EukaryoticGReg.txt

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Stephevette
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84360
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EukaryoticGReg.txt
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2011-05-08 23:10:20
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Splicing
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Final Exam Cards
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  1. Name three types of catalytic RNA splicing
    • Group I
    • Group II
    • RNase P
    • hammerhead

    Does this type of splicing require proteins?

    NO
  2. What are the in vitro requirements for splicing Group I intron from Tetrahymena?
    GTP (or guanosine) and Mg++ and not nuclear extracts (i.e., no proteins)
  3. Why are proteins required for activity of most RNA catalysts?
    Presumably to fold the RNA into the appropriate structure.
  4. True or False: Only a few RNAs are sufficient for catalytic activity alone?
    True
  5. How are Group I and II introns distinguished?
    By different structures and mechanisms of catalysis.
  6. What if perhaps a protein is required for Catalytic RNA splicing but it is resistant to removal by phenol and protease digestion how would you prove it?
    In vitro transcribed RNA using purified polymerase had same requirements and spliced at the same correct positions as in vivo and as purified RNA
  7. What is the size of the nt RNA genome of satellite tobacco ring spot virus?
    359nt
  8. What virus replicates by rolling circle generating tandem genome repeats?
    Satellite Tobacco Ring Spot Virus
  9. What virus folds into a hammerhead structure and is this active or inactive?
    Satellite Tobacco Ring Spot Virus and this structure is the ACTIVE form.
  10. True or False: Cleavage of Satellite Tobacco Ring Spot Virus is NOT self catalyzed.
    FALSE
  11. How can the cleavage of STRS Virus into monomers be reproduced?
    By using two short RNA oligos

  12. RNA oligo 02 on top RNA oligo 01 on bottom. What does this represent?
    Two RNA oligos reproducing catalytic activity of STRS virus.

    Hammerhead ribozymes made to function in trans (01 causes cleavage of 02 to P1 and P2).
  13. What are the types of alternative splicing?
    Simple and Complex
  14. Features of simple alternative splicing?
    one promoter, one splicing pattern and one polyadenylation site
  15. Features of complex alternative splicing
    multiple promoters, muliple splicing pattterns and poly (A) sites, and multiple opne reading frames (ORFs)
  16. During complex alternative splicing what is the consequence of multi promoters?
    Creates diff translation initiation condons (proteins have different N-termini)
  17. True or False: Vetebrate pre-mRNAs do not have incredible sequence complexity?
    FALSE
  18. How to avoid recognition of cryptic splice sites?
    The 5'SS: AG is well conserved followed by the third g is more you move down the sequence the less conserved the nucleotide sequence becomes. However, splice site sequences do not contain sufficient specificity to distinguish cryptic and bona fide splice sites.
  19. What are the four evidences of exon definition?
    • 1. Mutation of a 5' splice site results in exon skipping, not intron retention.
    • 2. Strengthening a 5' splice site boosts splicing of the upstream intron
    • 3. Exon length effects recognition (too large and too small are problems)
    • 4. Isolated exons assemble a complex indistinguishable from the A complex. Both found to contain U1 and U2 snRNPs and U2AF65/U2AF35.
  20. Mutation of a 5' splice site results in what?
    Exon skipping, not intron retention
  21. What causes intron retention?
    If splice sites are recognized as pairs across introns, a 5'ss mutation will cause the introns to be retained or use of the "next best" cryptic 5'ss.
  22. Exons are defined by what?
    Two splice sites and will be skipped if:

    One splice site is mutated or will use an internal cryptic 5'ss as a "defining" 5'ss.
  23. What does strengthening of a 5' splice site do?
    Boosts splicing of the upstream intron.

    Tested on a weak alternative exon
  24. What lessons are learned from strengthening a 5' splice site?
    • 1. Alternative exons have weak signals
    • 2. improving signals make them constitutive
    • 3. demonstrates functional interaction across exons
  25. True or False: Exon length affects exon recognition (too large and too small are problems)
    TRUE
  26. The mechanism of exon definition is partly known and involves a highly conserved family of proteins called _________?
    SR proteins
  27. Describe SR proteins
    They contain Serine-Arginine rich domain which is now called the RS domain.
  28. The Arginine Serine (RS) repeats of SR proteins act like ________?
    Molecular Velcro
  29. Components of teh spliceosome that recognize the 3' splice site and the 5' splice site contain_________.
    RS domains which interact with RS domains of SR proteins for cross-exon bridging
  30. What subunit of U2AF recognizes the 3' splice site?
    65kDa subunit
  31. What subunit of U1 snRNP recognizes the 5' splice site?
    70K
  32. Cross-intron bridging, involved in intron definition of vetebrate introns. What causes this?
    SR proteins via the same RS-RS domain interactions
  33. Yeast intron containing genes usually contain ______ intron so there is only intron definition and no ______ definition.
    • ONE
    • EXON
  34. SR proteins mediate interaction across exons to do what?
    define exons
  35. True or False: SR proteins are evolutionarily conserved protein family (Drosophila to humans)
    TRUE
  36. SR proteins contain 1 or 2 _______ and ________ domain of alternating arginines and serines.
    • RRMS
    • "RS"
  37. What is phosphorylated in SR proteins?
    Serines
  38. What does phosphorylation of SR cause?
    Affects intranuclear and nuclear; cytoplasmic distribution and spliceosome assembly.
  39. What is essential for splicing?
    SR proteins

    SR proteins also complement splicing deficient cytoplasmic extracts
  40. SR proteins promote binding of what proteins to pre-mRNA?
    • U1
    • U2AF
    • U4/5/6
  41. What regulates alternative splicing?
    SR proteins
  42. RS domains promote _________ interactions contributing to interactions across exons and introns.
    protein:protein
  43. SR proteins binds 5'__________ and exonic _____________.
    splice site and exonic splicing enhancers
  44. True or False: SR proteins do not function in nuclear to cytoplasmic transport and mRNA translation.
    FALSE
  45. True or False: Exons and introns contain auxiliary splicing elements.
    TRUE
  46. True or False: Most if not all exons utilize auxiliary cis-acting elements.
    TRUE
  47. Name four catergories of zuxiliary elements.
    • ESE=exon splicing enhancer
    • ESS=exon splicing silencer
    • ISE= intronic splicing enhancer
    • ISS= intronic splicing silencer
  48. What are the best characterized auxiliary splicing elements?
    ESEs
  49. True or False: Computational analysis indicates that most exons are likely to have ESEs.
    TRUE
  50. True or False: Mutations within some exons caused cryptic splice site selection.
    FALSE

    Exon skipping
  51. Describe ESEs
    Typically short (6-12nt) variable and found in multiple copies within exons.
  52. SR proteins bind to what and promote exon inclusion?
    ESEs
  53. Computational analysis has also identified large numbers of ESSs which do three things...
    • 1. block use of adjacent splice sites
    • 2. function in alternative splicing
    • suppress potential cryptic splice sites nearby

    ESSs bind to hnRNPs H and A1
  54. Exon recognition is a cumulative sum of several features:
    • 1. splice site strength (similarity to consensus)
    • 2. exon lenght (50-200 best)
    • 3. exonic element
    • 4. intronic element
    • 5. strength of competing splice sites
    • 6. secondary structure
  55. True or False: Different exons are recognized in different ways
    TRUE
  56. What is trans-splicing?
    Intermolecular splicing that joins exons from separate transcripts.
  57. What organsims trans-splice only and what does this mean?
    Trypanosomes

    All mRNAs contain teh same 39nt leader sequence at the 5' end.
  58. What organisms have both cis and trans splicing?
    Nematodes
  59. RNase H digestions in permeabilized Trypanosome cells demonstrated ___, ____ and ____ required for cis-splicing.
    U2, U4 and U6
  60. True or False: The mechanism of trans-splicing is essentially the same as cis-splicing.
    TRUE
  61. What are the similarites between trans and cis splicing?
    They use same two transesterification reactions.
  62. What are the differances between trans and cis splicing?
    Because there are two separate RNAs, the reaction produces a branched intermediate rather than a lariat in vitro.
  63. True or False: In nematodes, cis and trans-splicing occurs in the same pre-mRNA
    TRUE

    Trans and cis spliced introns distinguished by structure, not sequence
  64. True or False: Trans-splicing is used for coordinating gene regulation.
    TRUE
  65. About___% of genes in C. elegans are polycistronic?
    15%
  66. What sequence in required for SL2 trans-splicing?
    AAUAAA (3' forming signal)

    downstreams genes have different spliced leader SL2
  67. What is determinative for trans-splicing?
    Gene architecture rather than specific sequence
  68. What are two major types of 3' end formation for RNA polymerase II transcripts?
    • 1. Cleavage/polyadenylation (vast majority of genes)
    • 2. replication-dependent histone 3' end cleavage
  69. 3' end formation of the vast majority of Pol II (non-histone) genes is a three step process
    • 1. Cleavage of the pre-mRNA
    • 2. Polyadenylation (addition of ~200 adenosine residues to the mRNA 3' end)
    • 3. Transcription termination
  70. Three consensus sequence elements required for cleavage/polyadenylation:
    • 1. AAUAAA strongly conserved in vertebrates and absolutely required
    • 2. G/U-rich region downstream from cleavage site. Quite variable
    • 3. A few genes have upstream activating sequence (UAS) (mostly viral)
  71. Trans-acting factors required for polyadenylation.
    • 1. cleavage-polyadenylation specificity factor (CPSF)
    • 2. Cleavage-stimulatory factor (CstF)
    • 3. Cleavage factors I and II (CFI) and (CFII)
    • 4. Poly(A) polymerase (PAP)
    • 5. Poly (A) binding protein PABP
  72. Describe six specifics of CPSF
    • 1. 160kDA subunit binds directly to AAUAAA
    • 2. interacts with CstF, PAP and CFI
    • 3. binding of CPSF to RNA is weak, biochemical purification of stabilizing factor indentified CstF
    • 4. cooperative interactions with CstF the first step of polyadenylation complex assembly, forming stable complex that recruits other factors
    • 5. 73 kDa subunit proposed as endonuclease
    • 6. Required for both cleavage and polyadenylation
  73. Describe four specifics of CstF
    • 1. three subunits 77, 64 and 50 kDa
    • 2. 64 kDa protein binds the RNA; UV crosslinks to the G/U-rich region
    • 3. binding of CsfF to G/U rich region requires AAUAAA, reflecting the cooperative interaction with CPSF
    • 4. CstF 77kDa subunit interacts with the 160 kDA CPSF subunit
  74. What component interacts with the phosphorylated CTD.
    Cleavage factors I and II

    Called cleavage factos but now thought not to perform the endonuclease cut.
  75. What is PAP?
    Poly(A) polymerase that is the distributive addition of poly (A) tail following endonucleolytic cleavage.

    First 10-15 A's require factors bound to AAUAAA
  76. Whats the function of PABP?
    nuclear protein that binds poly (A) and mediates, in part, teh upper limit of ~200 A's. PABP binds to the growing poly(A) tail and interacts with CPSF and PAP stabilizing PAP binding and allowing rapid A addition. Length control results when these interactions are made difficult by long poly(A) tails.
  77. Be able to identify each and function.
    • Subunits 100, 30, 73 and 160 CPSF
    • Subunits 77, 64 and 50kDa CstF
    • Subunits 25/68 and ? CFII and II
    • Subunits 82 PAP
  78. What are the roles of the poly(A) tail?
    • 1. binds PABP
    • 2. increases mRNA stability. mRNAs are de-adenylated prior to degradation
    • 3. stimulates translation. PABP interacts with translation initiation factors (eIF4E/eIF4G), stimulates binding of 40S ribosomal subunit to initiate scanning for AUG codon.
    • 4. cytoplasmic polyadenylation is a mech to regulate translation of developmentally important proteins (short tail=no translation; long tail=translated)
  79. What to RNA when PABP and eIF4E were added?
    Circular RNA
  80. The polyadenylation complex consist of what components?
    CPSF, PAP, PABP
  81. Transcription termination is coupled to ________ in that _________________ are required.
    • 3' end formation
    • polyadenylation [poly (A)] signals are required
  82. After poly (A) tail is 10nt, what does PABP do?
    Stabilizes complex with CPSF and PAP, and converts PAP from distributive enzyme (dissociates after incorporation of each nt) to fully processive elongation to full poly (A) tail of 200-250nt.

    After 250nt, PAP becomes distributive again by feedback inhibition of PABP and CPSF
  83. True or False: Splicing enhances polyadenylation and polyadenylation enhances splicing
    TRUE
  84. What happens if you mutate the AAUAAA hexanucleotide?
    Inhibits removal of the last intron
  85. What happens if you mutate the 3' splice site of the last intron?
    inhibits polyadenylation
  86. TRUE or FALSE: The 3' splice site of the last exon enhances polyadenylation efficiency
    TRUE
  87. TRUE of FALSE: Mutation of polyadenylation signals reduce splicing efficiency of last intron
    TRUE
  88. True or False: RNA processing is cotranscriptional
    TRUE
  89. The transition from initiation to elongation during transcription is accompanied by phorphorylation of the CTD by _______, largely on Ser___. More phosphates are added, largely on Ser ___.
    • TFIIH
    • Ser5
    • Ser2
  90. The phosphorylated CTD serves as a docking platform for factors involved in ____, _____ and _____.
    splicing, capping and polyadenylation
  91. What is the spatial relationship between transcription and splicing within the nucleus?
    • The nuclear structures
    • 1. perichormatin fibers (PF)
    • 2. interchormatin granule clusters (IGC)
    • 3. coiled bodies (now called Cajal Bodies) (CB)
  92. Name two facts about perichromatin fibers (PF)
    • 1. contains bulk of newly synthesized pol II RNA by pulse-chase experiments
    • 2. immunofluroescent staining with antibodies to splicing components (SR proteins and snRNP proteins) show diffuse reticular staining that joins IGCs
  93. Name two facts about interchormatin granule clusters
    • 1. No nascent RNA labeled by pulse chase
    • 2. 20-50 immunoflurescent "speckles" per nucleus using antibodies to splicing components and some contain Pol II
  94. Name two facts about coiled bodies (now called Cajal Bodies) (CB)
    • 1. no nascent RNA labeled by pulse chase
    • 2. few intense foci using anti-SR protein antibodies, snRNP
  95. TRUE or FALSE: RNA processing occurs in perichromatin fibrils, probably co-transcriptionally.
    TRUE
  96. What is the function of PF and CB?
    Could be sites of storage, sites of pre-assembly or recycling.
  97. Antibodies to splicing factors stain IGC ("speckles") in quiescent nuclei. Speckles are thought to be ____________ for splicing and other factors.
    "storage areas"
  98. 3 main points about RNA editing
    • non-templated nts generated in mRNA
    • Mostly in noncoding regions
    • two types
    • -insertion/deletion of a base (in/del U)
    • -base modification (C->U, A->I)
  99. Describe the mitochondrial genome of trypanosomes.
    minicircles (encode gRNAs) and maxicircles (encode nonfunctional RNAs)
  100. What is the function of trypanosome gRNAs
    transfer sequence info to maxicircle RNA to restore ORF

    gRNAs are recycled to multiple mRNAs
  101. Trypansosome have weak RNA-RNA interactions so ________ RNP complex involved (proteins encoded in nuclear DNA)
    Editsome

    Up to 50% of RNA needs editing to be translatable.
  102. True or False: ADARs have the capacity for unaided site-specific RNA binding and editing.
    TRUE
  103. Name four RNase III proteins
    • Human Drosha
    • Human Pasha
    • Human Dicer
    • Ago proteins (in RISC)
  104. What is included in the miRNA microprocesser complex
    Drosha and Pasha
  105. Whats function of Pasha
    Stabilized Drosha (prot-prot) interaction. Single Negative Feedback Loop.
  106. What are the components of Ago protein (in RISC) and what does each do?
    • PAZ-docking for 3' end of small RNA
    • MID-anchors 5' terminal nt
    • PIWI-slicer activity (catalytic site)
  107. 3 rules for miRNA-mRNA interaction
    • 1. must have perfect bp-ing of highly conserved seed region (nts2-8)
    • 2. bulge/mismatches in central region of duplex prevents cleavage by Ago
    • 3. some complementarity in 3' region of miRNA to stabilize the interaction
  108. 3 ways miRNAs regulated?
    • 1. regulation at the processing step
    • 2. regulation by feedback loops
    • 3. change length of 3' UTRs
  109. What enzyme is responsible for 5'-3' mRNA degradation?
    XRN1
  110. What enzyme is responsible for 3'-5' mRNA degradation?
    Exosome which is a multi-protein complex cable of degrading various types of RNA.
  111. What is the enzyme responsible for mRNA decapping?
    DCP2
  112. What are the two mechanisms of mRNA decay?
    deadenylation and decapping
  113. Name four types of mRNA decay.
    • ARE-mediated decay
    • NMD
    • miRNA-mediated decay
    • Fos CRD-mediated decay
  114. What protein recruits CCR4?
    c-fos coding region determinant binds UNR which also binds PABP
  115. The exosome is found where and what does it do?
    • nucleus and cytoplasm
    • processing and degradation
  116. What do Ski proteins do?
    Unwinds secondary structures
  117. 2 ways mRNAs are localized?
    localized protection of mRNAs from degradation (bind proteins that protect/diffuse RNA degraded)

    Cytoskeletal transport of mRNAs (anchor proteins localize mRNAs transported along cytoskeleton)

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