Search

Abstract RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Background and objective: Previous germline studies on renal cell carcinoma (RCC) have usually pooled clear and non–clear cell RCCs and have not adequately accounted for population stratification, which might have led to an inaccurate estimation of genetic risk. Here, we aim to analyze the major germline drivers of RCC risk and clinically relevant but underexplored germline variant types. Methods: We first characterized germline pathogenic variants (PVs), cryptic splice variants, and copy number variants (CNVs) in 1436 unselected RCC patients. To evaluate the enrichment of PVs in RCC, we conducted a case-control study of 1356 RCC patients ancestry matched with 16 512 cancer-free controls using approaches accounting for population stratification and histological subtypes, followed by characterization of secondary somatic events. Key findings and limitations: Clear cell RCC patients (n = 976) exhibited a significant burden of PVs in VHL compared with controls (odds ratio [OR]: 39.1, p = 4.95e-05). Non–clear cell RCC patients (n = 380) carried enrichment of PVs in FH (OR: 77.9, p = 1.55e-08) and MET (OR: 1.98e11, p = 2.07e-05). In a CHEK2-focused analysis with European participants, clear cell RCC (n = 906) harbored nominal enrichment of low-penetrance CHEK2 variants—p.Ile157Thr (OR: 1.84, p = 0.049) and p.Ser428Phe (OR: 5.20, p = 0.045), while non–clear cell RCC (n = 295) exhibited nominal enrichment of CHEK2 loss of function PVs (OR: 3.51, p = 0.033). Patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset than patients without germline PVs (mean: 46.0 vs 60.2 yr, p

Abstract RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

Abstract Background Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors. Results We present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing. Conclusions FLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at https://github.com/YeoLab/FLARE .

Abstract Immune checkpoint blockade (ICB) therapies, which potentiate the body’s natural immune response against tumor cells, have shown immense promise in the treatment of various cancers. Currently, tumor mutational burden (TMB) and programmed death ligand 1 (PD-L1) expression are the primary biomarkers evaluated for clinical management of cancer patients across histologies. However, the wide range of responses has demonstrated that the specific molecular and genetic characteristics of each patient’s tumor and immune system must be considered to maximize treatment efficacy. Here, we review the various biological pathways and emerging biomarkers implicated in response to PD-(L)1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) therapies, including oncogenic signaling pathways, human leukocyte antigen (HLA) variability, mutation and neoantigen burden, microbiome composition, endogenous retroviruses (ERV), and deficiencies in chromatin remodeling and DNA damage repair (DDR) machinery. We also discuss several mechanisms that have been observed to confer resistance to ICB, such as loss of phosphatase and tensin homolog (PTEN), loss of major histocompatibility complex (MHC) I/II expression, and activation of the indoleamine 2,3-dioxygenase 1 (IDO1) and transforming growth factor beta (TGFβ) pathways. Clinical trials testing the combination of PD-(L)1 or CTLA-4 blockade with molecular mediators of these pathways are becoming more common and may hold promise for improving treatment efficacy and response. Ultimately, some of the genes and molecular mechanisms highlighted in this review may serve as novel biological targets or therapeutic vulnerabilities to improve clinical outcomes in patients.