mRNA Processing in Eukaryotes: Steps and Regulatory Roles

Eukaryotic mRNA processing converts primary RNA transcripts into mature messenger RNA capable of directing protein synthesis. This ensures genetic information is accurately expressed, influencing gene regulation and cellular function. Errors in mRNA processing can lead to diseases such as cancer and neurodegenerative disorders.

Key Steps In Eukaryotic mRNA Processing

Once a primary RNA transcript (pre-mRNA) is synthesized in the nucleus, it undergoes modifications to enhance stability, facilitate nuclear export, and ensure accurate protein production. The three major modifications—5′ capping, RNA splicing, and 3′ polyadenylation—play distinct roles in mRNA maturation.

5′ Capping

The first modification occurs co-transcriptionally when a 7-methylguanosine (m7G) cap is added to the 5′ end via a 5′-5′ triphosphate bond. This process involves three enzymatic steps: RNA triphosphatase removes the terminal phosphate, guanylyltransferase adds guanosine, and methyltransferase completes the reaction. The cap protects mRNA from degradation and facilitates recognition by the cap-binding complex (CBC), crucial for efficient ribosome recruitment. Defects in capping enzymes have been linked to neurological disorders by impairing mRNA stability and translation efficiency (Molecular Cell, 2020).

RNA Splicing

After capping, non-coding introns are removed through splicing, catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs) and associated proteins. Splicing occurs in steps: the 5′ splice site is cleaved, forming a lariat structure, followed by cleavage at the 3′ splice site and exon ligation. Errors in splicing can generate faulty transcripts, contributing to diseases such as spinal muscular atrophy (SMA) and cancers. Mutations in splicing factors like SF3B1 and U2AF1 disrupt exon recognition, leading to alternative splicing patterns that drive oncogenesis (Nature Communications, 2021).

3′ Polyadenylation

The final modification involves the addition of a poly(A) tail to the 3′ end. The pre-mRNA is cleaved at a conserved polyadenylation signal (AAUAAA), and adenine residues are added by poly(A) polymerase. Tail length, typically 50 to 250 nucleotides, influences mRNA stability and translational efficiency. Polyadenylation also aids nuclear export, ensuring mature mRNA reaches the cytoplasm. Disruptions in this process have been linked to neurodevelopmental syndromes and cancer. Mutations in the cleavage and polyadenylation specificity factor (CPSF) complex result in widespread defects in mRNA stability, affecting gene expression networks (Cell Reports, 2022).

Coordination Between Transcription And Processing

Eukaryotic mRNA synthesis is a coordinated process where transcription and processing occur simultaneously. The C-terminal domain (CTD) of RNA polymerase II (Pol II) recruits processing factors, ensuring efficient capping, splicing, and polyadenylation. The CTD contains heptapeptide repeats (YSPTSPS) that undergo phosphorylation at specific residues, guiding processing factor recruitment.

Early in transcription, Ser5 phosphorylation promotes capping machinery recruitment. As elongation progresses, Ser2 phosphorylation facilitates spliceosome assembly. Transcriptional kinetics influence splicing outcomes—slower elongation favors weak splice site recognition, promoting exon retention, while faster elongation can lead to exon skipping. Chromatin modifications, such as H3K36me3, further regulate co-transcriptional splicing by recruiting splicing regulators (Nature Structural & Molecular Biology, 2021).

Polyadenylation is closely linked to transcription termination, ensuring proper 3′ end formation before Pol II disengages. The cleavage and polyadenylation machinery interacts with Pol II and the CTD, positioning the poly(A) signal for precise cleavage. Defects in this coupling can cause premature termination or readthrough transcription, contributing to neurodevelopmental disorders. Mutations in the Pol II CTD phosphatase RPAP2 impair termination efficiency, leading to transcriptional dysregulation (Molecular Cell, 2022).

Regulatory Factors Influencing Splicing Patterns

Splicing is regulated by the sequence composition of pre-mRNA and the activity of RNA-binding proteins (RBPs). Strong splice sites promote constitutive splicing, while weaker sites require regulatory input. Exonic and intronic splicing enhancers (ESEs/ISEs) or silencers (ESSs/ISSs) recruit proteins that facilitate or inhibit spliceosome assembly.

RBPs such as serine/arginine-rich (SR) proteins enhance exon inclusion, whereas heterogeneous nuclear ribonucleoproteins (hnRNPs) often repress splicing. Their competitive interactions determine exon assembly in mature transcripts. For example, SRSF1 promotes exon inclusion, while hnRNP A1 antagonizes it. In myotonic dystrophy, sequestration of splicing regulators leads to widespread mis-splicing of muscle-related transcripts.

Extracellular signals also influence splicing by modifying RBP activity. Post-translational modifications such as phosphorylation alter binding affinities, allowing cells to adjust splicing patterns in response to environmental changes. Phosphorylation of SRSF1 by SRPK kinases enhances exon inclusion in apoptosis regulation, while stress pathways like MAPK signaling modify hnRNP activity to adapt gene expression under stress conditions.

Mechanisms Of Alternative Splicing

Alternative splicing generates transcriptomic diversity by allowing a single gene to produce multiple mRNA isoforms. Splice site strength, regulatory sequences, and splicing factors determine which exons are retained.

Exon skipping, the most common form in vertebrates, results in the omission of coding segments, altering protein function. Mutually exclusive exons ensure only one of two possible exons is incorporated, as seen in tropomyosin, which produces muscle-specific variants. Intron retention can lead to premature translation termination or altered RNA stability, while alternative 5′ or 3′ splice site usage adjusts exon boundaries, influencing protein interactions.

Functions Of mRNA Processing In Gene Regulation

mRNA processing controls gene expression by influencing stability, localization, and translation efficiency. The 5′ cap protects mRNA from degradation and facilitates ribosome binding. The poly(A) tail regulates transcript half-life—shorter tails reduce translation efficiency, while longer tails enhance mRNA longevity.

Splicing generates multiple protein isoforms from a single gene, expanding proteomic diversity. Alternative splicing can alter enzymatic activity, binding affinities, or subcellular localization. In neurons, alternative splicing of NEUREXIN influences synaptic connectivity and plasticity. Splicing can also regulate RNA stability by introducing premature stop codons that trigger nonsense-mediated decay (NMD), a quality control mechanism eliminating faulty transcripts. Misregulation of these processes contributes to diseases such as amyotrophic lateral sclerosis (ALS) and cancer.

RNA Editing Mechanisms

RNA editing introduces post-transcriptional modifications that alter coding sequences, splicing efficiency, or stability. The most well-characterized forms in humans are adenosine-to-inosine (A-to-I) and cytidine-to-uridine (C-to-U) conversions, mediated by ADAR and APOBEC enzymes, respectively. These modifications are abundant in the nervous system, influencing neurotransmission and synaptic plasticity.

A-to-I editing alters codon identity, affecting protein function. For example, editing of the GRIA2 transcript prevents calcium permeability in AMPA receptors, protecting neurons from excitotoxicity. Dysregulated ADAR activity has been linked to epilepsy and schizophrenia. C-to-U editing, though less common, plays a role in lipid metabolism, modifying the APOB transcript to generate functionally distinct proteins. Emerging research suggests RNA editing also influences immune regulation and cancer progression by affecting tumor growth and therapy response.