Bio 99 Final Lec 12

  1. Eukaryotic mRNAs uniquely
    undergo: (4)
    • 1) Addition of 5’ Cap
    • 2) Addition of Poly(A) tail
    • 3) Splicing
    • 4) Nuclear Export
  2. Pre-mRNA
    The immediate product of transcription
  3. mRNA
    fully processed transcript
  4. The 5' Cap
    What is it?
    Every transcript gets a modification made to it’s 5’ end shortly after transcription begins. This is the 5’ cap. Only Pol II transcripts (i.e. mRNA) are capped.
  5. The 5' Cap
    What is it for?
    • Protects 5’ end of transcript from degradation by 5’ to 3’ exonucleases
    • Molecular handle used by many proteins that bind to transcripts
    • Required for traditional translation (i.e.“capdependent translation”)
  6. The 5' Cap
    What's the capping molecule?
    • 7-Methylguanosine
    • It’s a GTP that is added backwards onto the 5’ phosphate of the transcript, then methylated at the 7 position. Sometimes the first two bases of the mRNA get methylated too
  7. How the 5’ cap is added
    How is the cap added?
    An enzyme called guanylyltransferase adds the cap. It’s also called the “capping enzyme”.
  8. How the 5’ cap is added
    How does it do it?
    Guanylyltransferase binds to the CTD of Pol II. As the mRNA leaves Pol II, the 5’ end sticks to the CTD, allowing guanylyltransferase to cap it.
  9. How the 5’ cap is added
    What happens next?
    After cap addition, the guanylyltransferase falls off, and a protein called CBC (cap-binding complex) binds to the cap. CBC and the 5’ end of the transcript remain tethered to the CTD
  10. Capped mRNA binds to ribosomes
    • Made capped and uncapped mRNA
    • Both types had 32P, but capped mRNA also had 3H
    • Mixed with ribosomes to allow binding
    • Ran through density gradient (ribosomes will sink to bottom)
    • If mRNA binds to ribosomes, it will sink to bottom
    • Could detect where capped mRNA ended up by 3H signal
  11. Polyadenylation
    What is it?
    A long tail of adenosines (about 80 to 250) added to the 3’ end of every mRNA. Also called a poly(A) tail.
  12. Polyadenylation
    What is it for?
    • Protects 3’ end from degradation
    • Helps recruit to ribosomes for translation
  13. Polyadenylation
    How is it added? (2 steps)
    • Step 1 – Pol II transcribes past the poly(A) addition site (AAUAAA). An enzyme attached to the CTD cuts the mRNA downstream of the AAUAAA. A GU-rich region downstream of the cut site is also involved.
    • Step 2 – polyadenylate polymerase (PAP) begins adding adenosines. Poly(A) binding protein (PABP) attaches to the tail, protecting it.
  14. Which is NOT true about 5’ capping?
    A. The cap is generated by guanylyltransferase
    B. The cap is a GTP added backwards to the 5’ phosphate of the
    first rNTP
    C. The cap is methylated at the 7 position
    D. A protein called CBC binds to the cap, tethering it to the CTD
    E. All are true
  15. Where exactly is the poly (A) addition site?

    A. )
  16. Splicing 
    What is it?
    Eukaryotic mRNA transcripts initially have a lot of extra sequence that doesn’t encode amino acids. These have to be removed before the mRNA can be translated.
  17. Exons
    parts of the mRNA that are present in the final transcript
  18. Introns
    regions in between exons that don’t encode amino acids and are removed by splicing
  19. Most exons encode
    amino acids, except: (2)
    • 5’ untranslated region (5’ UTR)- contains sequences that help initiate translation
    • 3’ UTR-  contains poly(A) addition site, some regulatory sequences
  20. Why have introns?
    Introns can contain sequences involved in gene regulation (e.g. intronic enhancers)
  21. Alternative splicing
    More than one type of mRNA can be produced from a single gene. Some exons are skipped. These alternatively spliced mRNAs are also called isoforms.
  22. Alternative processing
    Poly(A) site choice-
    • A gene can have more than one place for
    • poly(A) attachment
  23. One gene produces two protein products via alternative splicing ____
    poly(A) site choice
  24. How does the splicing machinery know where the introns and exons are?
    by sequences in the intron
  25. All introns contain:
    • 5’ splice site – typically GU, plus other sequence
    • 3’ splice site – typically AG, plus other sequence
    • Branch site – internal A nucleotide, just upstream of 3’ splice site
  26. Splicing Mechanism- Simple (3 steps)
    • 1) Branch point 2’OH attacks 5’ splice site
    • • Severs bond between exon and intron at 5’ site
    • • Attaches branch point to 5’ splice site on intron (both are used up)
    • 2) Severed 5’ exon is “activated” to attack the 3’ splice site
    • • 3’OH of 5’ exon is free to attack 3’ splice site
    • • Severs bond between exon and intron at 3’ site
    • • Attaches 5’ exon to 3’ exon
    • 3) Intron is released as a “lariat” shape
  27. What machinery does the splicing?
  28. snRNPs
    small nuclear ribonucleoproteins – complexes that form the spliceosome
  29. snRNAs
    small nuclear RNAs, the heart of the snRNPs, ~100-200 nucleotides
  30. 5 snRNPs are in the
    U1, U2, U4, U5, and U6 (no U3)
  31. Is U1 a snRNP or a
    • Both
    • The U1 snRNP contains the U1 snRNA + some
    • other proteins
  32. Splicing Mechanism, Complex Version: What you need to know (4 bullet points)
    • snRNPs facilitate each step of the splicing reaction
    • • snRNPs are recruited essentially in numerical order
    • • RNA sequences on the snRNAs interact with sequences on the 5’ and 3’ splice sites as well as the branch point
    • • The RNA of the snRNPs essentially catalyzes the splicing reaction
  33. Capping, polyadenlyation, and splicing all occur during _______
  34. All capping, polyadenlyation, and splicing involve the machinery binding to the ____. __' end of transcript remains tethered to ___ the whole time
    CTD; 5; CTD
  35. The nucleophile of the first step of spliceosome-mediated premRNA splicing is the:

    C. )
  36. Which part of the intron is the last one involved in splicing?

    A. )
  37. Which process occurs last?

    B. )
  38. Eukaryotic Transcription and Translation are
    separated by _______
  39. After processing, mRNAs must be _____ to the cytoplasm in order to be translated
  40. Some RNAs must be re-_____ back into the nucleus (like snRNAs after they assemble with proteins into snRNPs)
  41. After translated, mRNAs must be
    recycled and ______
  42. Non-protein-coding RNAs: ______ and _______ shittle RNA out of or into the nucleus
    • exportins
    • importins
  43. Export
    • Ran-GTP binds to exportin and RNA
    • • Complex exits nucleus
    • • Ran-GTP hydrolyzed to Ran-GDP,
    • releasing the RNA
    • o Exportin is shuttled back into nucleus to begin again
  44. Import
    • Ran-GDP binds to importin and RNA
    • • Complex re-enters nucleus
    • • Ran-GDP converts back to Ran-GTP, and releases RNA
  45. mRNA export
    • Uses TREX instead of exportin/Ran-GTP
    • • TREX recruited to Pol II
  46. Splicing helps target mRNAs for export
    • Splicing creates exon junction complexes (EJCs) at borders between exons
    • Export machinery uses EJCs to help target spliced transcripts
  47. Why use EJCs?
    To distinguish pre-mRNA from mRNA
  48. RNA Degradation
    • After translation, mRNA must be degraded and recycled
    • 5’ cap and poly(A) tail must first be removed Then, transcript chewed up by exoribonucleases
  49. Exosome
    complex of about 10 ribonucleases that degrade mRNAs
  50. P bodies (processing bodies)
    Unclear function. Storage place for mRNAs. Also involved in RNA degradation
  51. The transport receptor, exportin, transports:
    A. mRNA to the nucleus when associated with GDP-bound Ran
    B. noncoding RNA to the cytoplasm when associated with GTPbound Ran protein.
    C. noncoding RNA to the nucleus when associated with GDPbound Ran protein.
    D. mRNA to the cytoplasm when associated with GDP-bound Ran
  52. Which proteins are bound at the exon-exon borders and facilitate
    transport of the spliced mRNA from the nucleus to the cytoplasm?

    B. )
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Bio 99 Final Lec 12