mRNA Processing: Capping Splicing and Polyadenylation - Biochemistry

SECTION 1

Historical Context & Motivation

The discovery that eukaryotic genes are not simply collinear with their mRNA products ranks among the most surprising findings in molecular biology. For decades after the elucidation of the central dogma — DNA → RNA → protein — researchers assumed that transcription yielded a message ready for immediate translation, much as occurs in prokaryotes. The realization that eukaryotic pre-mRNA undergoes extensive co-transcriptional and post-transcriptional processing fundamentally reshaped our understanding of gene expression. Three principal modifications — 5′ capping, splicing, and 3′ polyadenylation — convert the primary transcript into a mature mRNA competent for nuclear export, ribosomal recognition, and regulated stability.

1975 • Discovery of the 5′ Cap

Furuichi, Shatkin, and colleagues identified the 7-methylguanosine (m⁷G) cap structure on eukaryotic mRNAs, revealing an unusual 5′–5′ triphosphate linkage distinct from standard 3′–5′ phosphodiester bonds.

1977 • Split Genes and Introns Discovered

Richard Roberts and Phillip Sharp independently demonstrated that adenovirus mRNA is encoded by non-contiguous DNA segments, revealing the existence of introns and exons. They received the 1993 Nobel Prize in Physiology or Medicine for this work.

1981 • Self-Splicing Introns

Thomas Cech discovered that Tetrahymena rRNA precursors could excise their own intron in the absence of protein, establishing catalytic RNA (ribozymes) and earning the 1989 Nobel Prize in Chemistry (shared with Sidney Altman).

1991–2001 • Spliceosome Architecture Elucidated

Biochemical and structural studies revealed the small nuclear ribonucleoprotein (snRNP) components of the spliceosome, establishing that the catalytic heart of splicing is RNA-based — a massive molecular machine rivaling the ribosome in complexity.

2015–Present • Cryo-EM Revolution

Near-atomic resolution cryo-electron microscopy structures of the spliceosome in multiple catalytic states have provided unprecedented mechanistic insight, confirming two-metal-ion catalysis and revealing conformational dynamics during intron removal.

These discoveries prompted a fundamental question that continues to drive research in gene expression: how do eukaryotic cells coordinate capping, splicing, and polyadenylation with ongoing transcription by RNA Polymerase II (Pol II) to ensure that every pre-mRNA is correctly processed before export to the cytoplasm? The answer, as we shall see, lies in an elegant coupling mechanism centered on the C-terminal domain (CTD) of Pol II, which serves as a dynamic landing pad for processing factors throughout the transcription cycle.

SECTION 2

Core Principles of mRNA Processing

Eukaryotic mRNA processing encompasses three chemically distinct yet temporally coordinated modifications that convert the primary transcript (pre-mRNA) into a mature messenger. Each modification serves multiple biological functions — from protecting the transcript against exonuclease degradation to directing efficient ribosome recruitment. Crucially, these events are not isolated; they are co-transcriptionally coupled through the phosphorylation state of the Pol II CTD, ensuring fidelity and efficiency in gene expression.

5′ Capping

Addition of a 7-methylguanosine (m⁷G) to the 5′ end via an unusual 5′–5′ triphosphate linkage. Occurs after ~20–30 nucleotides have been transcribed. Protects against 5′ exonucleases, facilitates ribosome binding via eIF4E, and promotes nuclear export.

Splicing

Removal of introns (non-coding intervening sequences) and ligation of exons (expressed sequences) by the spliceosome. Catalyzed by snRNAs via two transesterification reactions. Enables alternative splicing for proteome diversification.

3′ Polyadenylation

Endonucleolytic cleavage of the pre-mRNA downstream of the AAUAAA polyadenylation signal, followed by the addition of ~200 adenine residues by poly(A) polymerase (PAP). Enhances mRNA stability, nuclear export, and translational efficiency.

CTD Coordination

The heptad repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) of the Pol II C-terminal domain is differentially phosphorylated during transcription: Ser5-P recruits capping enzymes at initiation, while Ser2-P recruits splicing and polyadenylation factors during elongation and termination.

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KEY TAKEAWAY

Think of pre-mRNA processing as an editorial pipeline in a publishing house. The 5′ cap is like the cover page added to protect the manuscript. Splicing is the editor cutting out rough-draft sections (introns) and seamlessly joining the polished chapters (exons). The poly(A) tail is a binding that holds the final pages together and signals that the book is complete for shipping (export). All of this editing happens while the author (Pol II) is still writing, coordinated by a project manager (the CTD) whose changing marks (phosphorylation states) tell each editor exactly when to step in.

SECTION 3

Visual Overview of mRNA Processing

Overview of the three major mRNA processing steps. The primary transcript (top) contains exons (purple) separated by introns (dashed pink). Step 1 adds the m⁷G cap (cyan circle). Step 2 removes introns as lariat structures, joining the exons. Step 3 cleaves the 3′ end and appends the poly(A) tail. The mature mRNA (bottom, green border) is then export-competent.

The diagram above captures the ordered logic of mRNA maturation. Notice that each step modifies a different region of the transcript. The 5′ cap is attached co-transcriptionally, even before the first intron is fully synthesized. Splicing occurs predominantly co-transcriptionally as well, proceeding in a 5′-to-3′ direction as Pol II elongates through successive introns. Polyadenylation is the final processing event, triggered when Pol II transcribes past the polyadenylation signal near the 3′ end of the gene. These spatial and temporal relationships ensure that processing factors encounter their substrates in the correct order, minimizing errors and maximizing efficiency.

SECTION 4

Molecular Mechanisms of Each Processing Step

5′ Capping: Chemistry and Enzymology

The 5′ cap is installed in three sequential enzymatic reactions shortly after the nascent transcript emerges from the RNA exit channel of Pol II. The RNA triphosphatase first hydrolyzes the γ-phosphate from the 5′ triphosphate end of the pre-mRNA, converting it to a diphosphate. Next, guanylyltransferase transfers a GMP from GTP to the 5′ diphosphate via an unusual 5′–5′ triphosphate bond, forming GpppN. Finally, methyltransferase (using S-adenosylmethionine as the methyl donor) methylates the N-7 position of the guanine to produce m⁷GpppN — the cap 0 structure. Additional methylations at the 2′-O positions of the first and second nucleotides generate cap 1 and cap 2 structures, respectively, which are characteristic of higher eukaryotes.

CAPPING REACTION SUMMARY

pppN-RNA → ppN-RNA → GpppN-RNA → m⁷GpppN-RNA

Step 1: RNA triphosphatase removes γ-phosphate. Step 2: Guanylyltransferase adds GMP via 5′–5′ linkage. Step 3: Methyltransferase adds –CH₃ at N-7 of guanine using SAM as donor.

Splicing: The Two-Step Transesterification Mechanism

Intron removal is catalyzed by the spliceosome, a large ribonucleoprotein complex comprising five small nuclear RNAs (U1, U2, U4, U5, U6) and over 100 associated proteins. The spliceosome recognizes conserved sequence elements at each intron: the 5′ splice site (GU), the 3′ splice site (AG), the branch point adenosine (typically 18–40 nucleotides upstream of the 3′ splice site), and the polypyrimidine tract. The catalytic chemistry proceeds through two sequential transesterification reactions — SN2-type nucleophilic substitutions — that exchange phosphodiester bonds without the net input of energy.

FIRST TRANSESTERIFICATION

Branch-point A (2′-OH) attacks 5′ splice-site phosphodiester → Free exon 1 (3′-OH) + Lariat intermediate

The 2′-hydroxyl of the conserved branch-point adenosine performs a nucleophilic attack on the phosphorus at the 5′ splice site, severing exon 1 from the intron and forming a 2′–5′ phosphodiester bond (lariat structure).

SECOND TRANSESTERIFICATION

Free exon 1 (3′-OH) attacks 3′ splice-site phosphodiester → Ligated exons + Released lariat intron

The free 3′-OH of exon 1 attacks the phosphodiester bond at the 3′ splice site, joining exon 1 to exon 2 and releasing the intron in lariat form. The lariat is subsequently debranched and degraded.

3′ Polyadenylation: Cleavage and Tail Addition

Polyadenylation requires a multi-subunit machinery that includes CPSF (Cleavage and Polyadenylation Specificity Factor), which recognizes the AAUAAA hexamer, and CstF (Cleavage Stimulation Factor), which binds a GU-rich or U-rich downstream sequence element. Together with cleavage factors CF I and CF II, these proteins direct endonucleolytic cleavage of the pre-mRNA 10–30 nucleotides downstream of AAUAAA. Poly(A) polymerase (PAP) then processively adds approximately 200 adenylate residues to the new 3′ end, using ATP as a substrate. The growing tail is immediately bound by poly(A)-binding protein (PABPN1) in the nucleus, which stimulates processive elongation and determines the final tail length.

POLYADENYLATION REACTION

RNA-3′OH + n ATP → RNA-(AMP)ₙ + n PPᵢ

PAP catalyzes the template-independent addition of AMP residues (n ≈ 200 in mammals), releasing pyrophosphate (PP_i) with each addition. The reaction is driven forward by subsequent hydrolysis of PP_i by pyrophosphatase.

SECTION 5

Spliceosome Assembly & Alternative Splicing

The spliceosome assembles in a stepwise, highly ordered fashion on each intron, cycling through discrete complexes designated E, A, B, B*, and C. Understanding this assembly pathway is essential because it reveals multiple regulatory checkpoints at which the cell can control which exons are included in the mature mRNA, thereby enabling alternative splicing — a mechanism that allows a single gene to encode multiple protein isoforms.

Left: The stepwise assembly of the spliceosome on a pre-mRNA intron, progressing from the early (E) complex through catalytically active (B* and C) complexes. The branch point adenosine (yellow A) and conserved splice site dinucleotides (GU and AG) are indicated. Lower left: the four major patterns of alternative splicing that diversify the transcriptome.

Alternative splicing is remarkably prevalent in metazoan genomes. In humans, an estimated 95% of multi-exon genes undergo alternative splicing, enabling the roughly 20,000 protein-coding genes to generate well over 100,000 distinct mRNA isoforms. Regulation of splice site selection depends on cis-regulatory elements — exonic and intronic splicing enhancers (ESEs, ISEs) and silencers (ESSs, ISSs) — and trans-acting factors such as SR proteins and hnRNPs. SR proteins typically bind ESEs and recruit spliceosomal components to adjacent splice sites, promoting exon inclusion, whereas hnRNPs often bind silencer elements and repress splice site usage, promoting exon skipping. The combinatorial interplay of these factors provides exquisite tissue-specific and developmental control over the proteome.

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Clinical Connection

Mutations that disrupt splice sites or splicing regulatory elements are responsible for an estimated 15–50% of disease-causing mutations in humans. For example, certain forms of spinal muscular atrophy (SMA) result from aberrant exon skipping in the SMN2 gene. The FDA-approved antisense oligonucleotide drug nusinersen (Spinraza) corrects this splicing defect by masking an intronic splicing silencer, restoring production of full-length SMN protein.

SECTION 6

Worked Example: Tracing mRNA Processing

Consider a hypothetical human gene containing 4 exons and 3 introns. The genomic DNA encodes a primary transcript of 8,200 nucleotides. The exon lengths are 120 nt, 250 nt, 180 nt, and 350 nt. Determine the length of the mature mRNA (excluding the poly(A) tail), identify the components that must be removed, and calculate the approximate final transcript length including all processing modifications.

Processing a Four-Exon Pre-mRNA

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Step 1 — Identify the Exon and Intron Content

Total exon length = 120 + 250 + 180 + 350 = 900 nt. Since the primary transcript is 8,200 nt, the total intron content = 8,200 − 900 = 7,300 nt distributed among 3 introns. Note that introns constitute approximately 89% of the primary transcript — a ratio typical of mammalian genes.

Total intron content: 7,300 nt (89%)
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Step 2 — Apply 5′ Capping

The 5′ cap (m⁷GpppN) is added to the first nucleotide of the transcript. This does not change the nucleotide count of the coding sequence but adds one guanylate residue linked by the 5′–5′ triphosphate bridge. The capped 5′-UTR forms part of exon 1. Cap addition occurs when the transcript is only ~20–30 nt long.

Cap adds 1 m⁷G nucleotide to the 5′ end.
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Step 3 — Remove Introns by Splicing

The spliceosome removes all three introns through pairs of transesterification reactions, joining exon 1–exon 2, exon 2–exon 3, and exon 3–exon 4. Each intron is released as a lariat that is subsequently debranched and degraded. The spliced mRNA body now contains only the four exons: 120 + 250 + 180 + 350 = 900 nt. The number of exon–exon junctions created is 3 (one fewer than the number of exons).

mRNA body after splicing: 900 nt
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Step 4 — Cleave and Polyadenylate the 3′ End

CPSF recognizes the AAUAAA signal (located in the 3′-UTR within exon 4) and, together with CstF and cleavage factors, directs endonucleolytic cleavage 10–30 nt downstream. PAP then adds ~200 adenylate residues. The cleavage may remove a small number of nucleotides from the original 3′ end of the last exon (let us approximate this as negligible for this calculation). The poly(A) tail thus adds approximately 200 nt.

Poly(A) tail: ~200 A residues
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Step 5 — Calculate Final Mature mRNA Length

Mature mRNA ≈ 1 (cap) + 900 (spliced exons) + 200 (poly(A) tail) = ~1,101 nt. The mature message is therefore roughly 13.4% of the primary transcript length (1,101/8,200 × 100), dramatically illustrating the extent of mRNA processing in mammals. This mature mRNA is now decorated with the cap-binding complex (CBC) in the nucleus, has an exon junction complex (EJC) deposited ~20–24 nt upstream of each exon–exon junction, and is coated with poly(A)-binding proteins — all of which facilitate nuclear export and subsequent translation.

Mature mRNA: ~1,101 nt (≈13% of pre-mRNA)

SECTION 7

Comparing Processing Events: Functions and Consequences

Although capping, splicing, and polyadenylation are mechanistically distinct, they share a common organizational logic: each is recruited to the nascent transcript through interactions with the phosphorylated CTD of Pol II, each contributes to mRNA quality control, and each influences downstream events in the mRNA lifecycle. The following table compares the three processing events across key functional dimensions.

Feature	5′ Capping	Splicing	3′ Polyadenylation
Timing	Very early; after ~20–30 nt synthesized	Co-transcriptional; progressive as introns are synthesized	Late; after AAUAAA is transcribed
CTD Signal	Ser5-P (initiation)	Ser2-P + Ser5-P (elongation)	Ser2-P (late elongation/termination)
Key Enzymes/Factors	RNA triphosphatase, guanylyltransferase, methyltransferase	Spliceosome (U1, U2, U4, U5, U6 snRNPs + ~100 proteins)	CPSF, CstF, CF I/II, PAP, PABPN1
Chemistry	Hydrolysis + nucleotidyl transfer + methylation	Two transesterification reactions (S_N2)	Endonucleolytic cleavage + polymerization
Protection	Shields against 5′→3′ exonucleases (Xrn1/Xrn2)	Deposits EJCs for quality control (NMD)	Shields against 3′→5′ exonucleases (exosome)
Translation Role	Recruits eIF4E for ribosome binding	Determines open reading frame	Cooperates with cap via PABP–eIF4G interaction for mRNA circularization

Comparison of the three major mRNA processing events

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KEY TAKEAWAY

The three processing events are not independent assembly-line stations; they are more like an integrated manufacturing cell in which each workstation communicates with the others through a shared conveyor system — the CTD. Just as a modern factory uses real-time sensor data (phosphorylation marks) to coordinate robotic arms at different stations, the CTD dynamically recruits and releases processing factors to ensure that capping precedes splicing, which precedes polyadenylation. Defects at any station trigger quality control mechanisms (such as nonsense-mediated decay or nuclear retention) that prevent defective transcripts from reaching the ribosome.

SECTION 8

Connections to Advanced Theory & Regulation

The principles of mRNA processing presented here form the foundation for several advanced topics in molecular biology and biotechnology. Understanding how capping, splicing, and polyadenylation are regulated opens pathways to comprehending mRNA therapeutics, epitranscriptomics, and the mechanisms by which dysregulated RNA processing contributes to disease. The table below bridges basic concepts to their advanced extensions.

Basic Concept	Advanced Extension	Significance
m⁷G capping protects mRNA	Modified nucleotides in synthetic mRNA (N1-methylpseudouridine) reduce innate immune recognition	Basis of COVID-19 mRNA vaccine design (Pfizer/BioNTech, Moderna)
Spliceosome-mediated intron removal	Small molecule spliceosome modulators (e.g., pladienolides, E7107) target SF3B1	Experimental cancer therapeutics exploiting splicing vulnerabilities in tumors
Alternative splicing generates protein diversity	RNA-seq and long-read sequencing reveal full-length isoform landscapes	Transcriptome-wide splicing analysis in personalized medicine
Poly(A) tail controls mRNA stability	Cytoplasmic deadenylation by CCR4-NOT complex as a regulated decay pathway	Temporal control of gene expression during development and immune responses
CTD coordinates processing	Liquid–liquid phase separation of transcription and processing factors into nuclear condensates	Emerging model for gene regulation through biomolecular condensate dynamics

From basic mRNA processing to advanced frontiers

As you advance in molecular biology and biochemistry, you will encounter these themes repeatedly. The splicing code — the set of rules by which cis-elements and trans-factors dictate splice site selection — remains an active area of computational biology, with machine learning models now capable of predicting tissue-specific splicing patterns from primary sequence alone. Similarly, the regulation of poly(A) site selection (alternative polyadenylation, APA) has emerged as a widespread mechanism for changing the length and regulatory content of 3′-UTRs, with profound effects on microRNA-mediated regulation and mRNA localization. These advanced topics build directly on the mechanistic foundations covered in this lesson.

SECTION 9

Practice Problems

Question 1: CONCEPTUAL

Explain why the 5′ cap is connected to the pre-mRNA via a 5′–5′ triphosphate linkage rather than a standard 3′–5′ phosphodiester bond. What functional advantage does this unusual linkage confer?

Show answer

The 5′–5′ triphosphate linkage places the cap guanine in a reverse orientation relative to the rest of the transcript. This inverted orientation is critical because it renders the 5′ end of the mRNA resistant to degradation by conventional 5′→3′ exonucleases (such as Xrn1), which require a free 5′ monophosphate for catalysis and cannot cleave across a 5′–5′ bond. A standard 3′–5′ linkage would extend the chain normally and leave a cleavable 5′ end. Additionally, the unique geometry of the cap structure is specifically recognized by the nuclear cap-binding complex (CBC) and the cytoplasmic translation initiation factor eIF4E, enabling selective identification of properly processed mRNAs for export and ribosome recruitment.

Question 2: BASIC CALCULATION

A gene contains 5 exons (80 nt, 150 nt, 200 nt, 120 nt, and 300 nt) and 4 introns. The primary transcript is 12,000 nt. (a) What is the total intron content? (b) After processing (including a 250-nt poly(A) tail), what is the approximate length of the mature mRNA?

Show answer

(a) Total exon content = 80 + 150 + 200 + 120 + 300 = 850 nt. Total intron content = 12,000 − 850 = 11,150 nt, which constitutes approximately 92.9% of the primary transcript. (b) Mature mRNA = 1 (m⁷G cap nucleotide) + 850 (spliced exons) + 250 (poly(A) tail) ≈ 1,101 nt. The mature mRNA is therefore only about 9.2% of the primary transcript length. This dramatic reduction in size reflects the large intronic burden typical of mammalian genes and underscores the importance of accurate splicing.

Question 3: INTERMEDIATE

During splicing, the first transesterification produces a free exon 1 with a 3′-OH and a lariat intermediate. A researcher mutates the conserved branch point adenosine to a guanosine. Predict the effect on splicing and explain why, considering the chemical mechanism.

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The branch point adenosine provides the 2′-hydroxyl nucleophile that attacks the phosphodiester bond at the 5′ splice site in the first transesterification. Mutating the branch point adenosine to guanosine would disrupt base-pairing with U2 snRNA (which recognizes the branch point sequence by complementary pairing, bulging out the branch point residue). This impaired U2 binding would compromise A complex formation and likely block the first transesterification step entirely, as the bulged nucleotide must be precisely positioned in the spliceosome active site. The result would be intron retention in the mature mRNA. In some cases, cryptic branch points nearby might be activated at reduced efficiency, producing aberrantly spliced products. This scenario illustrates why branch point mutations are a significant class of pathogenic splicing mutations in human genetic disease.

Question 4: APPLIED

A pharmaceutical company is designing a synthetic mRNA to be delivered directly to the cytoplasm for therapeutic protein production. The mRNA will not be transcribed by Pol II. Which of the three processing modifications (capping, splicing, polyadenylation) must be incorporated in vitro, and how might each be achieved? Consider why splicing may or may not be necessary.

Show answer

All three modifications serve essential functions, but splicing can be bypassed by designing a cDNA template that already contains only the joined exon sequences (intron-free). For the 5′ cap, the company would use either enzymatic capping with vaccinia virus capping enzyme post-transcription or co-transcriptional capping using synthetic cap analogs such as CleanCap (a trinucleotide cap analog that initiates transcription with a pre-formed m⁷GpppA structure, yielding cap 1). For polyadenylation, a poly(A) sequence of ~100–150 adenines is typically encoded directly in the DNA template downstream of the 3′-UTR, so PAP treatment is unnecessary. The cap is particularly critical because without it, cytoplasmic 5′→3′ exonucleases would rapidly degrade the mRNA, and ribosomes would fail to initiate translation efficiently via the eIF4E-dependent pathway. Modified nucleotides such as N1-methylpseudouridine are also incorporated to reduce innate immune activation — this is precisely the strategy used in the Pfizer/BioNTech and Moderna COVID-19 mRNA vaccines.

Question 5: CRITICAL THINKING

Nonsense-mediated decay (NMD) degrades mRNAs containing premature termination codons (PTCs) if the PTC is located more than 50–55 nt upstream of an exon–exon junction marked by an exon junction complex (EJC). Suppose a mutation creates a PTC in exon 2 of a 4-exon gene. (a) Would this mRNA be targeted by NMD? Explain using the EJC model. (b) Now suppose the same PTC-containing mutation occurs in a single-exon gene. Would NMD be triggered? What does this reveal about the coupling between splicing and mRNA surveillance?

Show answer

(a) Yes. After splicing, EJCs are deposited ~20–24 nt upstream of each exon–exon junction. A 4-exon gene has 3 exon–exon junctions, generating EJCs at the exon 1–2, exon 2–3, and exon 3–4 boundaries. When a ribosome translating this mRNA encounters the PTC in exon 2, it terminates translation. Since the exon 2–3 and exon 3–4 EJCs lie downstream (more than 50–55 nt from the PTC), they will not have been displaced by the translating ribosome. The surveillance complex (UPF1 recruited at the premature stop codon interacts with UPF2/UPF3 at the downstream EJCs) triggers NMD, leading to rapid mRNA degradation. (b) No. A single-exon gene undergoes no splicing, so no EJCs are deposited. Without downstream EJCs, the canonical NMD pathway is not activated, even if the mRNA contains a PTC. This reveals that NMD is fundamentally coupled to splicing through the EJC: it is the physical mark left by the spliceosome that enables the cell to distinguish premature from normal stop codons. This coupling also explains why intronless genes (such as many histone genes) that acquire nonsense mutations may escape NMD, and why alternative NMD pathways (EJC-independent, involving long 3′-UTRs) have evolved as supplementary quality control mechanisms.

SUMMARY

Lesson Summary

Summary

Eukaryotic pre-mRNA processing transforms the primary transcript into a mature message through three coordinated modifications. 5′ capping adds a 7-methylguanosine via a 5′–5′ triphosphate linkage, protecting the transcript from exonucleolytic degradation and enabling ribosome recruitment through eIF4E binding. Splicing removes introns via two transesterification reactions catalyzed by the spliceosome, an RNA-based catalytic machine assembled from U1, U2, U4, U5, and U6 snRNPs. The first reaction generates a lariat intermediate; the second ligates the flanking exons. Alternative splicing allows a single gene to encode multiple protein isoforms, vastly expanding proteomic diversity.

3′ polyadenylation involves recognition of the AAUAAA signal by CPSF, endonucleolytic cleavage, and addition of ~200 adenylate residues by poly(A) polymerase. All three events are orchestrated by the C-terminal domain (CTD) of RNA Polymerase II, whose differential phosphorylation at Ser5 and Ser2 recruits the appropriate processing machinery at each stage of transcription. The resulting mature mRNA — capped, spliced, and polyadenylated — is then competent for nuclear export, quality control by nonsense-mediated decay, and efficient translation in the cytoplasm.