14.2 RNA processing: capping, splicing, and polyadenylation

RNA Processing: Capping, Splicing, and Polyadenylation

Before mRNA can leave the nucleus and be translated into protein, the cell modifies the pre-mRNA transcript in three key ways: it adds a protective cap to the 5' end, splices out non-coding sequences, and attaches a poly(A) tail to the 3' end. These processing steps are what convert a raw pre-mRNA into a mature, functional mRNA. Without them, the transcript would be degraded, stuck in the nucleus, or translated incorrectly.

Process of 5' Capping

Capping happens early, while transcription is still underway. Once RNA polymerase II has synthesized roughly the first 20–30 nucleotides, a set of enzymes modifies the 5' end of the nascent pre-mRNA in three sequential steps:

1. RNA triphosphatase removes the gamma (terminal) phosphate from the 5' end of the pre-mRNA.
2. Guanylyltransferase adds a GMP to the 5' end, forming an unusual 5'-5' triphosphate linkage (the phosphate bonds point in the reverse direction compared to normal internucleotide links).
3. Methyltransferase methylates the N7 position of the added guanine, producing the mature 7-methylguanosine (m7G) cap.

The 5' cap serves several functions:

• Stability: It shields the mRNA from 5'→3' exonucleolytic degradation by enzymes like the exosome complex.
• Nuclear export: It helps the mature mRNA pass through the nuclear pore complex into the cytoplasm.
• Translation initiation: The cap is recognized by the translation initiation factor eIF4E, which recruits the 40S ribosomal subunit. Without the cap, ribosomes can't efficiently find and bind the mRNA.

Mechanism of RNA Splicing

Splicing removes introns (non-coding intervening sequences) and joins the remaining exons (expressed sequences) to form a continuous coding message. This is carried out by the spliceosome, a large ribonucleoprotein complex made up of five small nuclear ribonucleoproteins (snRNPs): U1, U2, U4, U5, and U6, along with many associated proteins.

The splicing reaction itself proceeds through two transesterification steps:

1. The 2'-OH group of a conserved branch point adenosine within the intron attacks the 5' splice site. This cuts the RNA at that junction and forms a loop called a lariat structure, where the intron is connected back to itself.
2. The free 3'-OH of the upstream exon then attacks the 3' splice site, joining the two exons together and releasing the lariat intron for degradation.

A critical consequence of splicing is alternative splicing, which allows a single gene to produce multiple different mRNAs (and therefore multiple protein isoforms). For example, the CD44 gene can include or skip various exons to produce proteins with different functional domains, and tropomyosin pre-mRNA is spliced differently in skeletal muscle versus smooth muscle. This dramatically expands the coding potential of the genome.

Constitutive vs. Alternative Splicing

Constitutive splicing is the default: every exon in the pre-mRNA is included in the final mature mRNA, every time. Genes like β-actin are constitutively spliced, producing one consistent protein product.

Alternative splicing is selective. Specific exons can be skipped, included in a mutually exclusive fashion, or joined at alternative 5' or 3' splice sites. For instance, the FGFR2 gene uses mutually exclusive exons to produce receptor variants with different ligand-binding properties, and Bcl-x alternative splicing generates both pro-survival and pro-apoptotic protein isoforms from the same gene.

How does the cell decide which exons to include? Two layers of regulation work together:

• Cis-acting elements are sequences within the pre-mRNA itself, such as exonic or intronic splicing enhancers (ESE/ISE) and silencers (ESS/ISS).
• Trans-acting factors are proteins that bind those elements. SR proteins generally promote exon inclusion, while hnRNPs often promote exon skipping.

The balance between these factors determines the splicing pattern in a given cell type or developmental stage.

Significance of Polyadenylation

Polyadenylation adds a poly(A) tail to the 3' end of the mRNA. The process occurs in two steps:

1. The cleavage and polyadenylation specificity factor (CPSF) recognizes the polyadenylation signal sequence (AAUAAA) on the pre-mRNA and cleaves the transcript downstream of it.
2. Poly(A) polymerase (PAP) then adds approximately 200–250 adenosine residues to the new 3' end, forming the poly(A) tail.

The poly(A) tail has roles that parallel the 5' cap:

• Stability: It protects the mRNA from 3'→5' exonucleolytic degradation.
• Nuclear export: Like the cap, it facilitates transport through the nuclear pore complex.
• Translation efficiency: Poly(A) binding protein (PABP) coats the tail and interacts with eIF4G, which in turn contacts the cap-binding complex at the 5' end. This creates a closed-loop structure that circularizes the mRNA and strongly enhances ribosome recycling and translation.

Poly(A) tail length also acts as a regulatory signal. Deadenylation (shortening of the tail by complexes like CCR4-NOT) is often the first step in mRNA decay, since a shortened tail loses PABP protection and exposes the mRNA to degradation. Conversely, cytoplasmic polyadenylation can reactivate dormant mRNAs that had been stored with short tails. This mechanism is especially important during oocyte maturation and at neuronal synapses, where rapid, localized translation needs to be triggered without new transcription.