Source: Chapter 7 Review Sheet, Cell Biology, University of Florida
Difficulty: Intermediate | Prerequisites: Part 1 (Transcription basics), DNA/RNA structure, base pairing
Tags: mRNA processing, 5' cap, poly-A tail, spliceosome, snRNP, snRNA, introns, exons, alternative splicing, rRNA, tRNA, miRNA, lariat, gene expression, PCB 3023
After transcription produces a raw RNA transcript (pre-mRNA in eukaryotes), the cell is far from finished. The transcript must be processed before it can function: capped, tailed, and spliced. This part covers the different types of RNA involved in gene expression, how the eukaryotic mRNA is modified at both ends, how introns are removed by the spliceosome, and how alternative splicing lets a single gene produce multiple proteins. If you are coming in cold, make sure you understand transcription basics (Part 1) and the general structure of nucleic acids first.
Eukaryotic pre-mRNA is processed in three ways: a 5' cap is added, a poly-A tail is attached at the 3' end, and introns are spliced out by the spliceosome. Several types of RNA (mRNA, rRNA, tRNA, snRNA, miRNA) each play distinct roles in gene expression. Alternative splicing of exons allows one gene to encode multiple different proteins.
mRNA (messenger RNA)
The RNA molecule that carries the coding sequence from a gene to the ribosome, where it is translated into protein. Think of it as the portable copy of the gene's instructions.
rRNA (ribosomal RNA)
RNA that forms part of the ribosome's structure and catalyses peptide bond formation. It is both structural and enzymatic. The ribosome is mostly rRNA by mass.
tRNA (transfer RNA)
Small RNA molecules that carry amino acids to the ribosome during translation. Each tRNA has an anticodon that base-pairs with a specific mRNA codon, ensuring the correct amino acid is added to the growing polypeptide.
snRNA (small nuclear RNA)
RNA molecules found in the nucleus that combine with proteins to form snRNPs (small nuclear ribonucleoproteins). They are essential components of the spliceosome and help identify and remove introns from pre-mRNA.
miRNA (microRNA)
Small RNA molecules (roughly 21–23 nucleotides) that regulate gene expression post-transcriptionally. They bind to complementary sequences in mRNA, typically blocking translation or promoting mRNA degradation.
snRNP (small nuclear ribonucleoprotein, pronounced "snurp")
A complex of snRNA plus associated proteins. Multiple snRNPs assemble to form the spliceosome. The snRNA within each snRNP base-pairs with specific sequences in the pre-mRNA to locate intron boundaries.
Spliceosome
A large molecular machine made of snRNPs and other proteins that removes introns from pre-mRNA and joins the remaining exons together. In simple terms, it is the cell's editing suite for cutting out non-coding sequences.
Intron
A non-coding sequence within a gene that is transcribed into RNA but removed (spliced out) before translation. All vertebrate introns begin with GU and end with AG (the GU-AG rule).
Exon
A sequence within a gene that is retained in the mature mRNA after splicing. Exons contain the coding sequences that are translated into protein, plus the untranslated regions (UTRs) at each end.
5' cap
A modified guanine nucleotide (7-methylguanosine) added to the 5' end of a eukaryotic mRNA shortly after transcription begins. It protects the mRNA from degradation, aids ribosome recognition, and facilitates nuclear export.
Poly-A tail
A stretch of 100–250 adenine nucleotides added to the 3' end of a eukaryotic mRNA after a cleavage event. It protects the mRNA from exonuclease degradation and aids in nuclear export and translation efficiency.
Lariat
The looped intermediate structure formed during intron splicing. It is created when the 2'-OH of a specific adenine nucleotide (the branch point A) within the intron attacks the 5' splice site, forming an unusual 2'-5' phosphodiester bond. The two nucleotides covalently joined to form the lariat are the branch point adenine (A) and the guanine (G) at the 5' end of the intron.
Alternative splicing
A process by which different combinations of exons from the same pre-mRNA are joined together, producing multiple distinct mRNAs (and therefore multiple distinct proteins) from a single gene.
mRNA carries the genetic code from DNA to the ribosome. It is the template for protein synthesis.
rRNA is a structural and catalytic component of the ribosome. The large ribosomal subunit's rRNA catalyses peptide bond formation (it is a ribozyme).
tRNA acts as an adaptor, matching codons on the mRNA with the correct amino acids during translation.
snRNA functions within the spliceosome to identify intron-exon boundaries and catalyse splicing.
miRNA regulates gene expression by binding to target mRNAs and silencing them (blocking translation or triggering degradation).
All RNAs originate from transcription of DNA by RNA polymerase. Every RNA in the cell begins as a transcript of a DNA template.
Shortly after transcription begins (while the mRNA is still being made), a 7-methylguanosine cap is added to the 5' end of the transcript.
The cap is linked by an unusual 5'-5' triphosphate bridge (reversed orientation compared to normal nucleotide linkages).
Functions of the 5' cap:
Protects the mRNA from degradation by 5' exonucleases.
Required for efficient translation initiation (the ribosome recognises and binds the cap).
Assists in nuclear export of the mRNA.
Helps with intron splicing of the first intron.
The 3' end of the pre-mRNA is not simply where RNA polymerase stops. Instead, the transcript is cleaved at a specific signal sequence (AAUAAA in most cases), and then the enzyme poly-A polymerase adds a poly-A tail of roughly 100–250 adenine nucleotides.
Functions of the poly-A tail:
Protects the mRNA from 3' exonuclease degradation.
Aids in nuclear export.
Enhances translation efficiency.
The tail gradually shortens over the mRNA's lifetime; when it becomes too short, the mRNA is degraded. This provides a built-in expiry mechanism.
The spliceosome is assembled from five major snRNPs (U1, U2, U4, U5, U6) plus additional proteins.
Each snRNP contains snRNA (a small nuclear RNA) and associated proteins. The snRNA component is what base-pairs with the pre-mRNA to identify intron boundaries.
Conserved sequences at intron boundaries in vertebrates:
5' splice site: GU (the first two nucleotides of the intron)
3' splice site: AG (the last two nucleotides of the intron)
Branch point: a specific adenine (A) residue located 20–50 nucleotides upstream of the 3' splice site.
Splicing mechanism (simplified):
U1 snRNP binds the 5' splice site (GU). U2 snRNP binds the branch point A.
The remaining snRNPs join to form the complete spliceosome.
The 2'-OH of the branch point adenine attacks the phosphodiester bond at the 5' splice site, cutting the RNA there and forming the lariat loop.
The free 3'-OH of the upstream exon then attacks the 3' splice site, joining the two exons and releasing the intron as a lariat.
The two nucleotides covalently joined in the lariat are the branch point A and the G at the 5' end of the intron, linked by a 2'-5' phosphodiester bond.
Exons often (though not always) correspond to functional domains of a protein. A single exon may encode a discrete structural or functional unit, such as a DNA-binding domain or a catalytic domain.
Introns facilitate genetic recombination. Because crossing over during meiosis can occur within introns, exons from the same gene can be shuffled without disrupting the coding sequence of any individual exon. This is called exon shuffling.
Exon shuffling can produce new alleles of a gene (new combinations of existing exons) or even entirely new genes (when exons from different genes are brought together).
Introns therefore accelerate the evolution of new protein functions by allowing the modular recombination of protein domains.
By including or excluding certain exons during splicing, a single gene can produce multiple distinct mRNA molecules, each encoding a different protein (or protein variant).
This is a major reason why the number of proteins in a eukaryotic organism far exceeds the number of genes. Humans have roughly 20,000 genes but produce well over 100,000 distinct proteins.
Example: the Drosophila DSCAM gene can theoretically produce over 38,000 different mRNAs through alternative splicing.
Errors in splicing cause a number of human diseases. For instance, some forms of beta-thalassaemia result from mutations that disrupt normal splicing of the beta-globin gene. Understanding splicing mechanisms has also enabled the development of antisense oligonucleotide therapies (such as nusinersen for spinal muscular atrophy), which work by altering the splicing pattern of a target pre-mRNA.
Students often think introns are "junk DNA" with no function. Introns play a critical role in exon shuffling, gene regulation, and alternative splicing. They are non-coding, but that does not mean non-functional.
Students sometimes confuse the 5' cap with the start codon. The cap is a modified nucleotide added to the very beginning of the mRNA. The start codon (AUG) is located downstream, after the 5'-UTR.
Some students think the poly-A tail is encoded in the DNA. It is not. It is added enzymatically after the pre-mRNA is cleaved.
Students frequently mix up which nucleotides form the lariat. It is the branch point A within the intron joined to the G at the intron's 5' end, not the exon nucleotides.
⚠️ Know the functions of each RNA type (mRNA, rRNA, tRNA, snRNA, miRNA) and that all originate from DNA transcription.
⚠️ Be able to list the three processing steps for eukaryotic mRNA (5' cap, poly-A tail, splicing) and the function of each.
⚠️ Know the conserved intron sequences: GU at the 5' splice site, AG at the 3' splice site, and the branch point A.
⚠️ Know which two nucleotides are covalently joined to form the lariat (the branch point A and the 5' G of the intron).
⚠️ Understand how alternative splicing allows one gene to produce multiple polypeptides.
⚠️ Understand how exons relate to protein domains and how introns facilitate the evolution of new genes through exon shuffling.
True or False: The poly-A tail is encoded in the gene's DNA sequence. → False. It is added enzymatically by poly-A polymerase after cleavage of the pre-mRNA.
Fill in the blank: All vertebrate introns begin with _____ and end with _____. → GU ... AG.
True or False: snRNAs function by base-pairing with the pre-mRNA to identify intron boundaries. → True.
Fill in the blank: The unusual bond in the lariat is a - phosphodiester bond. → 2'-5'.
True or False: Alternative splicing is one reason eukaryotes can produce more proteins than they have genes. → True.
Q: Name the four major types of RNA involved in gene expression and state the role of each.
A: mRNA carries the coding sequence to the ribosome. rRNA is a structural and catalytic component of the ribosome. tRNA carries amino acids to the ribosome and matches them to mRNA codons. snRNA is part of the spliceosome and helps remove introns from pre-mRNA.
Q: What are the three processing modifications made to eukaryotic pre-mRNA, and what purpose does each serve?
A: (1) A 7-methylguanosine 5' cap protects from degradation, aids ribosome binding, and assists nuclear export. (2) A poly-A tail at the 3' end protects from degradation, aids export, and regulates mRNA lifespan. (3) Splicing removes introns and joins exons to produce the mature coding sequence.
Q: What components make up the spliceosome, and how does the snRNA contribute to intron recognition?
A: The spliceosome is composed of five snRNPs (U1, U2, U4, U5, U6) and additional proteins. Each snRNP contains snRNA that base-pairs with conserved sequences at the intron's splice sites and branch point, identifying the intron boundaries.
Q: Which two nucleotides are covalently joined to form a lariat, and what type of bond links them?
A: The branch point adenine (A) within the intron is joined to the guanine (G) at the 5' end of the intron via a 2'-5' phosphodiester bond.
Q: How can a single eukaryotic gene encode multiple distinct polypeptides?
A: Through alternative splicing. Different combinations of exons can be included or excluded during mRNA processing, producing different mature mRNAs from the same pre-mRNA, each of which is translated into a different polypeptide.
This material connects to transcription (Part 1), since mRNA processing occurs on the raw transcript produced during transcription. It links forward to translation (Parts 3–4), because only a fully processed mRNA (capped, tailed, spliced) is exported from the nucleus and translated at the ribosome. The concept of alternative splicing also connects to developmental biology and cell differentiation, where different cell types express different splice variants of the same gene.
mRNA processing, 5' cap, 7-methylguanosine, poly-A tail, polyadenylation, splicing, spliceosome, snRNP, snRNA, U1, U2, U4, U5, U6, intron, exon, GU-AG rule, branch point, lariat, 2'-5' phosphodiester bond, alternative splicing, exon shuffling, protein domains, miRNA, rRNA, tRNA, gene expression, pre-mRNA, eukaryotic mRNA processing, PCB 3023, cell biology