Functional genomics approaches for understanding the role of noncoding RNAS in human diseases

Dark matter matters: from noncoding RNA regulation to cancer immunity

ABSTRACT Long considered transcriptional noise, noncoding RNAs (ncRNAs), including microRNAs and long noncoding RNAs (lncRNAs), are now recognized as central regulators of cellular function, acting as scaffolds, structural elements, and regulators of gene expression. This expanding functional landscape is reshaping our understanding of cancer biology, immune regulation, and the limits of translation itself. Beyond gene regulation, lncRNAs may be implicated in the earliest stages of oncogenic transformation, orchestrating molecular reprogramming and remodeling of the immune microenvironment in premalignant lesions. Their functional analysis has further exposed an unexpected translational dimension with some lncRNAs harboring open reading frames encoding micropeptides. These cryptic micropeptides constitute an underexplored dimension of the immunopeptidome that could shape T-cell development and antitumor immunity, with implications for cancer immunosurveillance and therapeutic targeting. Collectively, these findings call for a revised molecular dogma in which the noncoding genome is recognized as a major regulator of cellular function, oncogenic transformation, and immune surveillance.

Beyond Junk DNA: Emerging Roles of Non-Coding RNAs in Cancer and Therapeutic Innovation

Non-coding RNAs (ncRNAs) are the biggest class of regulatory molecules in the human genome, which constitute ~98% of total transcripts, and were once considered as “transcriptional noise”. This review will focus on the newly identified functions of non-coding RNAs (ncRNAs) in cancer biology and therapeutic innovations. We review the existing knowledge on microRNAs, long non-coding RNAs, and circular RNAs in the context of their different experimental and computational studies, and we highlight their mechanisms of action in cancer hallmarks such as proliferation and resistance to apoptosis, metastasis, and metabolic reprogramming. We evaluate the translational landscape of ncRNA-based therapeutics by examining clinical trials in progress and completed, delivery methods, and emerging technologies. This review aims to separate fact from fiction and offer a balanced view on the facts, which will help guide future research and clinical development in the field of ncRNA oncology.

The emerging roles of non-coding RNAs in cancer.

Non-coding RNAs (ncRNAs) regulate gene expression through transcriptional, post-transcriptional, and epigenetic mechanisms, shaping hallmarks of cancer, including metastasis, therapy resistance, and relapse. Carcinogenesis arises when aberrant ncRNA networks initiate malignant transformation and sustain oncogenic changes through epigenetic modifications, shifts in cell identity, failures in genome protection, metabolic changes, and alterations in the tumour microenvironment. Environmental exposures, combined with chronic inflammation, reorganise these networks early on, leading to the formation of premalignant fields and persistent epigenetic changes. The four major ncRNA classes, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and PIWI-interacting RNAs (piRNAs) function as either oncogenes or tumour suppressors depending on the specific cancer type. Their stability, cell-type-specific expression, and presence in biofluids make them suitable candidates for biomarker discovery and liquid biopsy applications. Therapeutic strategies now include antisense oligonucleotides, small interfering RNAs, synthetic miRNA mimics, RNA aptamers, and aptamer-siRNA conjugates, which can either inhibit oncogenic ncRNAs or restore tumour-suppressive regulatory networks. CRISPR-based ncRNA modulation, including Cas9-mediated locus editing, CRISPR interference/activation, and Cas13-mediated transcript targeting, remains largely investigational because delivery, off-target activity, and an incomplete understanding of ncRNA context dependence continue to limit translation. High-throughput sequencing, single-cell transcriptomics, and computational modelling have accelerated the identification of cancer-related ncRNAs and elucidated their biological functions. This review examines how different types of ncRNAs contribute to cancer initiation, progression, and treatment resistance, and assesses their potential as diagnostic markers, prognostic factors, and therapeutic targets.

A cuffed CRISPR guide RNA for microRNA activity-dependent genome editing

Cells in multicellular eukaryotic systems are diverse biological units, with characteristics and functions determined by their molecular profiles. CRISPR–Cas9 genome editing has been widely used across biology to modulate gene expression and study gene function. However, there is currently no versatile and scalable method for editing a cell’s genome in response to endogenous cellular signals. Here, we report the engineering of a CRISPR guide RNA that efficiently confers genome editing in response to the catalytic activity of a target microRNA (miRNA) within a cell. miRNAs are short non-coding RNAs that are widely conserved across eukaryotes and can cleave their target RNA through almost perfect base pairing. In mammals, miRNAs are largely involved in development and homeostasis as well as disease progression and developmental disorders. To leverage these properties for genome editing, we developed a cuffed guide RNA (cgRNA) which is composed of a permutated order of sequence domains from the commonly used single guide RNA (sgRNA). These permutated domains were then concatenated with a miRNA target sequence, yielding a warped guide RNA that is inactive until cleaved by a complementary miRNA. We demonstrated that cgRNA enabled efficient miRNA activity-dependent genome editing in human and mouse cell lines. Biochemical and structural analyses revealed three stages of inhibition of the CRISPR genome-editing pathway for unprocessed cgRNA. Utilizing a lentiviral library of cgRNAs containing miRNA targets covering mouse genome-wide miRNAs, we identified miRNA cleavage activities and their sequence specificities in mouse embryonic stem cells and during smooth muscle cell differentiation. Furthermore, we showed that endogenous mRNA expression could be irreversibly recorded into a DNA sequence using a cgRNA targeted by a synthetic miRNA repeat. cgRNA is a simple, robust, miRNA activity-gated genome editing system that could facilitate the development of cell state-specific genome editing, the mapping of miRNA activity and gene expression landscapes, and the recording of molecularly determined cell states during the long-term progression of multicellular systems.

A Comprehensive Pipeline for Long Non-Coding RNA Discovery and Characterization in Cancer.

Alternative RNA Splicing in Cancer: Molecular Mechanisms, Functional Consequences, Biomarkers and Therapeutic Opportunities

Alternative pre-mRNA splicing is a central layer of gene regulation that enables a limited number of genes to generate a far larger and more context-dependent transcriptome and proteome. In cancer, splicing is disrupted by mutations in cis-regulatory sequences, recurrent lesions in spliceosome components, altered abundance or activity of RNA-binding proteins, and changes in transcription, chromatin, RNA modification, metabolism and stress signalling. These alterations are not merely by-products of malignant transformation. They can create oncogenic protein isoforms, eliminate tumour-suppressive products, remodel cellular identity, promote metastasis and drug resistance, and generate tumour-restricted peptides that are visible to the immune system. Large pan-cancer datasets, long-read sequencing, single-cell isoform profiling, proteogenomics and functional perturbation screens are now resolving this complexity at unprecedented scale. In parallel, multiple therapeutic strategies are advancing, including modulators of the SF3B complex, molecular glues that degrade RBM39, inhibitors of protein arginine methyltransferases and splicing kinases, splice-switching oligonucleotides, programmable RNA-targeting systems, and vaccines or T-cell receptors directed against splicing-derived neoantigens. This review integrates the molecular logic of splice-site selection with the cancer-specific mechanisms that perturb it, summarizes representative isoform switches across the hallmarks of cancer, evaluates emerging technologies and clinical biomarkers, and discusses the opportunities and constraints of translating splicing biology into precision oncology. Particular emphasis is placed on tumour specificity, intratumoural heterogeneity, proteomic validation, therapeutic windows and rational combination strategies.