Questions: RNA Splicing, Introns, Exons, and the Spliceosome

Question 1

A point mutation changes the first two nucleotides of an intron from GU to AU. What is the most likely consequence for the mRNA produced from this gene?

• A. Splicing proceeds normally, because the spliceosome recognizes introns by their internal sequences, not the terminal dinucleotides
• B. The intron is retained in the mature mRNA, disrupting the reading frame or introducing a premature stop codon
• C. The spliceosome switches to an alternative 5' splice site automatically, restoring normal splicing
• D. The poly-A tail cannot be added, preventing export of the mRNA from the nucleus

Answer: (B)

The GU at the 5' splice site is part of the nearly universal GU-AG rule and is essential for spliceosome recognition. The U1 snRNA base-pairs with the 5' splice site sequence; a GU→AU mutation disrupts this interaction and abolishes or severely reduces splicing at that site. The result is usually intron retention — the intron remains in the mRNA — which typically disrupts the reading frame or introduces a premature stop codon. Many human genetic diseases are caused precisely by such splice-site mutations.

Question 2

What is the catalytic heart of the spliceosome, and what category of enzyme does this make it?

• A. Large splicing proteins (SR proteins) that use ATP to cleave phosphodiester bonds and join exons
• B. Small nuclear RNAs (snRNAs U1, U2, U4, U5, U6) that position reactive groups and stabilize transition states, making the spliceosome a ribozyme
• C. RNA polymerase II, which catalyzes splicing co-transcriptionally using the same active site as transcription
• D. The branch-point adenosine itself, which acts as a protein cofactor in the cleavage reaction

Answer: (B)

The spliceosome's catalytic activity resides in its snRNA components, particularly U2 and U6, which form the active site that positions the reactive groups for both transesterification steps. This makes the spliceosome a ribozyme — an RNA molecule with catalytic activity. The associated proteins facilitate assembly, remodeling, and fidelity, but the chemistry is RNA-catalyzed. This was a significant discovery because it challenged the assumption that all biological catalysts are proteins.

Question 3

The two chemical steps of pre-mRNA splicing — lariat formation and exon joining — are transesterification reactions that do not require energy input from ATP hydrolysis because each step breaks one phosphodiester bond while forming another.

• T. True
• F. False

Answer: True

Transesterification exchanges one phosphoester linkage for another, keeping the total number of high-energy bonds constant. No net energy input or output is required for the chemical steps themselves. The energy balance is approximately neutral because a phosphodiester bond is broken and a new one is formed. Note that the spliceosome does require ATP hydrolysis by DEAD-box helicases to drive conformational rearrangements during assembly and activation, but the splicing chemistry itself — the two nucleophilic attacks — is energetically neutral.

Question 4

Introns are biologically inert 'junk sequences' that serve no function and are substantially degraded immediately after removal from the pre-mRNA.

• T. True
• F. False

Answer: False

This is a significant misconception. Many intron sequences have important regulatory functions: they harbor enhancers, silencers, and noncoding RNA genes (microRNAs, snoRNAs, lncRNAs often encoded within introns). Some introns are retained in the mature transcript as a form of gene regulation (intron retention). The capacity for alternative splicing — choosing which exons to include — is only possible because introns define the boundaries between exonic modules; this is how ~20,000 genes produce >100,000 protein isoforms. Introns are also evolutionarily useful as units that can be shuffled between genes (exon shuffling).

Question 5

Why does the co-transcriptional nature of splicing matter for gene expression regulation?

Model answer: Because splicing occurs while RNA polymerase II is still elongating the pre-mRNA, the cell can regulate splice site choices in response to cellular signals before the transcript is complete. The C-terminal domain of RNA Pol II recruits splicing factors to the nascent transcript, and transcription elongation rate influences which splice sites are recognized — slower elongation gives the spliceosome more time to commit to upstream splice sites. This coupling enables alternative splicing: different exons can be included or skipped depending on which splicing factors are present, allowing the same gene to produce different mRNAs in different cell types, developmental stages, or conditions.

Co-transcriptional splicing is what makes alternative splicing possible at scale. The ~94% of multi-exon human genes that undergo alternative splicing owe that capacity to the tight coupling between transcription and spliceosome assembly. This is why the human genome, with ~20,000 protein-coding genes, can produce a proteome of over 100,000 distinct protein variants — each gene is a modular toolkit, not a single blueprint.