Lecture 3 midterm 3

• mRNA capping


  Addition of 5' cap to mRNA
• Polyadenylation


  Addition of poly(A) tail to mRNA
• Parts of an mRNA

  • Exons
  • Introns
  • 5' UTR
  • 3' UTR
• mRNA splicing


  Removal of introns from pre-mRNA

  
• Spliceosome


  Machinery that facilitates the splicing process
• Nuclear export


  Steps for mRNA to be exported from the nucleus
• RNA degradation


  Steps for mRNA to be degraded and recycled
• Eukaryotic mRNAs uniquely undergo several key steps: 1) Addition of 5' Cap, 2) Addition of Poly(A) tail, 3) Splicing, 4) Nuclear Export

  
• The 5' cap protects the 5' end of the transcript from degradation by 5' to 3' exonucleases and serves as a molecular handle for proteins that bind to the transcript

  
• The 5' cap is a 7-Methylguanosine molecule added backwards onto the 5' phosphate of the transcript, then methylated at the 7 position

  
• How the 5' cap is added

  1. Guanylyltransferase enzyme binds to the CTD of RNA Pol II and adds the cap as the mRNA is being transcribed

  2. Cap-binding complex (CBC) then binds to the cap and remains tethered to the CTD

  
• The 5' cap helps recruit the mRNA to the ribosome for translation
• The poly(A) tail is a long tail of about 80-250 adenosines added to the 3' end of the mRNA
• How the poly(A) tail is added

  1. Pol II transcribes past the poly(A) addition site, where cleavage and polyadenylation factors cut the mRNA and polyadenylate polymerase adds the adenosines

  2. Poly(A) binding protein (PABP) then binds to the tail, protecting it

  
• The poly(A) tail protects the 3' end of the mRNA from degradation and helps recruit it to the ribosome for translation
• Exons


  Parts of the mRNA sequence that are present in the final transcript

  
• Introns


  Regions in between exons that don't encode amino acids and are removed by splicing
• Introns can contain sequences involved in gene regulation, and alternative splicing allows a single gene to produce multiple mRNA isoforms

  
• Splicing mechanism


  1. Branch point 2'OH attacks 5' splice site, severing bond between exon and intron

  2. Freed 5' exon attacks 3' splice site, severing bond and ligating exons

  3. Intron is released as a lariat

  
• Spliceosome


  Complex of small nuclear ribonucleoproteins (snRNPs) that facilitates the splicing process

  
• Nuclear export of mRNA

  1. Exportins and importins shuttle RNAs out of and into the nucleus

  2. mRNA export uses the TREX complex and is facilitated by exon junction complexes (EJCs) left behind from splicing

  
• RNA degradation


  1. 5' cap and poly(A) tail are first removed

  2. Transcript is then degraded by exoribonucleases in the exosome complex

  3. P-bodies may be involved in storage or degradation of mRNAs