L-GIREMI uncovers RNA editing sites in long-read RNA-seq.

Although long-read RNA-seq is increasingly applied to characterize full-length transcripts it can also enable detection of nucleotide variants, such as genetic mutations or RNA editing sites, which is significantly under-explored. Here, we present an in-depth study to detect and analyze RNA editing sites in long-read RNA-seq. Our new method, L-GIREMI, effectively handles sequencing errors and read biases. Applied to PacBio RNA-seq data, L-GIREMI affords a high accuracy in RNA editing identification. Additionally, our analysis uncovered novel insights about RNA editing occurrences in single molecules and double-stranded RNA structures. L-GIREMI provides a valuable means to study nucleotide variants in long-read RNA-seq.

LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity.

RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.

scAllele: A versatile tool for the detection and analysis of variants in scRNA-seq.

Single-cell RNA sequencing (scRNA-seq) data contain rich information at the gene, transcript, and nucleotide levels. Most analyses of scRNA-seq have focused on gene expression profiles, and it remains challenging to extract nucleotide variants and isoform-specific information. Here, we present scAllele, an integrative approach that detects single-nucleotide variants, insertions, deletions, and their allelic linkage with splicing patterns in scRNA-seq. We demonstrate that scAllele achieves better performance in identifying nucleotide variants than other commonly used tools. In addition, the read-specific variant calls by scAllele enables allele-specific splicing analysis, a unique feature not afforded by other methods. Applied to a lung cancer scRNA-seq dataset, scAllele identified variants with strong allelic linkage to alternative splicing, some of which are cancer specific and enriched in cancer-relevant pathways. scAllele represents a versatile tool to uncover multilayer information and previously unidentified biological insights from scRNA-seq data.

RNA editing in cancer impacts mRNA abundance in immune response pathways.

BACKGROUND: RNA editing generates modifications to the RNA sequences, thereby increasing protein diversity and shaping various layers of gene regulation. Recent studies have revealed global shifts in editing levels across many cancer types, as well as a few specific mechanisms implicating individual sites in tumorigenesis or metastasis. However, most tumor-associated sites, predominantly in noncoding regions, have unknown functional relevance. RESULTS: Here, we carry out integrative analysis of RNA editing profiles between epithelial and mesenchymal tumors, since epithelial-mesenchymal transition is a key paradigm for metastasis. We identify distinct editing patterns between epithelial and mesenchymal tumors in seven cancer types using TCGA data, an observation further supported by single-cell RNA sequencing data and ADAR perturbation experiments in cell culture. Through computational analyses and experimental validations, we show that differential editing sites between epithelial and mesenchymal phenotypes function by regulating mRNA abundance of their respective genes. Our analysis of RNA-binding proteins reveals ILF3 as a potential regulator of this process, supported by experimental validations. Consistent with the known roles of ILF3 in immune response, epithelial-mesenchymal differential editing sites are enriched in genes involved in immune and viral processes. The strongest target of editing-dependent ILF3 regulation is the transcript encoding PKR, a crucial player in immune and viral response. CONCLUSIONS: Our study reports widespread differences in RNA editing between epithelial and mesenchymal tumors and a novel mechanism of editing-dependent regulation of mRNA abundance. It reveals the broad impact of RNA editing in cancer and its relevance to cancer-related immune pathways.

Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA.

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs.

Regulation of RNA editing by RNA-binding proteins in human cells.

Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing.

RNA editing in nascent RNA affects pre-mRNA splicing.

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3' acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.