8.2 RNA Processing and Modification

mRNA Processing in Eukaryotes

Before a eukaryotic mRNA can leave the nucleus and be translated, it undergoes three major processing steps: 5' capping, 3' polyadenylation, and splicing. These modifications protect the transcript from degradation, help it get exported from the nucleus, and remove non-coding sequences so only the correct reading frame reaches the ribosome.

Steps of Eukaryotic mRNA Processing

5' Capping

This is the first modification, and it happens co-transcriptionally (while RNA polymerase II is still transcribing). A 7-methylguanosine (m7G) cap is added to the 5' end of the pre-mRNA through an unusual 5'-to-5' triphosphate linkage.

The cap serves several functions:

• Protects the mRNA from degradation by 5' exonucleases
• Facilitates nuclear export
• Promotes translation initiation by recruiting eIF4E (eukaryotic initiation factor 4E), which is part of the cap-binding complex that helps the ribosome find the mRNA

3' Polyadenylation

At the 3' end, a poly(A) tail of roughly 150–250 adenine residues is added. This process requires:

1. Recognition of the polyadenylation signal (consensus sequence AAUAAA), located 10–30 nucleotides upstream of the cleavage site
2. The cleavage and polyadenylation specificity factor (CPSF) complex binds the signal and cleaves the pre-mRNA
3. Poly(A) polymerase (PAP) then adds the string of adenines without a DNA template

The poly(A) tail protects the mRNA from 3' exonuclease degradation, facilitates nuclear export, and enhances translation efficiency. Over time in the cytoplasm, the tail gradually shortens, which is one way cells control mRNA lifespan.

Splicing

Splicing removes non-coding introns and joins the remaining exons together. It takes place in the nucleus and is catalyzed by the spliceosome, a large ribonucleoprotein complex.

Four sequence elements within the intron are required for splicing:

• 5' splice site (GU dinucleotide at the intron's start)
• 3' splice site (AG dinucleotide at the intron's end)
• Branch point sequence (BPS) containing a conserved adenine residue
• Polypyrimidine tract between the BPS and the 3' splice site

Role of snRNPs in Splicing

The spliceosome is built from snRNPs (small nuclear ribonucleoproteins, pronounced "snurps"). Each snRNP contains one small nuclear RNA (snRNA) and associated proteins (Sm or LSm proteins). Five snRNPs participate: U1, U2, U4, U5, and U6.

Spliceosome assembly and catalysis proceed in an ordered sequence:

1. U1 snRNP binds the 5' splice site through base-pairing between U1 snRNA and the GU-containing sequence.
2. U2 snRNP binds the branch point sequence. The conserved adenine at the branch point bulges out, which is critical for the first catalytic step.
3. The U4/U6·U5 tri-snRNP complex joins, forming the fully assembled spliceosome.
4. U6 snRNP replaces U1 at the 5' splice site, and U4 is released. U6 and U2 now base-pair with each other, bringing the 5' splice site and branch point into close proximity.
5. The first transesterification reaction occurs: the 2'-OH of the branch point adenine attacks the 5' splice site, forming a lariat intermediate.
6. U5 snRNP helps align the two exons, and the second transesterification reaction joins them together while releasing the lariat intron.

The key concept here is that snRNAs are doing much of the catalytic work. The spliceosome is essentially a ribozyme, with the RNA components catalyzing the splicing reactions.

Constitutive vs. Alternative Splicing

Constitutive splicing is straightforward: every transcript of a given gene is spliced the same way, producing a single mRNA isoform and therefore a single protein product. The same introns are always removed, and the same exons are always joined.

Alternative splicing is far more interesting. Different transcripts from the same gene can be spliced in different patterns, producing multiple mRNA isoforms from one gene. This is a major source of protein diversity in eukaryotes.

The main types of alternative splicing events:

• Exon skipping — an exon that's normally included gets left out
• Intron retention — an intron that's normally removed stays in the mature mRNA
• Alternative 5' splice site — a different GU site is used, changing where the intron begins
• Alternative 3' splice site — a different AG site is used, changing where the intron ends

These different isoforms can encode proteins with altered functions, stability, or subcellular localization. A classic example: the Drosophila Dscam gene can produce over 38,000 mRNA isoforms through alternative splicing.

Alternative splicing is regulated by two categories of elements working together:

• Cis-acting regulatory elements on the pre-mRNA itself: exonic/intronic splicing enhancers (ESEs/ISEs) and exonic/intronic splicing silencers (ESSs/ISSs)
• Trans-acting factors that bind those elements: SR proteins generally promote exon inclusion by binding enhancers, while hnRNPs often promote exon skipping by binding silencers

The balance between these activating and repressing factors determines which splice pattern is used in a given cell type or developmental stage.

RNA Modifications and Their Significance

Beyond the three core processing steps, RNA molecules carry a wide range of chemical modifications that fine-tune their behavior. This field, sometimes called epitranscriptomics, has revealed that RNA modifications regulate stability, translation efficiency, structure, and even coding potential. These modifications are found not just on mRNA but also on tRNA, rRNA, and snRNA.

Types and Significance of RNA Modifications

N6-methyladenosine (m6A)

This is the most abundant internal modification in eukaryotic mRNA. A methyl group is added to the nitrogen at position 6 of adenine.

m6A is dynamically regulated by three classes of proteins:

• Writers (METTL3/METTL14 complex) install the modification
• Erasers (FTO, ALKBH5) remove it
• Readers (YTH domain-containing proteins) recognize m6A and mediate its downstream effects

This writer/eraser/reader system means m6A is reversible, which is why it can act as a regulatory signal rather than a permanent mark. m6A influences mRNA stability, translation efficiency, and splicing, and plays roles in cell differentiation, development, and stress responses.

Pseudouridine ($\Psi$)

Pseudouridine is an isomer of uridine where the uracil base is rotated and attached to the ribose through a carbon-carbon bond instead of the usual nitrogen-carbon bond. This subtle change is catalyzed by pseudouridine synthases (PUS enzymes).

$\Psi$ is found across many RNA types (mRNA, tRNA, rRNA, snRNA) and enhances RNA stability by increasing base-stacking interactions. It also influences RNA structure and function, which is why it was incorporated into the modified mRNA used in certain COVID-era vaccines to reduce immune detection.

5-methylcytosine (m5C)

A methyl group is added to position 5 of cytosine by RNA methyltransferases, primarily NSUN2 and DNMT2. This modification occurs in tRNA, rRNA, and mRNA.

m5C regulates RNA stability and translation. In tRNA specifically, m5C can influence tRNA cleavage, affecting the production of tRNA-derived fragments that have their own regulatory roles.

Inosine (I)

Inosine is generated when ADAR enzymes (adenosine deaminases acting on RNA) convert adenosine to inosine by removing an amino group. This is called A-to-I editing.

The translational machinery reads inosine as guanosine, so A-to-I editing in coding regions can change the amino acid sequence of the resulting protein. In non-coding regions, it can alter RNA secondary structure and stability, or affect microRNA targeting. This modification occurs in both coding and non-coding regions of mRNA.

2'-O-methylation (Nm)

A methyl group is added to the 2'-hydroxyl of the ribose sugar. In rRNA, this is typically guided by snoRNAs (small nucleolar RNAs) that base-pair with the target site and recruit the methyltransferase fibrillarin. Stand-alone methyltransferases can also catalyze this modification independently.

2'-O-methylation is found in rRNA, tRNA, snRNA, and the mRNA 5' cap structure. It enhances RNA stability, influences RNA folding, and protects against nuclease degradation. The modification also helps distinguish "self" RNA from foreign RNA in innate immune signaling.