RNA splicing and processing

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Molecular biology
of genes

RNA splicing and processing

From gene
to mRNA.

In eukaryotes, RNAs transcribed from their genes require further processing to become mature and functional. Splicing often occurs as the pre-mRNA is still being transcribed.

Interrupted genes

01

Exons and introns

In an interrupted gene, the sequences retained in the mature RNA, the exons, are separated by intervening sequences called introns. Introns are transcribed into RNA along with the exons but are then removed by splicing, and the exons are joined in the mature RNA.

02

In all eukaryotes

Interrupted genes are found in all groups of eukaryotes. Most genes in multicellular eukaryotes are interrupted; only a few genes in unicellular eukaryotes, such as yeast, are.

03

Short exons, long introns

Genes vary widely in the numbers and lengths of their introns, but a typical mammalian gene has 7–8 exons. The exons are relatively short, only around 100–200 bp, and the introns are long, approximately 1 kb.

Exons
7–8
An exon
100–200 bp
An intron
≈1 kb
A gene of eight exons and seven introns, and its mRNA once the introns are removed, drawn to the same scale.

Processed in the nucleus

Splicing occurs in the nucleus, along with other modifications to the new RNA. The transcript is capped at the 5′ end, has its introns removed and is polyadenylated at the 3′ end; it is then transported through nuclear pores to the cytoplasm, where it is translated into protein.

The spliceosome

The splicing reaction occurs within a large complex, the spliceosome, which is larger than a ribosome. It forms by the sequential assembly of snRNPs and other proteins onto the pre-mRNA, and its primary role is to bring the 5′ and 3′ splice sites together before any cleavage occurs.

It has a mass of about 12 MDa. Five snRNPs, each a single snRNA with its proteins, account for almost half of it; the rest are splicing factors, needed to assemble it, bind the pre-mRNA and create its catalytic centre, and proteins involved in other stages of gene expression.

Alternative splicing

For the majority of genes in multicellular eukaryotes, one pre-mRNA gives several distinct mRNAs: over 90% of mammalian genes are estimated to undergo alternative splicing. The modes can be used individually or in combination for a single pre-mRNA.

Explore

Scene · 8 steps

The 5′ cap

RNA polymerase II pauses with the capping enzyme, a G is added in reverse by a 5′–5′ link and methylated at N7, the cap stops a 5′–3′ exonuclease that degrades an uncapped RNA, and CBP20/80 and eIF4E hold it.

Play ↗ Scene · 7 steps

Splice sites

The exon–intron junctions, the GU–AG rule and each site's consensus as a logo along the pre-mRNA, the branch site, a point mutation that stops splicing, and minor introns.

Play ↗ Experiment · 7 steps

Splice sites in pairs

Predict whether a hybrid SV40–β-globin intron is spliced and which cryptic site replaces a mutated branch site, and watch splice sites leave RNA polymerase II in 5′ to 3′ order.

Play ↗ Scene · 8 steps

Splicing proceeds through a lariat

Atom by atom in the spliceosome's core: the branch-site A's 2′-OH attacks the 5′ splice site, making a lariat with a 2′–5′ bond; exon 1's freed 3′-OH joins the exons; the lariat is then debranched and degraded.

Play ↗ Scene · 7 steps

snRNPs

Small RNAs with their proteins: the five snRNPs, U4/U6 joined with U5, the Sm and Lsm rings, and the snRNPs in a spliceosome.

Play ↗ Scene · 8 steps

The spliceosome cycle

Eight cryo-EM structures in one frame: U1 and U2 mark the intron, the tri-snRNP joins, U1 and U4 leave, the lariat forms, the exons are joined and the snRNPs are freed.

Play ↗ Experiment · 7 steps

U1 and the 5′ splice site

U1 snRNA pairs with the 5′ splice site base by base; predict what a mutant splice site, and a compensating mutation in U1, do to splicing.

Play ↗ Scene · 7 steps

The commitment complex

U1, U2AF and BBP/SF1 commit the pre-mRNA to splicing, SR proteins bridge the intron, and splice sites are paired by intron or exon definition.

Play ↗ Experiment · 8 steps

Alternative splicing

One pre-mRNA spliced six ways: predict which mRNA each choice of splice sites gives, then watch the introns loop out and the exons join.

Play ↗ Scene · 8 steps

Isoforms

What alternative splicing does: CaMKIIδ kinases sent to the cytoplasm, nucleus or membrane, Bcl-xL and Bcl-xS with opposite fates, and an mRNA degraded because of a premature stop codon.

Play ↗ Library · 17 molecules

The molecules, one by one

The capping enzyme and the cap-binding proteins, the snRNPs and their rings, the splice-site factors, a spliceosome, the debranching enzyme and the proteins alternative splicing makes, each in 3D.

Open ↗ Reading

The notes

RNA splicing and processing in full: the cap, splice sites, the lariat, snRNAs and the spliceosome, committing the pre-mRNA, and alternative splicing.

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