Lecture 25: RNA splicing

Question 1

gene structure eubacteria vs. eukaryotes

Answer 1

• eukaryotes has introns
• most human genome is repetitive
  *

Question 2

Intron discovery

Answer 2

• found in viral and eukaryotic genomes at same time
  
  • adenovirus: Phillip Sharp
  • Hemoglobin: Alec Jeffries
    *

Question 3

general Splicing process

Answer 3

• genomic DNA to pre-mRNA to spliced mRNA
  
  • splicing must maintain ORF or nonsense proteins will be produced
    *

Question 4

sequencing markers for position of splicing

Answer 4

• 5’ splice site: donor site GU
• 3’ splice site: acceptor site AG

Question 5

Structure of spliced intron

Answer 5

• intron removed in lariat form
  
  • lariat formed by transesterification
• exons are joined by second transesterification

Question 6

Spliceosome: function, structure, process

Answer 6

• carries out RNA splicing
• catalyzed by snRNP (small nuclear ribonuecloeproteins particles)
  
  • complexes of RNA and protein
  • RNA components - uridine rich
  • each snRNA associated with 10 or more proteins
• cotranscriptional splicing: transcription of mRNA at same time as splicing
  
  • splicing factors loaded on phosphorylated CTD of RNA poly II
• process
  
  1. U1 snRNA recognizes 5’ (donor) splice site.
  2. BBP recognizes branch point
  3. U2AF recognizes polypyrimidine tract
  4. U2 snRNA recognizes branch point.

U2 binding causes release of BBP

    * U2 auxiliary factor: complex of two proteins
5. BInding of U4/U6 and U5 completes final spliceosome. Release of U2AF
    * trisnRNP - U4 and U6 are tightly linking with matching RNA.
        * recruit U5 (a catalytic components)
6. Rearrangement releases U1 from 5' splice site
7. U4 released from U6
8. U6 catalyzes frist transesterification rxn * exonic splicing enhancers *

Question 7

Lariat structure

Answer 7

• formed by spliceosome
• 2’ to 5’ bond
  
  • when broken, the intron is degraded

Question 8

exonic splicing enhancers (spliceosome)

Answer 8

• ehancers in exons
• bind serine-arginine rich proteins
  
  • they attract U1 snRNP to 5’ splice site and U2AF to 3’ splice sites

Question 9

Spliceosome Cotranscriptional splicing

Answer 9

• splicing at same time at mRNA transcription

Question 10

constitutive and alternative splicing

Answer 10

• consititutive splicing: two or more splice variants always made
• alternative splicing: regulated - splice variants only in specific cell times or at specific times of development
  
  • variety of related proteins synthesized from one gene
  • exon shuffling: exons usually encode diff parts of protein - combined by DNA rearrangements for new proteins
• *

Question 11

Gene Evolution with Introns

Answer 11

• introns speed up evolution with exon shuffling
• exon duplication
  
  1. duplciation of exon
  2. mutation of the duplicated domain
  3. changed proteins
• exon shuffling
  
  • exons mixed and matched
• splicing mutations
  
  • cis - disruption of:
    
    • alternative
    • constitutive
  • trans - disruption of:
    
    • basal factors
    • regulatory factors
    • eg mutations in snRNPs
• eg tryosinase related protein (TYRP1)
  
  • causes different colored cats