Non-coding RNA plays key roles in regulating various biological functions across different cell types and tissues, such as gene expression, maintaining chromatin structure, RNA processing, protein synthesis etc. Dysregulation of ncRNA has been linked to several diseases, including cancers, cardiovascular, neurodegenerative, and infectious diseases. ncRNAs play an important role as potential biomarkers and therapeutic targets for several disease diagnostics.

Over the past four decades, regulatory ncRNAs, particularly small RNAs and long non-coding RNAs (lncRNAs) have become recognized as key players in gene regulation². In recent years, while there have been significant advances in understanding pathways involving small RNAs, the mechanism of action of many lncRNAs remains still unclear. Despite the gap in understanding, key discoveries regarding biogenesis, functions, and the role of lncRNA in diseases have significantly advanced the field. The growing understanding of their functions will open new avenues for therapeutic interventions, especially in the context of gene regulation and disease treatment.

Types of non-coding RNA

ncRNAs are broadly categorized into three main classes: long non-coding RNAs, circular RNAs (circRNAs) and small non-coding RNAs, and play vital roles in gene expression regulation and have efficient involvement in epigenetic control³. They also help in triggering cellular processes like growth and differentiation. Here is an overview of the different types of ncRNAs:

Long non-coding RNA (lncRNA)

lncRNAs are defined as a non-coding transcript longer than 200 nucleotides transcribed primarily by RNA polymerase II⁴. They play an essential role in cell differentiation, development, and various physiological processes. Although they do not have protein-coding functions, they are often associated with other functions such as chromatin-remodeling, transcriptional regulation, epigenetic modification, RNA splicing and processing etc, linking their expression to the spatial regulation of gene expression and cellular organization.

Despite their rapid evolution and cell-type specificity, lncRNAs exhibit modular structures and functional repeats, posing challenges for their classification and study. Yet they hold great promises for understanding development, cell biology, and disease. They include several important classes:

Intergenic lncRNAs and intronic lncRNAs:

• Transcribed from regions of the genome located between protein-coding genes.
• Non-overlapping with any known protein-coding gene distinguishes them from other types of lncRNAs, such as antisense or intronic lncRNAs. Intronic lncRNAs are transcribed from the introns of the protein-coding genes.
• Both intergenic and intronic lncRNAs might be regulated by transcription activation mechanisms. It also may have different poly(A) modifications that help them to perform functions at different cellular locations.
• Involved in regulating the expression of nearby genes and can act in both cis (on the same chromosome) and trans (on different chromosomes) to influence gene activity.
• Regulates processes such as development, differentiation, and cellular responses to external signals, and their dysregulation has been implicated in various diseases, including cancers and neurodegenerative disorders⁵.

Sense and antisense lncRNAs

• Sense lncRNAs are transcribed from the sense strand of protein-coding genes and may overlap with or cover the entire sequence of these genes. Antisense lncRNAs, on the other hand, originate from the antisense strand and can overlap exons, reside in introns, or span entire sense genes.
• Both appear to exhibit mRNA-like features such as 3′ polyadenylation and 5′ capping.
• These lncRNAs can be multi-exonic in nature.
• While most lncRNAs lack protein-coding potential, some sense lncRNAs, like SRA and ENOD40, function both as RNA and protein-coding genes, challenging traditional gene classification⁵.
• Antisense lncRNAs regulate cancer cell behaviors by promoting or suppressing proliferation, migration, invasion, and chemo-radiosensitivity. They achieve this through epigenetic, transcriptional, post-transcriptional, and translational mechanisms6.

Promoter-associated RNAs (PATs):

• Transcribed from regions adjacent to gene promoters, often overlapping with them allowing them to interact directly with the transcription machinery.
• Influence the activity of nearby promoters, thereby enhancing or repressing transcription depending on their interactions with transcription factors and other regulatory proteins.
• Serve as scaffolds for the assembly of protein complexes involved in transcriptional regulation and chromatin remodeling.
• PATs can influence histone modifications by interacting with chromatin-modifying complexes and DNA methylation patterns at promoter regions, thereby affecting gene expression. For example, short RNAs of 50–200 nt in length originate from the promoters of PcG target genes in primary T cells and embryonic stem cells. Components of the PcG complex bind to stem loop structures of these RNAs and mediate transcriptional repression in cis.

Enhancer RNAs (eRNAs):

• Produced from enhancer regions, which are cis-regulatory elements that can be located far from the genes they regulate and can function independently of their orientation.
• Facilitates the formation of loops between enhancers and their target promoters bringing transcriptional machinery into proximity with the promoter, enhancing gene transcription.
• Interacts with various transcription factors and co-regulators, thereby enhancing or stabilizing the binding of these proteins to DNA.
• Enhancer RNAs (eRNAs), can bind directly to transcriptional co-activators like CBP/p300. This interaction enhances the acetyltransferase activity of these co-activators, leading to increased histone acetylation and promotion of an open chromatin state conducive to transcriptional activation⁷.
• Dysregulation of eRNA expression has been implicated in various diseases, including cancer and neurological disorders.

Circular RNAs (circRNAs)

• Formed through a process known as back-splicing, where a downstream splice donor is joined to an upstream splice acceptor.
• Regulated by specific RNA structures, reverse complementary sequences in flanking introns, and RNA-binding proteins (RBPs) that facilitate the back-splicing process.
• The unique structure of circRNAs provides them with enhanced stability and resistance to degradation by linear RNA decay machineries.
• For example, ciRS-7 is a circRNA in the brain that sequesters other molecules, such as miR-7, a microRNA involved in various cellular processes8. By sequestering miR-7, ciRS-7 modulates the expression of miR-7 target genes, influencing neuronal development and function.
• In cancer, circRNAs can modulate gene expression9. For example, circ-Foxo3 has been shown to inhibit cell proliferation and induce cell cycle arrest in breast cancer by forming a complex with p21 and CDK2, thereby suppressing their activity.

Small non-coding RNA (sncRNA)

sncRNAs are a class of non-coding RNA molecules that are typically less than 200 nucleotides10. They do not encode proteins but regulate gene expression at the transcriptional, post-transcriptional, and translational levels. They are also involved in gene silencing, RNA processing, chromatin remodeling, and maintaining genomic stability. Key subtypes include:

MicroRNA (miRNA)

• miRNAs are small non-coding RNAs (typically 18-24 nucleotides) that regulate gene expression post-transcriptionally¹⁰.  The canonical mechanism involves the seed region of the miRNA (nucleotides 2–8) binding to the complementary sequences in the 3' untranslated region (UTR) of target mRNAs, leading to either degradation of the mRNA or inhibition of its translation¹¹. This process occurs through the RNA-induced silencing complex (RISC), which incorporates the mature miRNA (guide strand) and directs the silencing of the target mRNA.
• Controls a wide array of biological functions, including development, apoptosis, immune response, and cell cycle control.
• Influences different pathways like glycolysis, lipid metabolism, and amino acid synthesis¹². In cancer cells, miRNAs such as miR-375 and miR-143 modulate metabolic processes by targeting key enzymes and transporters. Thus, it supports the altered metabolic states characteristic of tumors.
• Plays a significant role in cellular responses to stress, including DNA damage, oxidative stress, and nutrient deprivation. For example, p53, a tumor suppressor protein, regulates the expression of specific miRNAs upon DNA damage, promoting cell cycle arrest and apoptosis¹³.

Dysregulation of miRNAs has been implicated in neurodegenerative diseases (NDs) like amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Huntington’s disease (HD), and Parkinson’s disease (PD).

Small interfering RNA (siRNA)

• Small interfering RNAs (siRNAs), 20–25 nucleotides in length, induce gene silencing by forming RNA-induced silencing complexes (RISC) to cleave and degrade the targeted mRNA molecules, thereby inhibiting their translation and regulating gene expression post-transcriptionally¹⁴.
• siRNA acts as a powerful tool for gene silencing¹⁵. However, their unintended interactions with non-target mRNAs, known as off-target effects, can pose significant challenges. These effects primarily arise from partial complementarity between the siRNA and unintended mRNA targets, potentially causing unintended biological consequences.
• Introducing chemical modifications to siRNAs can enhance their stability and specificity. For example, 2'-O-methylation of the guide strand has been shown to decrease miRNA-like off-target effects without compromising gene silencing efficacy¹⁵.
• In cancer therapy, it inhibits overexpressed target mRNAs and can be combined with anticancer agents to overcome drug resistance.
• Regulates stem cell self-renewal through complex transcriptional networks and can be studied on a genome-wide scale using siRNA libraries.

Piwi-interacting RNA (piRNA)

• Piwi-interacting RNAs  (piRNAs) are small RNAs (approximately 24-31 nucleotides) enriched in germline tissues, where they interact with P-element-induced wimpy testis (PIWI) proteins to silence and prevent insertional activity of transposable elements (TEs) through DNA and histone modification, protecting genomic integrity¹⁰.
• piRNA can be categorized into primary piRNAs and secondary piRNAs based on their biogenesis pathways. These secondary piRNAs can target the primary piRNA precursor transcripts to generate more secondary piRNAs, resulting in a piRNA-specific "ping-pong" loop.
• Initially thought to be exclusive to germline cells, piRNAs have also been identified in somatic tissues, including the brain and blood, with emerging roles in neurodegenerative diseases such as Parkinson's and Alzheimer's.

Small nucleolar RNA (snoRNA): involvement in RNA modification

• snoRNAs are essential for the proper processing and modification of rRNA, ensuring the proper assembly and function of ribosomes¹⁰.
• They modify RNA by guiding methylation (C/D box snoRNAs) and pseudouridylation (H/ACA snoRNAs) and primarily target rRNA to enhance ribosome functionality.
• Their biogenesis involves the formation of snoRNP complexes through distinct assembly pathways and processing in the nucleolus, enabling their roles in rRNA modification, snRNA guidance, and regulation of alternative splicing.
• Dysregulation of specific snoRNAs has been linked to early Alzheimer’s pathology and autism spectrum disorder (ASD), highlighting their potential in early diagnosis and understanding of these conditions.

Small nuclear RNA (snRNA): essential role in RNA splicing

• snRNAs are uridine-rich RNA molecules essential for the spliceosome, which removes non-coding introns from pre-mRNA to produce functional mRNA¹⁰.

• The spliceosome consists of five snRNAs: U1, U2, U4, U5, and U6. Each one has a distinct role in the splicing process.

  • U1 snRNA recognizes and binds to the 5' splice site of pre-mRNA, initiating spliceosome assembly.
  • U2 snRNA binds to the branch point sequence of the intron, facilitating the formation of the lariat structure during splicing.
  • U4 snRNA associates with U6 snRNA to form the U4/U6 di-snRNP
  • U5 snRNA interacts with both exons, flanking the intron and aligning them for ligation.
  • U6 snRNA participates in the catalytic core of the spliceosome, facilitating the splicing reaction¹⁶.

• Dysregulation of snRNAs, particularly U1 snRNP, has been linked to neurodegenerative diseases like Alzheimer’s, where they contribute to RNA splicing defects and the accumulation of unspliced RNA, potentially driving disease pathology.

Housekeeping non-coding RNAs

• Housekeeping non-coding RNAs (ncRNAs), such as rRNAs, tRNAs, snRNAs, snoRNAs, and telomerase RNAs, have been extensively studied due to their essential roles in cell viability, including protein synthesis, RNA splicing, and RNA modifications¹⁷.
• They play essential, constitutive roles in the basic cellular functions required for maintaining cell viability and homeostasis.
• In addition to their core functions, some housekeeping ncRNAs, such as tRNA-derived RNA fragments (tRFs) and translation-interfering tRNAs (tiRNAs), have been identified as regulatory molecules involved in cellular stress responses by inhibiting translation.

Transfer RNA (tRNA)

• tRNAs are the most abundant type of small non-coding RNAs, playing a central role in translation10. Still, recent research highlights their involvement in broader regulatory functions through tRNA-derived fragments (tRFs) and tRNA-halves. Acts as an adapter molecule, helping translate the genetic information encoded in mRNA into functional proteins by carrying specific amino acids to the ribosome.
• tRNAs are transcribed as precursor molecules and undergo extensive post-transcriptional modifications before being processed into mature tRNAs, which then undergo cleavage to produce tRNA halves and tRFs that contribute to stress responses and gene regulation.
• tRFs, including those linked to neurodegenerative diseases like ALS and Parkinson's disease, are associated with cellular stress and dysfunction. For instance, mutations in the angiogenin CREB-binding protein / E1A-associated protein p300 (ANG) gene, which influences tRF production, have been linked to ALS¹⁸. These mutations may disrupt normal tRF functions, contributing to motor neuron degeneration observed in ALS patients.

Ribosomal RNA

• Ribosomal RNA (rRNA) is a vital housekeeping ncRNA that plays an essential role in protein synthesis within all living cells¹⁰.
• It is a significant component of ribosomes, where it facilitates the translation of messenger RNA (mRNA) into proteins by providing a scaffold for ribosomal proteins and catalyzing peptide bond formation during translation.
• The ribosomes are made from two subunits in both prokaryotes and eukaryotes. Bacterial ribosomes, such as those of Escherichia coli, consist of a small subunit (SSU) with 16S rRNA and 21 ribosomal proteins and a large subunit (LSU) containing 5S and 23S rRNAs along with 33 ribosomal proteins19. These subunits work together to facilitate protein synthesis.
• Eukaryotic ribosomes are larger and more complex than bacterial ribosomes, featuring expansion segments (ES) and additional ribosomal proteins. The 80S ribosome consists of more than 5,500 nucleotides of rRNA, including 18S rRNA in the small subunit (SSU) and 5S, 5.8S, and 25S rRNA in the large subunit (LSU). It also contains 80 ribosomal proteins (79 in yeast), contributing to its structural and functional complexity¹⁹.
• RNA polymerase I (Pol I) is primarily responsible for transcribing the large rRNA precursor, which includes the 28S, 18S, and 5.8S rRNAs in the nucleolus. However, the 5S rRNA gene is transcribed by RNA polymerase III (Pol III) outside the nucleolus²⁰.
• rRNA is constitutively expressed, highly conserved across species, and important for cellular viability, making it a fundamental housekeeping RNA necessary for basic cellular functions.

Biogenesis and processing of non-coding RNAs

Advancements in proteomics and translation technologies have revealed that some lncRNAs can encode small proteins and micropeptides²¹. These peptides are encoded by small or short open reading frames (sORFs) and can range in length from tens to over a hundred amino acids. There is growing evidence that peptides derived from lncRNAs have specific biological functions and can act as oncogenic drivers or tumor suppressors. They play important roles in various cancer-related processes, such as transcriptional regulation, post-transcriptional regulation, translation and post-translational regulation, signal transduction, and cancer metabolism.

They are transcribed by RNA polymerase II, undergoing similar processing as mRNAs, such as capping, splicing, and polyadenylation.

They are expressed in a tissue-specific manner, usually at low levels, exhibit low evolutionary conservation, and typically lack open reading frames. lncRNAs are gene expression modulators through diverse mechanisms, including chromatin regulation, transcription factor recruitment, chromatin folding, and influencing mRNA processing, translation, and degradation.

Transcription and initial processing

ncRNAs are transcribed from DNA but do not encode proteins, and their transcription follows a process involving RNA polymerase II (for miRNA and siRNA) or RNA polymerase III (for non-coding Y RNA)^22,23.

• The transcription begins with RNA polymerase binding to the gene's promoter, it involves two processing stages in the nucleus: Drosha (an RNase III enzyme and DGCR8 cleave the primary RNA transcript (pri-miRNA) into a precursor (pre-miRNA).
• Pri-miRNAs are often greater than 1 kb in length and can be embedded within other genes or transcribed independently from intergenic regions by RNA polymerase II.
• The pre-miRNA is then exported from the nucleus to the cytoplasm by exportin-5 protein for further processing by the Dicer.
• Dicer cleaves the pre-miRNA hairpin loop, producing a double-stranded RNA molecule that consists of the miRNA (the strand that is typically retained for function) and the passenger strand (which is usually degraded).
• TAR RNA-binding protein (TRBP) is often involved in facilitating the binding of Dicer to the pre-miRNA and the subsequent cleavage.
• The mature miRNA strand is then incorporated into the RISC, where the miRNA guides the complex to its target mRNA by base pairing with complementary sequences in the 3' untranslated region (3' UTR) of the mRNA. This interaction typically leads to either the degradation of the mRNA or inhibition of its translation, depending on the degree of complementarity between the miRNA and its target.

Post-transcriptional modifications

Post-transcriptional modifications are essential for ncRNAs to perform their specialized roles in gene regulation, protein synthesis, RNA processing, and various other cellular processes²⁴. Without these post-transcriptional modifications, ncRNAs would be unable to carry out their functions effectively, leading to defects in cellular function and potentially contributing to disease.

The post-transcriptional modifications that occur are 5' capping that protects RNA and aids in its export and processing, splicing that removes introns from snRNAs, lncRNAs, and other ncRNAs, polyadenylation that adds a poly-A tail to specific ncRNAs, enhancing stability and export, RNA editing, methylation and uridylation. In plants, non-coding RNAs like miRNAs and siRNAs, are key in gene regulation and stress responses, with their biogenesis and activity influenced by these modifications.
Structural studies have revealed that post-transcriptional modifications are important for the function of small ncRNAs, as they interact with specific proteins to form high molecular weight complexes.

Functions of non-coding RNA

Non-coding RNAs have a wide range of functions in regulating gene expression and cellular processes, with some acting locally and others at distance²⁴. These RNAs come in various forms, such as spliced, unspliced, and circular, and can have dual roles, including coding and regulatory functions, influencing both developmental biology and cell architecture. Despite the vast number of lncRNAs cataloged, many of their functions remain underexplored, and future research is needed to fully understand their roles in diverse biological contexts.

Gene regulation and expression modulation

Non-coding RNAs act as key regulators of gene expression across various biological processes, including development, stress responses, and disease²⁵. These regulatory ncRNAs modulate gene expression at the transcriptional and post-transcriptional levels. With increasing evidence of their roles in diverse cellular functions, ncRNAs are emerging as vital players in the regulation of vascular biology and other physiological pathways.

RNA interference (RNAi)

RNAi is a conserved biological process that responds to double-stranded RNA, silencing gene expression and providing resistance to both parasitic and pathogenic nucleic acids²⁶. This natural mechanism has vast potential for experimental biology, therapeutic interventions, agriculture, and other fields.

It is important for regulating gene expression in response to viral infections, transposons, and other parasitic nucleic acids. RNAi serves as a vital component of the host defense system against exogenous and endogenous 'parasitic' nucleic acids, such as viral genome replication intermediates²⁶. Thus, it protects the cell from viral infections. RNAi not only protects against viruses by degrading viral RNA but also plays a role in the regulation of gene expression through the action of microRNAs (miRNAs).
The adaptive nature of RNAi in response to various genetic threats highlights its evolutionary importance. It is a sequence-specific RNA degradation mechanism triggered by long double-stranded RNA (dsRNA), which RNase III Dicer processes into small interfering RNA (siRNA) duplexes²⁷. It can be extended in some organisms through an amplification loop involving RNA-dependent RNA polymerase, and its high specificity makes it a promising tool in molecular biology.

Competing endogenous RNA (ceRNA)

ceRNAs are both coding and non-coding that share common microRNA recognition elements (MREs) and compete for binding to the same microRNAs²⁸. This competition allows ceRNAs to relieve each other from microRNA repression, leading to an increase in each other's expression levels.

The discovery of ceRNAs has broadened our understanding of gene regulation, showing how different RNA molecules interact through microRNAs to create complex regulatory networks and emphasizing the importance of studying these interactions to fully understand gene expression and function.

Specialized functions of long non-coding RNAs

lncRNAs are involved in different cellular processes like the scaffolding of nuclear bodies or promoting signaling pathways organizing the nuclear architecture by facilitating the assembly of macromolecular complexes involved in gene regulation and chromatin remodeling²⁹.

Some lncRNAs function as molecular scaffolds, facilitating the binding of RNA-binding proteins (RBPs) or ribonucleoproteins to mRNA, thereby influencing mRNA stability and protein synthesis. This regulation can impact tumor progression and contribute to therapeutic resistance.

Xist (X-inactive specific transcript) is a lncRNA) of about 15,000 nucleotides that play an important role in mediating X chromosome inactivation (XCI) by coating the X chromosome from which it is expressed³⁰.

Xist's function is primarily in initiating XCI and contributing to the maintenance of silencing, causing structural changes in the chromosome, such as the formation of poorly structured megadomains. Xist RNA coats the chromosome and excludes transcriptional machinery factors like RNA Pol II. Gene silencing occurs via X chromosome inactivation, which is primarily regulated by the lncRNA XIST. This facilitates the recruitment of repressive complexes to silence one X chromosome.

Roles of non-coding RNA in biological processes

lncRNA plays a variety of roles in gene regulation, including gene imprinting, chromatin modification, and apoptosis, while circRNAs regulate miRNA functions, splicing, and protein translation³¹.

Additionally, tRNAs contribute to protein synthesis and gene expression regulation through their involvement in translation and interactions with RNA-binding proteins.

Other non-coding RNAs, such as snRNAs, snoRNAs, and piRNAs, are essential for RNA processing, ribosome maturation, and genome stability, with piRNAs also regulating gene transcription in germ and stem cells and ncRNAs influencing stem cell differentiation.

Maintenance of cellular structures

ncRNAs, especially transfer RNAs (tRNAs) play a central role in protein synthesis by translating mRNA codons into the appropriate amino acids during translation. Although they do not encode proteins themselves, their function is vital for cellular machinery.

Cell differentiation and development

Non-coding RNAs play essential roles in regulating gene expression during differentiation and development³². The diversity and abundance of lncRNAs increase in more complex organisms, highlighting their evolutionary significance in controlling developmental processes. These lncRNAs are involved in different functions like cell differentiation, dosage compensation, genomic imprinting, and organogenesis, especially in mammals.

lncRNAs also guide stem cell differentiation by modulating signaling pathways and gene expression profiles³³. For example, LincU stabilizes dual-specificity phosphatase 9 (DUSP9), inhibiting the MAPK/ERK pathway and maintaining pluripotency in mouse embryonic stem cells^34,35.

Brain activity and neuroplasticity

Sequencing results reveal that potentially 90% of the genome may be transcribed by non-coding RNAs. These ncRNAs, which correlate with organismal complexity, regulate processes like transcription, epigenetics, and gene silencing, making them central to understanding molecular evolution and brain function³⁶. They play an important role in regulating gene expression and epigenetic control elements that drive the complexity and cognitive development of the human brain.

They influence key aspects of neuroscience, such as chromatin modification, transcriptional regulation, alternative splicing, RNA editing, and translation, contributing to neuroplasticity and function in brain activity. They even contribute to the proliferation and migration of neural stem cells for generating new neurons throughout life³⁷.

For instance, the lncRNA Bdnf-AS regulates the expression of the brain-derived neurotrophic factor (BDNF) gene, which is essential for neuronal survival, differentiation, and plasticity³⁸.

Immune responses and signaling

The ncRNAs play essential roles in regulating gene expression in the immune system, particularly during inflammatory responses and immune cell activation³⁹. They are involved in key immune functions such as producing inflammatory molecules and regulating T cell development and differentiation. These lncRNAs are emerging as vital immune-related regulators with significant impacts on physiological and pathological processes, including autoimmune diseases.

• Cellular stress response: lncRNAs and other ncRNAs help cells respond to stress by regulating pathways involved in cell survival, apoptosis, and repair. For example, specific lncRNAs can modulate the stress response by interacting with proteins that control cell-cycle checkpoints or DNA damage repair mechanisms.
• Defense against viruses and transposons: Both siRNAs and piRNAs play essential roles in protecting the genome by silencing viral RNAs and transposons. This prevents the integration of viral DNA into the host genome or the mobilization of mobile genetic elements.

Non-coding RNA in disease

ncRNAs play important roles as regulators in various biological processes, with increasing evidence linking their dysregulation to a wide range of human diseases such as cancer, cardiovascular disorders, developmental disorders and neurological conditions⁴⁰. Recent discoveries are paving the way for the development of novel therapeutic strategies targeting ncRNAs.

Cancer

ncRNAs are a major regulator of cellular activities in cancer41. Each type of ncRNA has specific mechanisms of either promoting or suppressing tumors. Here are some of the examples.

miRNAs play key roles in cancer research.  Although some miRNAs are increased, most miRNAs are repressed in cancers relative to normal tissue counterparts. In agreement with these observations, the global depletion of miRNAs by knockdown of the miRNA processing machinery stimulated cell transformation and tumorigenesis in vivo. This suggests that miRNA expression changes are more than just the outcome of tumorigenesis but actively contribute to cancer growth. Despite the overall decline in miRNA expression in cancers, many miRNAs are up-regulated, some of which have oncogenic roles.

circRNAs can act as either tumor suppressors, such as circCDYL and circFOXO3, or oncogenes, such as circCCAC1 and circHIPK3, influencing cancer progression through mechanisms like miRNA sponging and signaling regulation.

lncRNAs, like HOTTIP, lnc-TCF7, and EPIC1, promote tumor growth, while tumor-suppressive lncRNAs like Pvt1b, DIRC3, and SATB2-AS1 inhibit cancer progression by modulating gene expression and transcriptional complexes.

piRNAs are expressed in the germline and regulate cancer progression by influencing processes such as cell proliferation, apoptosis, and transposable element repression.

Neurological disorders

• miRNAs and lncRNAs regulate neuronal differentiation, survival, and stress response and, therefore, play an important role in neurological disorders. In schizophrenia, lncRNAs are involved in alternative splicing and neuronal function, contributing to disease pathology.
• In autism spectrum disorder (ASD), specific lncRNAs are upregulated, disrupting neuronal structure and affecting neurodevelopment.
• In Alzheimer’s disease, lncRNAs contribute to the production of amyloid plaques and are involved in brain degeneration. Furthermore, brain-specific miRNA, miR-124 regulates neuronal differentiation and survival. In Alzheimer’s disease and Parkinson’s disease, miR-124 expression is altered, affecting neurogenesis and synaptic plasticity. In Alzheimer's disease, siRNAs are being developed to silence the amyloid precursor protein (APP) gene, reducing the production of amyloid-beta plaques, which are also associated with neurodegeneration.
• Huntington’s disease involves the dysregulation of lncRNAs that influence transcription and neuronal health.
• Parkinson’s disease is associated with altered lncRNA expression, leading to dopamine loss and synaptic dysfunction⁴².

Cardiovascular diseases

ncRNAs, particularly miRNAs and lncRNAs, are vital regulators in cardiovascular diseases, influencing processes like post-infarction injury, cardiac remodeling, and atherosclerosis⁴³.

• miRNAs can promote or inhibit cardiomyocyte death, regulate neovascularization, and control fibrosis. Therapies targeting specific miRNAs have shown promise in conditions like myocarditis and atherosclerosis.
• lncRNAs, such as those linked to cardiac hypertrophy, heart failure, and neointimal formation, play vital roles in regulating cardiac structure and function.
• Studies have also identified roles for ncRNAs in processes like endothelial recovery, cholesterol metabolism, and inflammatory responses, laying the foundation for novel treatment strategies.

Autoimmune diseases and diabetes

ncRNAs play a significant role in the pathogenesis of autoimmune diseases by regulating gene expression and impacting immune responses⁴⁴.

• In rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, and other autoimmune conditions, altered lncRNA expression contributes to inflammation, tissue damage, and disease progression. These molecules show potential as diagnostic biomarkers and therapeutic targets.
• Further, lncRNAs focus on the involvement in diabetes pathogenesis, influencing pancreatic β-cell differentiation and responding to hyperglycemia while contributing to complications through inflammation, fibrosis, and oxidative stress⁴⁵.
• Overexpression of miR-155 has been linked to several autoimmune diseases. miRNA plays a role in immune tolerance and is downregulated in diseases like lupus. Their expression is highly individual-specific, and genetic variations within lncRNA loci may help identify individuals at elevated risk for diabetes and its complications. Emerging technologies and therapeutic strategies targeting lncRNAs, including their use as non-invasive biomarkers, show potential for early detection and treatment of diabetic complications.

Non-coding RNAs as biomarkers

ncRNAs, once thought to be non-functional, are now recognized for their important role in cancer pathogenesis and their potential in biomarker discovery. Further, these regulatory RNAs are connected to neuropsychiatric and neurodegenerative diseases and hold potential as biomarkers and therapeutic targets⁴⁶.

Diagnostic potential

miRNAs are gaining attention as promising biomarkers for liquid biopsy due to their dysregulated expression in various cancers and neurodegenerative diseases, aided by advances in sequencing and RNA quantification technologies⁴⁷.

ncRNAs also show potential as biomarkers for cancer diagnosis⁴⁸. It also acts as an important marker for managing ischemic heart disease, offering insights into personalized treatments.

These biomarkers could improve early detection, monitor disease progression, and guide therapeutic interventions. Challenges remain in fully understanding and translating non-coding RNA biomarkers into clinical applications.

CRISPR and gene editing applications

The CRISPR-Cas9 system, a groundbreaking gene-editing technology, has opened new possibilities for targeting ncRNAs in cancer therapy⁴⁹. By editing ncRNAs such as miRNAs, long ncRNAs, and circRNAs, CRISPR/Cas9 has the potential to regulate cancer-related genes and pathways. Knockdown of specific genes of interest using CRISPR-Cas9 can help in understanding the role of ncRNA in cancer and other chronic diseases⁵⁰.

The ability to precisely edit ncRNAs using CRISPR/Cas9 could lead to more effective, personalized therapies by directly targeting the molecular mechanisms driving cancer progression and resistance. Despite challenges such as off-target effects, CRISPR/Cas9-mediated ncRNA editing holds promise for precision oncology and advancing cancer treatment.

For instance, research focusing on lncRNAs, like UCA1, is implicated in cancer progression⁵¹. Studies have demonstrated that CRISPR/Cas9-mediated inhibition of UCA1 can suppress malignant phenotypes in bladder cancer cells.

Methods for studying non-coding RNA

LncRNAs are vital to numerous biological processes, although a lot of gaps in understanding persist due to their complex nature52. Advances in high-throughput sequencing and bioinformatics have accelerated lncRNA research, focusing on novel lncRNA identification, functional characterization, and mechanistic studies. Experimental methods and computational approaches for these areas provide a comprehensive framework for genome-wide lncRNA research strategies.

Sequencing technologies

Advancements in RNA-sequencing technologies, from early Sanger sequencing to third- generation long-read methods, have greatly enhanced the ability to study complex transcriptomes and identify novel transcripts⁵².

Synteny analysis in mammals is a useful method for identifying orthologous lncRNAs by examining their conserved positions in the genome, even when sequence similarities are low⁵³.

This approach has revealed that many lncRNAs, such as natural antisense transcripts (NATs) are conserved not through the sequence but through transcriptional activity, which is often important for their function in regulating gene expression.

Emerging techniques, such as single-cell RNA sequencing, further refine transcriptome annotation with improved accuracy. These technologies, combined with extensive RNA-seq databases like gene expression omnibus (GEO) and sequence read archive (SRA), that have enabled large-scale genomic and transcriptomic projects, providing a strong foundation for the biological annotation of lncRNAs.

Bioinformatics tools

Mammalian genomes are extensively transcribed, producing numerous ncRNAs with functional roles, including lncRNAs that interact with nucleic acids and proteins.

Computational tools like FEELnc and CPAT excel at distinguishing coding from non-coding RNAs, while ASSA, RIblast, LASTAL, and Triplexator are the most effective for predicting RNA-RNA and RNA-DNA interactions54. FEELnc, an alignment-free tool using a random forest model, classifies lncRNAs with high accuracy based on features like multi-k-mer frequencies and relaxed open reading frames⁵⁵.

This is an outperforming tool for predicting lncRNA interactions on benchmark datasets. However, challenges remain in accurately predicting short RNA-RNA interactions, highlighting the need for continued improvement in bioinformatics tools for ncRNA analysis.

Functional studies

Mammalian cells produce thousands of large intergenic non-coding RNAs (lincRNAs), whose functional significance has often been debated. Chromatin-state mapping reveals ~1,600 highly conserved lincRNAs with diverse roles in processes such as stem cell pluripotency and cell proliferation, supported by functional validation for many lincRNAs⁵⁶.

Specific lincRNAs are regulated by key transcription factors like p53, NFκB, Sox2, Oct4, and Nanog, highlighting their importance in various biological processes. ncRNA-associated proteins like recombinant human TP73-AS1 protein can also be used in different immunoassays like ELISA and western blotting.

Current research and future directions

Since the discovery of various RNA species, ncRNAs have been recognized as key players in gene regulation, with small RNAs well-understood and lncRNAs remaining more diverse and yet to be discovered. Future directions in lncRNA research should focus on further elucidating the diverse and less understood mechanisms, drawing from the well-established knowledge of small RNA pathways.

Recent discoveries

Recent discoveries highlight the important role of lncRNAs in regulating gene expression and signaling pathways involved in the inflammatory response57. lncRNAs have been identified as key players in various inflammatory diseases, including cardiovascular disease, osteoarthritis, and asthma, influencing macrophage polarization and disease progression. While the understanding of lncRNAs is still developing, their potential as therapeutic targets for inflammatory diseases offers promising clinical applications.

Emerging techniques

Emerging techniques in ncRNA research have significantly advanced the understanding of their roles in biological systems.

• High-throughput sequencing, including RNA-seq and single-cell RNA-seq, enables the identification and quantification of ncRNAs^58.
• Specialized methods like ribonuclease R treatment enhance the detection of challenging classes like circRNAs⁵⁹.
• Structural analysis methods like cryogenic electron microscopy (cryo-EM) and selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), along with CRISPR-based tools, allow for precise manipulation and functional studies of ncRNAs^60,61.
• NMR spectroscopy is a valuable technique for studying the structural dynamics of ncRNAs due to their high flexibility and polymorphic nature⁶⁰.
• Additionally, techniques such as methylated RNA immunoprecipitation sequencing (MeRIP-Seq) for RNA modifications, live-cell imaging systems, and computational tools are integral to advancing ncRNA research and understanding their roles in health and disease⁶².

Integrating computational predictions with wet-lab validation is important for understanding how ncRNAs regulate genes, which influence drug efficacy and safety⁶³. Advancements in bioinformatics tools, databases, and experimental technologies are necessary to enhance the accuracy and efficiency of ncRNA target identification and characterization in toxicology research.

Challenges and opportunities

Advancements in high-throughput sequencing have enabled the exploration of ncRNA evolution and the prediction of novel ncRNAs across species, but significant challenges remain. These include difficulties in identifying species-conserved or species-specific ncRNAs and predicting their functional roles.

For circRNAs, challenges like low expression levels and the absence of poly(A) tails hinder detection and quantification, requiring improved sequencing methods and functional research techniques to fully understand their biological roles.

Other challenges in studying lncRNAs lie in identifying their interactions with effector proteins and determining their target specificities, as these processes involve complex RNA-protein assemblies and short nucleotide complementarity29.

Advanced techniques like individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), RNA antisense purification and mass spectrometry (RAP-MS), and a general repository for interaction datasets (GRID)-seq are helping to map these interactions and uncover the roles of lncRNAs in regulating chromatin, RNA, and cytoplasmic functions.

FAQs

What are the main functions of non-coding RNAs?

Non-coding RNAs (ncRNAs) play important roles in regulating gene expression at various levels, including transcriptional and post-transcriptional control. They can act as guides, scaffolds, or catalysts in cellular processes, such as RNA splicing, translation regulation, and chromatin remodeling. Some well-known examples include microRNAs, which aid in modulation of mRNA stability, and long non-coding RNAs, which can influence chromatin structure and gene silencing.

How do microRNAs and snoRNAs differ from other non-coding RNAs?

MicroRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) differ from other non-coding RNAs primarily in their specific functions and mechanisms of action. miRNAs regulate gene expression by binding to target mRNAs, leading to their degradation or translation repression, thereby controlling cellular processes like development and differentiation. In contrast, snoRNAs are involved in the modification and processing of other RNAs, such as rRNA, tRNA, and snRNA, and play a role in maintaining the integrity of the ribosome and other cellular machinery.

Are there any known diseases associated with non-coding RNA dysfunction?

Yes, dysfunctions in non-coding RNAs have been linked to a variety of diseases, including cancers, neurological disorders, and autoimmune and cardiovascular diseases. For example, mutations in microRNAs or their regulatory pathways can lead to uncontrolled cell growth, contributing to cancer development. Additionally, long non-coding RNAs have been implicated in diseases like Alzheimer's and Parkinson's, where their dysregulation affects gene expression and cellular function in the brain.

How are non-coding RNAs discovered and identified in genomes?

Non-coding RNAs (ncRNAs) are discovered and identified in genomes through a combination of computational and experimental approaches. Bioinformatics tools are used to predict potential ncRNA sequences by analyzing genomic regions that do not code for proteins but are conserved across species or exhibit structural motifs typical of ncRNAs. Experimental methods, such as high-throughput RNA sequencing (RNA-Seq), allow researchers to identify ncRNAs based on their transcriptional activity and expression patterns in different cell types or conditions.

References

1. Nemeth K, Bayraktar R, Ferracin M et al. Non-coding RNAs in disease: from mechanisms to therapeutics. Nature Reviews Genetics. 25, (2023).
2. Chen LL, Kim VN. Small and long non-coding RNAs: Past, present, and future. Cell. 187(23), (2024).
3. He X, Cai L, Tang H et al. Epigenetic modifications in radiation-induced non-targeted effects and their clinical significance. Biochimica et Biophysica Acta (BBA) - General Subjects. 1867, (2023).
4. Mattick JS, Amaral PP, Carninci P et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nature Reviews Molecular Cell Biology. 24, (2023).
5. Ma L, Bajic VB, Zhang Z. On the classification of long non-coding RNAs. RNA Biology. 10(6), (2013).
6. Liu B, Xiang W, Liu J et al. The regulatory role of antisense lncRNAs in cancer. Cancer Cell International. 21(1), (2021).
7. Bose et al. RNA Binding to CBP Stimulates Histone Acetylation and Transcription. Cell. 2017;168(1-2):135-149.e22. doi:10.1016/j.cell.2016.12.020
8. Hansen TB, Jensen TI, Clausen BH et al. Natural RNA circles function as efficient microRNA sponges. Nature. 495, (2013).
9. Zhu LP, He YJ, Hou JC et al. The role of circRNAs in cancers. Bioscience Reports. 37, (2017).
10. Watson CN, Belli A, Di Pietro V. Small Non-coding RNAs: New Class of Biomarkers and Potential Therapeutic Targets in Neurodegenerative Disease. Frontiers in Genetics. 10, (2019).
11. Chipman LB, Pasquinelli AE. miRNA Targeting: Growing beyond the Seed. Trends in Genetics. 35(3), (2019).
12. Chen B, Li H, Zeng X et al. Roles of microRNA on cancer cell metabolism. Journal of Translational Medicine. 10, (2012).
13. Leung AKL, Sharp PA. MicroRNA Functions in Stress Responses. Molecular Cell. 40(2), (2010).
14. Jin JO, Kim G, Hwang J et al. Nucleic acid nanotechnology for cancer treatment. Biochimica Et Biophysica Acta - Reviews On Cancer. 1874(1), (2020).
15. Neumeier J, Meister G. siRNA Specificity: RNAi Mechanisms and Strategies to Reduce Off-Target Effects. Frontiers in Plant Science. 11, (2021).
16. Will CL, Luhrmann R. Spliceosome Structure and Function. Cold Spring Harbor Perspectives in Biology. 3(7), (2010).
17. Poliseno L, Lanza M, Pandolfi PP et al. Coding, or non-coding, that is the question. Cell Research (2024).
18. Mathew BA, Katta M, Ludhiadch A, et al. Role of tRNA-Derived Fragments in Neurological Disorders: a Review. Molecular Neurobiology 60(2), (2022).
19. Wilson DN, Cate JH. The Structure and Function of the Eukaryotic Ribosome. Cold Spring Harbor Perspectives in Biology 4(5) (2012).
20. Cooper GM. Eukaryotic RNA Polymerases and General Transcription Factors. The Cell: A Molecular Approach (2nd edition). (2000).
21. Zhang, Y. LncRNA-encoded peptides in cancer. J Hematol Oncol 17, 66 (2024).
22. Dykes IM, Emanueli C. Transcriptional and Post-transcriptional Gene Regulation by Long Non-coding RNA. Genomics, Proteomics & Bioinformatics 15(3) (2017).
23. Billmeier M, Green D, Hall AE, et al. Mechanistic insights into non-coding Y RNA processing. RNA Biology 19(1), (2022).
24. Treiber T, Treiber N, Meister G. Regulation of microRNA biogenesis and its crosstalk with other cellular pathways. Nature Reviews Molecular Cell Biology 20(1) (2018).
25. Kaikkonen MU, Lam MTY, Glass CK. Non-coding RNAs as regulators of gene expression and epigenetics. Cardiovascular Research 90(3) (2011).
26. Zhou R, Rana TM. RNA-based mechanisms regulating host-virus interactions. Immunological Reviews 253(1) (2013).
27. Svoboda P. Key Mechanistic Principles and Considerations Concerning RNA Interference. Frontiers in Plant Science 11 (2020).
28. Poliseno L, Pandolfi PP. PTEN ceRNA networks in human cancer. Methods 77-78 (2015).
29. Mattick JS, Amaral PP, Carninci P, et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nature Reviews Molecular Cell Biology 24, (2023).
30. Cable J, Heard E, Hirose T, et al. Noncoding RNAs: biology and applications-a Keystone Symposia report. Annals of the New York Academy of Sciences 1506(1), (2021).
31. Chen X, Xie W, Zhang M, et al. The Emerging Role of Non-Coding RNAs in Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells. Frontiers in Cell and Developmental Biology 10, (2022).
32. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development. Nature Reviews Genetics 15(1) (2013).
33. Chen J, Wang Y, Wang C, et al. LncRNA functions as a new emerging epigenetic factor in determining the fate of stem cells. Frontiers in Genetics, 11, (2020).
34. Jiapaer Z, et al. LincU Preserves Naïve Pluripotency by Restricting ERK Activity in Embryonic Stem Cells. Stem Cell Reports. 11(2),395-409(2018).
35. LincU Preserves Naïve Pluripotency by Restricting ERK Activity in Embryonic Stem Cells (2018).
36. Spadaro PA, Bredy TW. Emerging role of non-coding RNA in neural plasticity, cognitive function, and neuropsychiatric disorders. Frontiers in Genetics 3 (2012).
37. Zhao Y, Liu H, Zhang Q, et al. The functions of long non-coding RNAs in neural stem cell proliferation and differentiation. Cell & Bioscience 10(1), (2020).
38. Bohnsack JP, Teppen T, Kyzar EJ, et al. The lncRNA BDNF-AS is an epigenetic regulator in the human amygdala in early onset alcohol use disorders. Translational Psychiatry, 9, (2019).
39. Ghahramani Almanghadim H, Karimi B, Valizadeh S, Ghaedi K. Biological functions and affected signaling pathways by Long Non-Coding RNAs in the immune system. Non-coding RNA Research 10 (2025).
40. Lekka E, Hall J. Noncoding RNAs in disease. FEBS Letters 592, (2018).
41. Yan H, Bu P. Non-coding RNA in cancer. Essays in Biochemistry 65(4), (2021).
42. Li L, Zhuang Y, Zhao X, et al. Long Non-coding RNA in Neuronal Development and Neurological Disorders. Frontiers in Genetics 9, (2019).
43. Poller W, Dimmeler S, Heymans S, et al. Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives. European Heart Journal 39(29), (2017).
44. Zou Y, Xu H. Involvement of long noncoding RNAs in the pathogenesis of autoimmune diseases. Journal of Translational Autoimmunity 3, 100044 (2020).
45. Leung A, Natarajan R. Long Noncoding RNAs in Diabetes and Diabetic Complications. Antioxidants & Redox Signaling 29(11), (2018).
46. Guennewig B, Cooper AA. The Central Role of Noncoding RNA in the Brain. International Review of Neurobiology 153, 53–94 (2014).
47. Toden S, Goel A. Non-coding RNAs as liquid biopsy biomarkers in cancer. British Journal of Cancer 126(3), 351–360 (2022).
48. Caporali A, Anwar M, Devaux Y, et al. Non-coding RNAs as therapeutic targets and biomarkers in ischaemic heart disease. Nature Reviews Cardiology 21(8), 556–573 (2024).
49. Yang J, Meng X, Pan J, et al. CRISPR/Cas9-mediated noncoding RNA editing in human cancers. RNA Biology 15(1), 35–43 (2017).
50. da Silva Santos R, Pinheiro DP, Teixeira LPR, et al. CRISPR/Cas9 small promoter deletion in H19 lncRNA is associated with altered cell morphology and proliferation. Scientific Reports 11(1), (2021).
51. Ghafouri-Fard, S., Taheri, M. UCA1 long non-coding RNA: An update on its roles in malignant behavior of cancers. Biomedicine & Pharmacotherapy. 120, (2019).
52. Tao S, Hou Y, Diao L, et al. Long noncoding RNA study: Genome-wide approaches. Genes & Diseases 10(6), (2023).
53. Szcześniak MW, Kubiak MR, Wanowska E, Makałowska I. Comparative genomics in the search for conserved long noncoding RNAs. Essays in Biochemistry 65(4), (2021).
54. Antonov I, Mazurov E, Borodovsky M, et al. Prediction of lncRNAs and their interactions with nucleic acids: benchmarking bioinformatics tools. Briefings in Bioinformatics, 20(2), (2018).
55. Wucher V, Legeai F, Hédan B, et al. FEELnc: a tool for long non-coding RNA annotation and its application to the dog transcriptome. Nucleic Acids Research, 45(8), (2017).
56. Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature, 458(7235), (2009).
57. Zhang Y, Liu H, Niu M, et al. Roles of long noncoding RNAs in human inflammatory diseases. Cell Death Discovery, 10(1), (2024).
58. D’Agostino N, Li W, Wang D. High-throughput transcriptomics. Scientific Reports, 12(1), (2022).
59. Wang Y, Wang J, Gruninger RJ, et al. Assessment of different enrichment methods revealed the optimal approach to identify bovine circRnas. RNA Biology, 21(1), (2024).
60. Rocca R, Grillone K, Citriniti EL, et al. Targeting non-coding RNAs: Perspectives and challenges of in-silico approaches. European Journal of Medicinal Chemistry, 261, (2023).
61. Martin S, Blankenship C, Rausch JW, et al. Using SHAPE-MaP to probe small molecule-RNA interactions. Methods, (2019).
62. Rong D, Sun G, Wu F, et al. Epigenetics: Roles and therapeutic implications of non-coding RNA modifications in human cancers. Molecular Therapy - Nucleic Acids, 25, (2021).
63. Li D, Chen M, Hong H, et al. Integrative approaches for studying the role of noncoding RNAs in influencing drug efficacy and toxicity. Expert Opinion on Drug Metabolism & Toxicology, 18(2), (2022).