RNA processing

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RNA processing
2012-05-12 08:53:05
RNA processing miska

RNA processing
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  1. 5'cap: structure
    • m7G5'ppp5'N... (eukaryotes)
    • p5'N (prokaryotes)
  2. 3' poly(A)tail: length
  3. 5'cap: roles (4)
    • Protects RNA from 5' endonucleases
    • Increases intron splicing efficiency
    • Required for export to cytoplasm
    • Required for efficient translation initiation
  4. Exceptions to 3' poly (A) tail
    histone mRNA, some viral mRNA
  5. Roles of poly (A) tail
    • Protects mRNA from 3'exonucleases
    • Controls degradation rate
    • Enhances rate of translation
  6. Termination of transcription in eukaryotes
    • No precise end
    • pre-mRNA cleaved between AAUAAA and GU/U rich sequence
    • poly(A) polymerase adds ~250 As
  7. Difference between genomic and cDNA libraries
    • genomic: from whole genomes: harder to make and maintain, more detailed, used to determine geen structure, characterise sequences that regulate transcription, express bacterial proteins
    • cDNA: from mRNA (using reverse transcriptase with poly-T primer) - determine RNA sequence, express eukaryotic proteins, characterise regulatory sequences in UTRs
  8. Molecules required for splicing
    • snRNPs (small nuclear ribonucleoprotein particles)
    • With snRNAs eg U1, U2 (uridine rich)
    • Transcribed by RNA pol II (except U6 from pol III)
  9. Splice site consensus sequence
    • Introns: begin with 5' GU
    • end with A at branch point, then polypyridmine tract, then AG
  10. Base pairings between consensus sequences and snRNAs
    • U1: GU
    • U2: branch point A
    • U2AF: polypyrimidine tract
  11. Splicing process
  12. Modes of alternative splicing
    • Use alternative 5'/3' splice sites
    • Splice/don'tsplive
    • Exon skipping
    • May introduce termination codons/change reading frames
  13. Example of gene with many splicing sites, can cause cancer
    • WT1 - Wilms tumour suppressor gene
    • >20 isoforms
    • Wilms tumour: most common solid tumour of childhood
    • WT1: TF, recognises GC/TC rich, 2 domains, which truncated forms lack
  14. 2 domains in WT1
    • N terminal activation: pro and glu rich
    • C terminal DNA binding: 4 Zn fingers
  15. Example of RNA editing
    • Hydrolytic deaminations: C->U or A-> I
    • can affect splicing/introduce a premature stop codon/increase coding capacity
  16. Example of C->U editing (bad)
    • NF1 encodes neurofibromin, a tumour suppressor, contains GTPase activating protein (GAP) which interacts with Ras.
    • Editing: CGA -> UGA at GAP domain
    • Inactivates tumour suppressor function