central regulatory hub upon which multiple signals converge. Thus, the ASR is an essential mediator of the cellular rewiring that occurs following cytokine exposure, via the translational regulation of this process.

immunology↗

bioRxiv2021

SETD2 negatively regulates cell size through its catalytic activity and SRI domain

Cell size varies between cell types but is tightly regulated by cell-intrinsic and extrinsic mechanisms. Cell-size control is important for cell function and changes in cell size are frequently observed in cancer cells. Here we uncover a non-canonical role of SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription regulation, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Although most of SETD2s cellular functions have been linked to this activity, several non-histone substrates of SETD2 have recently been identified - some of which have been linked to novel functions of SETD2 beyond chromatin regulation. Using multiple, independent perturbation strategies we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that this function is dependent on the catalytic activity of SETD2 but independent of H3K36 methylation. Paradoxically, ectopic overexpression of a decoy SRI domain also increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

cell biology↗

bioRxiv2020

Ribo-ODDR: Ribo-seq focused Oligo Design pipeline for experiment-specific Depletion of Ribosomal RNAs

Ribosome profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet, several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of ribosomal RNA (rRNA) fragments, which frequently make up more than 90% of sequencing reads if not depleted. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, such kits may perform suboptimally in Ribo-seq. There is therefore potential to significantly increase the information that can be gleaned from Ribo-seq experiments. Here we show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a "one-size-fits-all" approach may result in inefficient rRNA depletion. In order to overcome this, it is possible to use custom-designed biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. We have developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We show that Ribo-ODDR designed oligos lead to a significant increase in rRNA depletion, and increased sequencing depth as a result, providing substantial information that would otherwise have been lost. Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR

bioinformatics↗