D. Molecular Genetics · Molecular GeneticsRNA Processing in Eukaryotes: Caps, Tails and Splicing
D3.3 D3.2 D2.1 A2.2About 65 minutes
Learning goals- I can describe how a eukaryotic pre-mRNA is processed: 5′ capping, 3′ polyadenylation and splicing.
- I can explain the roles of the cap, the poly-A tail and the spliceosome.
- I can calculate the length of a mature mRNA and the protein it codes for from exon and intron data.
- I can explain how alternative splicing lets one gene code for several proteins, and give a medical application.
Words to know
pre-mRNA
The primary transcript made by RNA polymerase II, before processing.
5′ cap
A modified guanine nucleotide (7-methylguanosine) added to the 5′ end of pre-mRNA; it protects the mRNA and helps ribosomes bind.
poly-A tail
A string of adenine nucleotides (often about 200) added to the 3′ end of pre-mRNA.
exon
A part of a gene that is kept in the mature mRNA (it is "expressed").
intron
A part of a gene that is transcribed but removed from the pre-mRNA by splicing (an "intervening" sequence).
splicing
Removing introns and joining exons together.
spliceosome
A large complex of small nuclear RNAs (snRNAs) and proteins that carries out splicing.
alternative splicing
Joining different combinations of exons from the same pre-mRNA to produce different mRNAs.
untranslated region (UTR)
A part of the mature mRNA before the start codon (5′ UTR) or after the stop codon (3′ UTR) that is not translated.
Your genes are mostly interruptions
In 1977, Phillip Sharp and Richard Roberts made a surprising discovery: in many genes of eukaryotes and their viruses, the coding sequence is broken into pieces separated by long stretches that do not appear in the final mRNA. They shared the 1993 Nobel Prize in Physiology or Medicine for these "split genes".
The human gene for the muscle protein dystrophin is about 2.2 million base pairs long, but its mature mRNA is only about 14 000 nucleotides. More than 99% of the gene is removed after transcription. Why would a cell make a huge RNA and throw most of it away? This lesson explains how processing works and why it is useful.
Remember from the last lesson
In eukaryotes, RNA polymerase II transcribes protein-coding genes in the nucleus. Its product is a pre-mRNA (primary transcript). Bacteria do not process their mRNA in these ways: their mRNA is translated even while it is being made.
Step 1: the 5′ cap
While the pre-mRNA is still being made, an enzyme adds a modified guanine nucleotide, 7-methylguanosine, to its 5′ end. It is attached backwards (by an unusual 5′-to-5′ link), so it is not a normal nucleotide in the chain. The cap:
- protects the mRNA from enzymes that chew RNA from the 5′ end;
- helps the mRNA get exported through the nuclear pores;
- is the signal the small ribosomal subunit recognizes to start translation.
Step 2: the poly-A tail
At the end of the gene, RNA polymerase II copies the signal AAUAAA. Proteins cut the RNA a short distance downstream, and the enzyme poly-A polymerase adds a poly-A tail of adenine nucleotides (often about 200) to the new 3′ end. The tail:
- protects the 3′ end and helps set how long the mRNA lasts in the cytoplasm (the tail slowly shortens, and when it is short the mRNA is broken down);
- helps with export and translation.
Step 3: splicing
Introns (intervening sequences) are removed and exons (expressed sequences) are joined. Splicing is done by the spliceosome, a large complex made of proteins and small nuclear RNAs (snRNAs). The snRNAs recognize the ends of each intron by base pairing:
- Almost every intron begins with GU (at its 5′ end) and ends with AG (at its 3′ end).
- The spliceosome cuts at the 5′ end of the intron, loops the intron into a shape called a lariat, cuts at the 3′ end, and joins the two exons with a new phosphodiester bond. The lariat is broken down.
Splicing must be exact: removing even one extra nucleotide would shift the reading frame of everything downstream.
Why split genes? Alternative splicing
Humans have only about 20 000 protein-coding genes, yet make many more different proteins. One reason is alternative splicing: cells can include or skip particular exons, so one gene gives several related mRNAs and proteins. Different tissues, or the same tissue at different times, can make different versions. About 95% of human genes that have more than one exon are alternatively spliced.
An extreme example: a fruit-fly gene called Dscam, which helps nerve cells recognize each other, could in theory produce 38 016 different mRNAs by alternative splicing.
Alternative splicing is also a level of gene control, which you will study in Controlling Genes in Eukaryotes.
Not all of the mRNA is translated
The mature mRNA has a 5′ untranslated region (5′ UTR) between the cap and the start codon, the coding sequence, and a 3′ UTR between the stop codon and the poly-A tail. The UTRs help control when and how much the mRNA is translated. Note: UTRs are parts of exons. Exons are not the same thing as "coding sequence".
| Processing step | Where on the RNA | Main jobs |
|---|
| 5′ cap (7-methylguanosine) | 5′ end | protects from breakdown; export; ribosome binding |
| Poly-A tail | 3′ end | protects; controls lifetime; export; translation |
| Splicing (spliceosome) | intron-exon boundaries (GU...AG) | removes introns; joins exons; alternative splicing |
Worked exampleExample 1: How long is the mature mRNA and the protein?
A gene's pre-mRNA has (in order) exon 1: 210 nucleotides, intron 1: 950, exon 2: 135, intron 2: 2400, exon 3: 300. The 5′ UTR is the first 60 nucleotides of exon 1, and the 3′ UTR is the last 135 nucleotides of exon 3. Ignore the cap and tail.
(a) How long is the mature mRNA? (b) How many codons are translated, including the stop codon? (c) How many amino acids are in the protein?
- (a) Add only the exons: 210 + 135 + 300 = 645 nucleotides.
- (b) Coding sequence = 645 − 60 (5′ UTR) − 135 (3′ UTR) = 450 nucleotides. 450 ÷ 3 = 150 codons.
- (c) The last codon is the stop codon, which does not code for an amino acid: 150 − 1 = 149 amino acids.
- Check: the pre-mRNA was 3995 nucleotides; about 84% of it was intron.
(a) 645 nucleotides; (b) 150 codons; (c) 149 amino acids.
Worked exampleExample 2: Spot the splice sites
This short (made-up) pre-mRNA section contains one intron. The exons are in capitals and the intron in lower case:
5′-...GCAAGguaaguc...cuuuucagGAUGC...-3′
What will the joined sequence be at the exon-exon junction, and why are the bases "gu" and "ag" important?
- The intron begins with gu and ends with ag; these are recognized by the spliceosome's snRNAs.
- Everything from the g of gu to the g of ag (inclusive) is removed.
- The exon ending ...GCAAG is joined directly to the exon starting GAUGC...: ...GCAAGGAUGC...
The junction reads ...GCAAGGAUGC...; GU and AG mark the intron's two ends so the spliceosome cuts in exactly the right places.
RNA processing in medicine
- Spinal muscular atrophy (SMA) is a serious inherited disease in which the SMN1 gene does not work. A similar backup gene, SMN2, usually skips one exon when it is spliced, so it makes very little working protein. The drug nusinersen is a short, modified strand of nucleic acid that binds the SMN2 pre-mRNA and makes the spliceosome include that exon, so more working protein is made. It is approved in Canada.
- mRNA vaccines, such as the COVID-19 vaccines used in Canada, are designed like a mature mRNA: they have a cap, UTRs, a coding sequence and a poly-A tail so cells will translate them. Katalin Karikó and Drew Weissman won the 2023 Nobel Prize in Physiology or Medicine for discovering a change to the nucleosides that stops the immune system from destroying such mRNA before it is used.
Just for funThe human dystrophin gene is so long that making one pre-mRNA from it is estimated to take about 16 hours. That's a full school day, plus homework, plus a hockey game, for one RNA.
Hands-on activitySplice a sentence
You need: a strip of paper about 60 cm long (tape shorter strips together), a marker, scissors (use them carefully) and tape.
- Write this "pre-mRNA" along the strip in capital letters, with no spaces:
CAPTHEBIGGUXXQZJKWAGREDFOXGUPLMNOPQRSAGATETHEJAMTAIL
- Find each intron: it starts with GU and ends with AG. (Careful: the first intron starts at the GU after "THEBIG".)
- Cut out each intron exactly from its G of GU to its G of AG, and tape the exons together.
- Read your mature "mRNA" and put in spaces.
- Alternative splicing: make a second copy, but this time also remove the exon "REDFOX" (as if it were skipped). What changes in the meaning?
What to look for: after splicing you should get CAP THE BIG ... and the full answer is CAPTHEBIGREDFOXATETHEJAMTAIL: "(cap) THE BIG RED FOX ATE THE JAM (tail)". Skipping an exon gives "THE BIG ATE THE JAM", a different "protein". If you cut one letter off in the wrong place, the words after it come out garbled, just like a reading-frame shift.
Your turn
1Name the three processing steps for eukaryotic pre-mRNA and the end (5′, 3′ or middle) each affects.
5′ cap (5′ end), poly-A tail (3′ end), splicing (removes introns from the middle).
2Give two functions of the 5′ cap.
Any two: protects mRNA from breakdown at the 5′ end; helps export from the nucleus; is recognized by the small ribosomal subunit to start translation.
3What are the first two and last two nucleotides of almost every intron?
Begins with GU, ends with AG.
4What is the spliceosome made of?
Small nuclear RNAs (snRNAs) and proteins.
5A pre-mRNA has exons of 120, 90 and 180 nucleotides and introns of 500 and 800. How long is the mature mRNA (ignoring the cap and tail)?
120 + 90 + 180 = 390 nucleotides.
6How does alternative splicing allow about 20 000 human genes to produce many more kinds of protein?
Different combinations of a gene's exons can be joined, giving different mRNAs and therefore different proteins from the same gene, in different tissues or conditions.
More practice: warm-up
1Which part of a gene is kept in the mature mRNA: exons or introns?
2Which enzyme adds the poly-A tail?
A modified guanine nucleotide, 7-methylguanosine.
4Do bacteria splice their mRNA the way eukaryotes do?
No; bacterial mRNA is not capped, tailed or spliced like eukaryotic mRNA.
5What shape does a removed intron form?
A lariat (a loop with a tail).
More practice: on your own
1Explain why a mistake of one nucleotide at a splice site could ruin the whole protein.
Codons are read in groups of three. Removing one nucleotide too many or too few shifts the reading frame, so every codon after the junction is read wrongly, usually giving a useless protein and often an early stop codon.
2A mature mRNA is 1200 nucleotides long without its tail. The 5′ UTR is 75 nucleotides and the 3′ UTR is 228 nucleotides. How many amino acids does the protein have?
Coding = 1200 − 75 − 228 = 897 nucleotides = 299 codons, including the stop codon, so 298 amino acids.
3Why is a long poly-A tail associated with an mRNA that is translated for longer?
The tail is slowly shortened in the cytoplasm, and the mRNA is broken down once the tail is short. A longer tail means the mRNA survives longer, so more protein can be made from it.
4A student says, "Exons are the parts of the mRNA that code for protein." Correct the statement.
Exons are the parts kept in the mature mRNA. They include the coding sequence and the 5′ and 3′ untranslated regions, which are not translated.
5Explain how nusinersen helps people with spinal muscular atrophy.
It binds the SMN2 pre-mRNA and changes splicing so that a normally skipped exon is included. The resulting mRNA codes for a working SMN protein, partly making up for the missing SMN1 gene.
Challenge
1Human insulin is made in bacteria by genetic engineering. Explain why scientists insert a DNA copy of the mature mRNA (called cDNA) rather than the human gene from a chromosome.
The chromosomal gene contains introns. Bacteria have no spliceosome, so they cannot remove introns; the introns would be translated, giving a wrong protein. A cDNA copy, made from mature mRNA with reverse transcriptase, has no introns, so bacteria can translate it correctly. (You will see this in The Biotechnology Toolkit.)
2Researchers find that in liver cells a gene's mRNA contains exons 1, 2, 3 and 5, while in muscle cells it contains exons 1, 2, 4 and 5. Suggest how the two cells produce different mRNAs from the same DNA, and predict what is similar about the two proteins.
Both cells transcribe the same pre-mRNA, but tissue-specific splicing proteins direct the spliceosome to include exon 3 in liver and exon 4 in muscle (alternative splicing). The two proteins share the parts coded by exons 1, 2 and 5, so they probably have similar overall structure and some shared function, with one region different, perhaps adapting the protein to each tissue (assuming the exons keep the reading frame).
3A mutation changes the GU at the start of intron 2 of a gene to AU. Predict two possible outcomes for the mRNA.
The spliceosome can no longer recognize that splice site. Possible outcomes: (1) intron 2 is kept in the mRNA (intron retention), adding extra codons, often including a stop codon or a frameshift; (2) the spliceosome uses a different nearby GU (a cryptic splice site) or skips exon 2 or 3, giving a shortened or altered protein. Either way the protein is likely abnormal.
Quiz
1. Which modification is added to the 5′ end of a eukaryotic pre-mRNA?
3. A pre-mRNA has exons of 150, 300 and 210 nucleotides and introns of 1000 and 400 nucleotides. How long is the mature mRNA (no cap or tail)?
4. What carries out splicing?
5. Which statement best describes alternative splicing?
6. A mature mRNA has a 5′ UTR of 50, a coding sequence of 600 and a 3′ UTR of 150 nucleotides. How many amino acids are in the protein?
Big idea (D3.3, D3.2): in eukaryotes the first RNA copy of a gene is edited before use: a cap goes on the 5′ end, a poly-A tail on the 3′ end, and introns are spliced out. Alternative splicing lets one gene make several proteins.
How to help: Ask your teen to explain the "splice a sentence" activity to you and then relate each part (cut, join, cap, tail) to the real process.
Common difficulties:
- Thinking introns are not transcribed (they are, then removed).
- Thinking exons = coding sequence (exons also include the UTRs).
- Forgetting to subtract the stop codon when counting amino acids.
- Applying splicing to bacteria (bacteria do not splice their mRNA this way).
The activity uses scissors; supervise as needed.