RNA Biology: Non-Coding RNAs and RNA Processing

Introduction to RNA Biology

For decades RNA was seen primarily as an intermediary between DNA blueprints and protein products. The discovery that most of the human genome is transcribed yet only 2% encodes proteins revealed a vast landscape of non-coding RNA (ncRNA) species with diverse regulatory functions. ncRNAs including microRNAs, long non-coding RNAs, circular RNAs, PIWI-interacting RNAs, and small nucleolar RNAs regulate gene expression at transcriptional, post-transcriptional, and translational levels, playing fundamental roles in development, cellular identity, and disease.

RNA biology also encompasses the intricate processing of pre-mRNA into mature mRNA: 5' capping, 3' polyadenylation, splicing of introns, and RNA editing. Alternative splicing—selection of different exon combinations from a single pre-mRNA—dramatically expands proteome diversity: approximately 95% of human genes produce multiple splice isoforms. Post-transcriptional modification of RNA nucleotides (RNA methylation, pseudouridylation) adds further regulatory layers—the epitranscriptome—with roles in translation efficiency and RNA stability.

Small Non-Coding RNAs

MicroRNAs

MicroRNAs (miRNAs) are ~22-nucleotide RNAs processed from hairpin precursors by Drosha and Dicer ribonucleases. miRNAs are incorporated into RISC (RNA-induced silencing complex) containing Argonaute proteins, which bind complementary sequences mainly in the 3'UTR of target mRNAs, causing mRNA destabilisation and translational repression. Each miRNA can target hundreds of mRNAs; the human genome encodes over 2000 miRNAs collectively targeting most protein-coding genes. miR-21 is the most commonly upregulated miRNA in cancers; miR-122 is essential for HCV replication—miravirsen (anti-miR-122 locked nucleic acid) demonstrated suppression of HCV viraemia in clinical trials.

PIWI-Interacting RNAs

PIWI-interacting RNAs (piRNAs) are 26-32 nucleotide RNAs expressed in germline cells. They associate with PIWI-clade Argonaute proteins (MILI, MIWI in mice) and function primarily to silence transposable elements—genomic parasites that could otherwise cause insertional mutations and genome instability in the germline. piRNAs guide PIWI proteins to complementary transposon transcripts for cleavage and also establish heritable DNA methylation silencing. Disruption of piRNA biogenesis causes transposon derepression and male infertility in mice. piRNA pathways represent a sophisticated germline genome defence system evolved to protect genome integrity across generations.

Long Non-Coding RNAs

lncRNA Functions in Gene Regulation

Long non-coding RNAs (lncRNAs) are generally defined as ncRNAs of 200+ nucleotides with no protein-coding potential. The human genome encodes tens of thousands of lncRNAs; most have low expression, high tissue specificity, and evolutionary conservation of function despite modest sequence conservation. lncRNAs regulate gene expression through diverse mechanisms: XIST coats the inactive X chromosome recruiting Polycomb repressive complexes for silencing; HOTAIR recruits PRC2 to repress homeobox gene loci; NEAT1 forms nuclear paraspeckles; enhancer RNAs are transcribed from active enhancers and may facilitate enhancer-promoter looping. lncRNA dysregulation is pervasive in cancer with both oncogenic and tumour-suppressive roles.

Circular RNAs

Circular RNAs (circRNAs) arise from non-canonical back-splicing events generating covalently closed circular RNA molecules resistant to exonucleases. CDR1as (ciRS-7) contains over 70 miR-7 sponge sites, potentially sequestering miR-7 from its targets in neurons. Other circRNAs regulate splicing by competing for splicing factors, translate into proteins using internal ribosome entry sequences, or simply serve as stable molecular sponges. The CDR1as circRNA is highly expressed in the brain; its disruption impairs midbrain development in mice. Circular RNA abundance in exosomes makes them attractive as biomarkers—their stability in biofluids outlasts linear RNAs.

Alternative Splicing

Alternative splicing estimates suggest more than 90,000 distinct splice isoforms are produced from human genes—vastly amplifying protein diversity from ~20,000 genes. SR proteins promote exon inclusion; hnRNPs often promote skipping—their relative concentrations in a cell type determine the splicing outcome. Tissue-specific splicing (neurexin alternative exons generating thousands of isoforms for neuronal circuit wiring) and developmentally regulated splicing (MBNL1/CUGBP regulation of cardiac and skeletal muscle exons) highlight how alternative splicing drives phenotypic complexity. Aberrant splicing occurs in cancer (splice site mutations, splicing factor mutations in myeloid malignancies) and neurological disease.

Examples and Applications

Example 1: RNA Interference Therapeutics

siRNA (small interfering RNA) of 21-23 nucleotides with perfect complementarity to target mRNA directs RISC-mediated cleavage and degradation—RNA interference with high specificity and potency. The challenge of siRNA delivery to target tissues has been addressed by lipid nanoparticles (patisiran, FDA-approved 2018 for hereditary transthyretin amyloidosis), GalNAc conjugates for hepatocyte targeting (inclisiran reducing LDL-cholesterol by silencing PCSK9 mRNA), and antibody conjugates. siRNA drugs for rare hereditary liver diseases, hypercholesterolaemia, and haematological conditions have transformed treatment options.

Example 2: Antisense Oligonucleotides in Disease

Antisense oligonucleotides (ASOs) bind complementary target RNA through Watson-Crick base pairing, redirecting splicing or inducing RNase H-mediated degradation. Nusinersen (Spinraza) corrects SMN2 splicing to produce functional SMN protein in spinal muscular atrophy, transforming a uniformly fatal disease into one where many children sit and walk. Eteplirsen skips exon 51 of dystrophin pre-mRNA in Duchenne muscular dystrophy restoring a truncated but functional protein. Inotersen silences TTR mRNA in transthyretin amyloidosis. ASO drugs demonstrate how understanding RNA splicing and processing directly enables curative or disease-modifying therapies.

Example 3: m6A RNA Methylation

N6-methyladenosine (m6A) is the most abundant internal mRNA modification, added by the METTL3/14 writer complex, removed by ALKBH5/FTO eraser enzymes, and recognised by YTH domain reader proteins affecting mRNA stability, translation, splicing, and export. m6A marks enriched near stop codons and 3'UTRs affect mRNA decay; YTHDF2 reader promotes decay of marked transcripts. m6A is critical for development (Mettl3 knockout is embryonic lethal), haematopoiesis, and circadian rhythm. Dysregulation of m6A machinery occurs in multiple cancer types—METTL3 amplification promotes translation of oncogene mRNAs in AML. The epitranscriptome represents a new layer of gene regulation with therapeutic opportunities.

Example 4: Splicing Factor Mutations in Myeloid Cancers

Recurrent somatic mutations in core splicing factors—SF3B1, SRSF2, U2AF1, ZRSR2—occur in 50-60% of myelodysplastic syndromes (MDS) and 5-10% of AML. SF3B1 mutations cause aberrant 3' splice site selection producing alternative transcripts with premature stop codons or altered protein function from hundreds of genes. Understanding how these mutations cause global splicing dysregulation leading to haematopoietic failure guides development of splicing modulators (H3B-8800) as therapeutic candidates. Splicing factor mutations are essentially always heterozygous, suggesting that full disruption of splicing is lethal, constraining therapeutic targeting.

Example 5: miRNA in Cardiovascular Disease

miR-208a is encoded in the intron of alpha-MHC and upregulated in cardiac hypertrophy; it targets thyroid hormone receptor beta (an anti-hypertrophic gene). Anti-miR-208a reduced pathological cardiac hypertrophy in rodent models. miR-122 constitutes 70% of liver miRNA; its loss disorders hepatic lipid metabolism. Circulating miRNAs (miR-21, miR-499) are elevated in plasma after myocardial infarction as biomarkers. miRNA-based therapies (miRNA mimics and inhibitors) offer tissue-targeted approaches; challenges include delivery specificity, off-target effects, and the numerous targets each miRNA regulates simultaneously.

Example 6: XIST and X Chromosome Inactivation

In female mammals, one X chromosome is epigenetically inactivated early in development to achieve dosage compensation with males. XIST lncRNA (~17 kb) is transcribed exclusively from the inactive X and coats it in cis, recruiting Polycomb repressive complexes (PRC2 depositing H3K27me3), HDAC complexes, and other silencing factors that propagate heterochromatic silencing across the entire chromosome. Reactivation of XIST in iPSC derivation often causes loss of X inactivation—a technical challenge for disease modelling. Targeting XIST to specifically silence autosomal genes for gene therapy applications (chromosome therapy) is being explored for Down syndrome and X-linked diseases.

Example 7: RNA Vaccines

Successful mRNA COVID-19 vaccines validated RNA as a therapeutic modality. mRNA encoding the antigen is delivered in lipid nanoparticles; cells translate the mRNA producing antigen that stimulates immune response without genomic integration. Modified nucleosides (Pseudouridine, N1-methyl-pseudouridine) reduce innate immune activation while improving translation efficiency—key innovations enabling effective mRNA vaccines. The platform is highly flexible; candidate mRNA vaccines against influenza (multivalent, cross-protective), RSV, HIV, and personal neoantigen cancer vaccines (with Moderna and BioNTech) are in clinical development.

Example 8: Riboswitches in Bacteria

Riboswitches are structured RNA elements in bacterial mRNA 5'UTRs that bind small molecules directly, changing secondary structure to regulate gene expression without protein co-factors—RNA acting both as sensor and regulatory switch. TPP (thiamine pyrophosphate) riboswitches regulate genes of thiamine biosynthesis; SAM riboswitches regulate methionine-related genes; purine riboswitches regulate nucleotide metabolism. Riboswitches provided the first evidence for the RNA world hypothesis—that ancient RNA molecules performing both catalytic and regulatory functions preceded protein-dependent gene regulation. Riboswitches are promising antibiotic targets, with compounds binding bacterial riboswitches lacking human homologues entering early development.

Try it live

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