RNA Splicing

Question 1

mRNA in prokary. are a direct copy of the DNA gene sequence. TRUE OR FALSE

Answer 1

TRUE

Question 2

mRNA in eukary. are a direct copy of the DNA gene sequence. TRUE OR FALSE

Answer 2

FALSE, splicing is required in eukary. to remove non coding intervening sequences known as introns.

Question 3

Why must introns be removed from mRNA?

Answer 3

1. Often contain stop codons that would prematurely terminate translation.
2. May shift the translational reading frame of downstream exons.

Question 4

By what mechanism does splicing of introns occur?

Answer 4

The “spliceosome” (an RNA-protein complex of ~300 proteins and 5 small RNAs) catalyses the splicing of introns from pre-mRNAs.

Question 5

Composition of the spliceosome

Answer 5

-5 snRNPs (snRNA plus associated protein factors / small nuclear ribonucleoproteins)
U1, 
U2, 
U4 + U6,
U5, 
U4 + U5 + U6
- ~200 polypeptides

Question 6

How are introns recognized?

Answer 6

Splice Site consensus sequences
Most introns have the same general structure

Exon1 5’ Splice site Branch Point 3’ Splice Site Exon2

The branch point is an invariant A residue.

RNA-RNA base pairing occurs between the intron branch point and intron-exon junctions and specific regions of complementary RNA sequence in the trans-acting snRNA components of the spliceosome snRNP subunits

U1 snRNA base pairs across the 5’ exon-intron junction
U2 snRNA base pairs across the intron branch point

U2AF (U2 Auxiliary factor) facilitates U2 binding

Question 7

The Spliceosome Cycle

Answer 7

• Occurs in a step-wise fashion and proceeds through a series of intermediate complexes as the separate snRNP subunits and additional protein splicing factors are recruited onto the pre-mRNA
• The assembly of spliceosomes occurs on nascent pre-mRNA
• A separate spliceosome assembles independently on each intron
• Introns need not be removed in a 5’ to 3’ order

Question 8

Splicing a pre-mRNA involves two/three, simultaneous/sequential reactions, both/all of which are catalysed by the Spliceosome

Answer 8

Splicing a pre-mRNA involves TWO, SEQUENTIAL reactions, BOTH of which are catalysed by the Spliceosome.

Question 9

Splicing Step 1

Answer 9

Splicing Step 1 is a ‘trans-esterification’ reaction where the 2’ hydroxyl group of the branch site A residue attacks the phosphodiester bond between exon 1 and the 5’ end of the intron
Produces a free exon1. and a “lariat” intron attached to exon2.

Question 10

Splicing Step 2

Answer 10

Splicing Step 2 is another ‘trans-esterification’ reaction where the 3’ hydroxyl group of the terminal ribose of the free 5’ exon attacks the phosphodiester bond between the 3’ end of the intron and the adjacent exon.
Causes excision of the “lariat” intron, leaving spliced mRNA

Question 11

Protein Isoforms

Answer 11

Protein isoforms are groups of proteins that share similar structures and often have similar and/or related functions.
Protein Isoforms can be generated either from separate, homologous genes, or via Alternative Splicing.

Question 12

Consequences of Alternative Splicing

Answer 12

Alternative splicing many leave intact or remove sequences that affect:
Nucleic acid binding/recogniton
Protein targeting/stability
Protein-protein interactions
Post-translational modifications and associated activities

Question 13

Splicing Mutations and Disease

Answer 13

~15% of human diseases are caused by splicing mutations.
Causes of mis-splicing: Mutations in trans-acting elements, Mutations in cis-acting elements, Changes in the posttranslational modifications of splicing factors etc.

Altered ratios of alternatively spliced isoforms affects many cancers (anti-apoptotic isoforms, proto-oncogenes etc.)

Question 14

Regulation of Alternative Splicing

Answer 14

Sequence elements within exons and introns control splice site selection: (Can be either intronic or exonic for both)

• Splicing Enhancers (Promote exon definition)
• Splicing Silencers

Exon Splicing Enhancers (ESE) bind to proteins rich in Ser and Arg.