The subcellular distribution of miRNA isoforms, tRNA-derived fragments, and rRNA-derived fragments depends on nucleotide sequence and cell type.

BACKGROUND: MicroRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent most of the small non-coding RNAs (sncRNAs) found in cells. Members of these three classes modulate messenger RNA (mRNA) and protein abundance and are dysregulated in diseases. Experimental studies to date have assumed that the subcellular distribution of these molecules is well-understood, independent of cell type, and the same for all isoforms of a sncRNA. RESULTS: We tested these assumptions by investigating the subcellular distribution of isomiRs, tRFs, and rRFs in biological replicates from three cell lines from the same tissue and same-sex donors that model the same cancer subtype. In each cell line, we profiled the isomiRs, tRFs, and rRFs in the nucleus, cytoplasm, whole mitochondrion (MT), mitoplast (MP), and whole cell. Using a rigorous mathematical model we developed, we accounted for cross-fraction contamination and technical errors and adjusted the measured abundances accordingly. Analyses of the adjusted abundances show that isomiRs, tRFs, and rRFs exhibit complex patterns of subcellular distributions. These patterns depend on each sncRNA's exact sequence and the cell type. Even in the same cell line, isoforms of the same sncRNA whose sequences differ by a few nucleotides (nts) can have different subcellular distributions. CONCLUSIONS: SncRNAs with similar sequences have different subcellular distributions within and across cell lines, suggesting that each isoform could have a different function. Future computational and experimental studies of isomiRs, tRFs, and rRFs will need to distinguish among each molecule's various isoforms and account for differences in each isoform's subcellular distribution in the cell line at hand. While the findings add to a growing body of evidence that isomiRs, tRFs, rRFs, tRNAs, and rRNAs follow complex intracellular trafficking rules, further investigation is needed to exclude alternative explanations for the observed subcellular distribution of sncRNAs.

R2Dtool: integration and visualization of isoform-resolved RNA features.

MOTIVATION: Long-read RNA sequencing enables the mapping of RNA modifications, structures, and protein-interaction sites at the resolution of individual transcript isoforms. To understand the functions of these RNA features, it is critical to analyze them in the context of transcriptomic and genomic annotations, such as open reading frames and splice junctions. RESULTS: We have developed R2Dtool, a bioinformatics tool that integrates transcript-mapped information with transcript and genome annotations, allowing for the isoform-resolved analytics and graphical representation of RNA features in their genomic context. We illustrate R2Dtool's capability to integrate and expedite RNA feature analysis using epitranscriptomics data. R2Dtool facilitates the comprehensive analysis and interpretation of alternative transcript isoforms. AVAILABILITY AND IMPLEMENTATION: R2Dtool is freely available under the MIT license at github.com/comprna/R2Dtool.

In silico RNA isoform screening to identify potential cancer driver exons with therapeutic applications.

Alternative splicing is crucial for cancer progression and can be targeted pharmacologically, yet identifying driver exons genome-wide remains challenging. We propose identifying such exons by associating statistically gene-level cancer dependencies from knockdown viability screens with splicing profiles and gene expression. Our models predict the effects of splicing perturbations on cell proliferation from transcriptomic data, enabling in silico RNA screening and prioritizing targets for splicing-based therapies. We identified 1,073 exons impacting cell proliferation, many from genes not previously linked to cancer. Experimental validation confirms their influence on proliferation, especially in highly proliferative cancer cell lines. Integrating pharmacological screens with splicing dependencies highlights the potential driver exons affecting drug sensitivity. Our models also allow predicting treatment outcomes from tumor transcriptomes, suggesting applications in precision oncology. This study presents an approach to identifying cancer driver exon and their therapeutic potential, emphasizing alternative splicing as a cancer target.

Single-cell long-read targeted sequencing reveals transcriptional variation in ovarian cancer.

Single-cell RNA sequencing predominantly employs short-read sequencing to characterize cell types, states and dynamics; however, it is inadequate for comprehensive characterization of RNA isoforms. Long-read sequencing technologies enable single-cell RNA isoform detection but are hampered by lower throughput and unintended sequencing of artifacts. Here we develop Single-cell Targeted Isoform Long-Read Sequencing (scTaILoR-seq), a hybridization capture method which targets over a thousand genes of interest, improving the median number of on-target transcripts per cell by 29-fold. We use scTaILoR-seq to identify and quantify RNA isoforms from ovarian cancer cell lines and primary tumors, yielding 10,796 single-cell transcriptomes. Using long-read variant calling we reveal associations of expressed single nucleotide variants (SNVs) with alternative transcript structures. Phasing of SNVs across transcripts enables the measurement of allelic imbalance within distinct cell populations. Overall, scTaILoR-seq is a long-read targeted RNA sequencing method and analytical framework for exploring transcriptional variation at single-cell resolution.

High-throughput single-molecule long-read RNA sequencing analysis of tissue-specific genes and isoforms in lettuce (Lactuca sativa L.).

Lettuce is one of the most widely cultivated and consumed dicotyledonous vegetables globally. Despite the availability of its reference genome sequence, lettuce gene annotation remains incomplete, impeding comprehensive research and the broad application of genomic resources. Long-read RNA isoform sequencing (Iso-Seq) offers substantial advantages for analyzing RNA alternative splicing and aiding gene annotation, yet it faces throughput limitations. We present the HIT-ISOseq method tailored for bulk sample analysis, significantly enhancing RNA sequencing throughput on the PacBio platform by concatenating cDNA. Here we show, HIT-ISOseq generates 3-4 cDNA molecules per CCS read in lettuce, yielding 15.7 million long reads per PacBio Sequel II SMRT Cell 8 M. We validate its effectiveness in analyzing six lettuce tissue samples, including roots, stems, and leaves, revealing tissue-specific gene expression patterns and RNA isoforms. Leveraging diverse tissue long-read RNA sequencing, we refine the transcript annotation of the lettuce reference genome, expanding its GO and KEGG annotation repertoire. Collectively, this study serves as a foundational reference for genome annotation and the analysis of multi-sample isoform expression, utilizing high-throughput long-read transcriptome sequencing.

Long-read RNA sequencing identifies region- and sex-specific C57BL/6J mouse brain mRNA isoform expression and usage.

Alternative splicing (AS) contributes to the biological heterogeneity between species, sexes, tissues, and cell types. Many diseases are either caused by alterations in AS or by alterations to AS. Therefore, measuring AS accurately and efficiently is critical for assessing molecular phenotypes, including those associated with disease. Long-read sequencing enables more accurate quantification of differentially spliced isoform expression than short-read sequencing approaches, and third-generation platforms facilitate high-throughput experiments. To assess differences in AS across the cerebellum, cortex, hippocampus, and striatum by sex, we generated and analyzed Oxford Nanopore Technologies (ONT) long-read RNA sequencing (lrRNA-Seq) C57BL/6J mouse brain cDNA libraries. From > 85 million reads that passed quality control metrics, we calculated differential gene expression (DGE), differential transcript expression (DTE), and differential transcript usage (DTU) across brain regions and by sex. We found significant DGE, DTE, and DTU across brain regions and that the cerebellum had the most differences compared to the other three regions. Additionally, we found region-specific differential splicing between sexes, with the most sex differences in DTU in the cortex and no DTU in the hippocampus. We also report on two distinct patterns of sex DTU we observed, sex-divergent and sex-specific, that could potentially help explain sex differences in the prevalence and prognosis of various neurological and psychiatric disorders in future studies. Finally, we built a Shiny web application for researchers to explore the data further. Our study provides a resource for the community; it underscores the importance of AS in biological heterogeneity and the utility of long-read sequencing to better understand AS in the brain.

Incautious design of shRNAs for stable overexpression of miRNAs could result in generation of undesired isomiRs.

shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.

IsoVis - a webserver for visualization and annotation of alternative RNA isoforms.

Genes commonly express multiple RNA products (RNA isoforms), which differ in exonic content and can have different functions. Making sense of the plethora of known and novel RNA isoforms being identified by transcriptomic approaches requires a user-friendly way to visualize gene isoforms and how they differ in exonic content, expression levels and potential functions. Here we introduce IsoVis, a freely available webserver that accepts user-supplied transcriptomic data and visualizes the expressed isoforms in a clear, intuitive manner. IsoVis contains numerous features, including the ability to visualize all RNA isoforms of a gene and their expression levels; the annotation of known isoforms from external databases; mapping of protein domains and features to exons, allowing changes to protein sequence and function between isoforms to be established; and extensive species compatibility. Datasets visualised on IsoVis remain private to the user, allowing analysis of sensitive data. IsoVis visualisations can be downloaded to create publication-ready figures. The IsoVis webserver enables researchers to perform isoform analyses without requiring programming skills, is free to use, and available at https://isomix.org/isovis/.

Uncovering the dynamics and consequences of RNA isoform changes during neuronal differentiation.

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.

RNA Isoforms as Broad Targets for Cancer Immunotherapy.

While immunotherapy is typically reserved for cancer patients with a high mutational burden, neoantigens produced from post-transcriptional regulation provide a possible untapped reservoir of common immunogenic targets for new targeted cancer therapies. In this review, we describe new and emerging technologies, unconventional molecular targets and challenges for the precision immune targeting of diverse malignancies. In particular, we focus on the unique potential of targeting alternative mRNA isoforms as a source for broadly presented neoantigens and cell surface proteins. Finally, we discuss emerging challenges for alternative isoform immune targeting, with an emphasis in silico prioritization and high-throughput target validation.

Comprehensive assessment of mRNA isoform detection methods for long-read sequencing data.

The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length of sequencing to several kilobases, thereby facilitating the identification of alternative splicing events and isoform expressions. Recently, numerous computational tools for isoform detection using long-read sequencing data have been developed. Nevertheless, there remains a deficiency in comparative studies that systemically evaluate the performance of these tools, which are implemented with different algorithms, under various simulations that encompass potential influencing factors. In this study, we conducted a benchmark analysis of thirteen methods implemented in nine tools capable of identifying isoform structures from long-read RNA-seq data. We evaluated their performances using simulated data, which represented diverse sequencing platforms generated by an in-house simulator, RNA sequins (sequencing spike-ins) data, as well as experimental data. Our findings demonstrate IsoQuant as a highly effective tool for isoform detection with LRS, with Bambu and StringTie2 also exhibiting strong performance. These results offer valuable guidance for future research on alternative splicing analysis and the ongoing improvement of tools for isoform detection using LRS data.

Single-cell long-read sequencing-based mapping reveals specialized splicing patterns in developing and adult mouse and human brain.

RNA isoforms influence cell identity and function. However, a comprehensive brain isoform map was lacking. We analyze single-cell RNA isoforms across brain regions, cell subtypes, developmental time points and species. For 72% of genes, full-length isoform expression varies along one or more axes. Splicing, transcription start and polyadenylation sites vary strongly between cell types, influence protein architecture and associate with disease-linked variation. Additionally, neurotransmitter transport and synapse turnover genes harbor cell-type variability across anatomical regions. Regulation of cell-type-specific splicing is pronounced in the postnatal day 21-to-postnatal day 28 adolescent transition. Developmental isoform regulation is stronger than regional regulation for the same cell type. Cell-type-specific isoform regulation in mice is mostly maintained in the human hippocampus, allowing extrapolation to the human brain. Conversely, the human brain harbors additional cell-type specificity, suggesting gain-of-function isoforms. Together, this detailed single-cell atlas of full-length isoform regulation across development, anatomical regions and species reveals an unappreciated degree of isoform variability across multiple axes.

Isoform-specific RNA structure determination using Nano-DMS-MaP.

RNA structure determination is essential to understand how RNA carries out its diverse biological functions. In cells, RNA isoforms are readily expressed with partial variations within their sequences due, for example, to alternative splicing, heterogeneity in the transcription start site, RNA processing or differential termination/polyadenylation. Nanopore dimethyl sulfate mutational profiling (Nano-DMS-MaP) is a method for in situ isoform-specific RNA structure determination. Unlike similar methods that rely on short sequencing reads, Nano-DMS-MaP employs nanopore sequencing to resolve the structures of long and highly similar RNA molecules to reveal their previously hidden structural differences. This Protocol describes the development and applications of Nano-DMS-MaP and outlines the main considerations for designing and implementing a successful experiment: from bench to data analysis. In cell probing experiments can be carried out by an experienced molecular biologist in 3-4 d. Data analysis requires good knowledge of command line tools and Python scripts and requires a further 3-5 d.

FL-circAS: an integrative resource and analysis for full-length sequences and alternative splicing of circular RNAs with nanopore sequencing.

Circular RNAs (circRNAs) are RNA molecules with a continuous loop structure characterized by back-splice junctions (BSJs). While analyses of short-read RNA sequencing have identified millions of BSJ events, it is inherently challenging to determine exact full-length sequences and alternatively spliced (AS) isoforms of circRNAs. Recent advances in nanopore long-read sequencing with circRNA enrichment bring an unprecedented opportunity for investigating the issues. Here, we developed FL-circAS (https://cosbi.ee.ncku.edu.tw/FL-circAS/), which collected such long-read sequencing data of 20 cell lines/tissues and thereby identified 884 636 BSJs with 1 853 692 full-length circRNA isoforms in human and 115 173 BSJs with 135 617 full-length circRNA isoforms in mouse. FL-circAS also provides multiple circRNA features. For circRNA expression, FL-circAS calculates expression levels for each circRNA isoform, cell line/tissue specificity at both the BSJ and isoform levels, and AS entropy for each BSJ across samples. For circRNA biogenesis, FL-circAS identifies reverse complementary sequences and RNA binding protein (RBP) binding sites residing in flanking sequences of BSJs. For functional patterns, FL-circAS identifies potential microRNA/RBP binding sites and several types of evidence for circRNA translation on each full-length circRNA isoform. FL-circAS provides user-friendly interfaces for browsing, searching, analyzing, and downloading data, serving as the first resource for discovering full-length circRNAs at the isoform level.

Molecular analysis of the human cytoglobin mRNA isoforms.

Multiple functions have been proposed for the ubiquitously expressed vertebrate globin cytoglobin (Cygb), including nitric oxide (NO) metabolism, lipid peroxidation/signalling, superoxide dismutase activity, reactive oxygen/nitrogen species (RONS) scavenging, regulation of blood pressure, antifibrosis, and both tumour suppressor and oncogenic effects. Since alternative splicing can expand the biological roles of a gene, we investigated whether this mechanism contributes to the functional diversity of Cygb. By mining of cDNA data and molecular analysis, we identified five alternative mRNA isoforms for the human CYGB gene (V-1 to V-5). Comprehensive RNA-seq analyses of public datasets from human tissues and cells confirmed that the canonical CYGB V-1 isoform is the primary CYGB transcript in the majority of analysed datasets. Interestingly, we revealed that isoform V-3 represented the predominant CYGB variant in hepatoblastoma (HB) cell lines and in the majority of analysed normal and HB liver tissues. CYGB V-3 mRNA is transcribed from an alternate upstream promoter and hypothetically encodes a N-terminally truncated CYGB protein, which is not recognized by some antibodies used in published studies. Little to no transcriptional evidence was found for the other CYGB isoforms. Comparative transcriptomics and flow cytometry on CYGB+/+ and gene-edited CYGB-/- HepG2 HB cells did not unveil a knockout phenotype and, thus, a potential function for CYGB V-3. Our study reveals that the CYGB gene is transcriptionally more complex than previously described as it expresses alternative mRNA isoforms of unknown function. Additional experimental data are needed to clarify the biological meaning of those alternative CYGB transcripts.

High-throughput RNA isoform sequencing using programmed cDNA concatenation.

Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.

Regulation and function of alternative polyadenylation in development and differentiation.

Alternative processing of nascent mRNAs is widespread in eukaryotic organisms and greatly impacts the output of gene expression. Specifically, alternative cleavage and polyadenylation (APA) is a co-transcriptional molecular process that switches the polyadenylation site (PAS) at which a nascent mRNA is cleaved, resulting in mRNA isoforms with different 3'UTR length and content. APA can potentially affect mRNA translation efficiency, localization, stability, and mRNA seeded protein-protein interactions. APA naturally occurs during development and cellular differentiation, with around 70% of human genes displaying APA in particular tissues and cell types. For example, neurons tend to express mRNAs with long 3'UTRs due to preferential processing at PASs more distal than other PASs used in other cell types. In addition, changes in APA mark a variety of pathological states, including many types of cancer, in which mRNAs are preferentially cleaved at more proximal PASs, causing expression of mRNA isoforms with short 3'UTRs. Although APA has been widely reported, both the function of APA in development and the mechanisms that regulate the choice of 3'end cut sites in normal and pathogenic conditions are still poorly understood. In this review, we summarize current understanding of how APA is regulated during development and cellular differentiation and how the resulting change in 3'UTR content affects multiple aspects of gene expression. With APA being a widespread phenomenon, the advent of cutting-edge scientific techniques and the pressing need for in-vivo studies, there has never been a better time to delve into the intricate mechanisms of alternative cleavage and polyadenylation.