Exam 3 (Part 1)

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1. mRNA surveillance mechanisms can attack mRNAs that are broken or not polyAdenylated. Describe amethod that you could use that would cause a cell to produce lots of broken mRNA with a particularsequence. (The mRNA should be an endogenous RNA, not one that is added to the cells.)

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You could target an siRNA to the middle of an mRNA—would result in cleavage of the mRNA. You could use ribozymes that bind to a specific sequence of the mRNA and also have endonucleaseactivity (like the self-splicing introns in prokaryotes)

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2. What is the "Last Exon Rule? Explain how Nonsense Mediated Decay mechanisms have contributed tothis rule.

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This rule refers to the observation that stop codons (not PTCs) in most genes are located in the lastexon or 50nt from the end of the penultimate exon (less than about 50nt upstream from the lastintron). NMD mechanisms use the EJC as a marker to degrade mRNAs with a stop codon more than50nt upstream of an EJC. Thus NMD would essentially select against the survival of mRNAs with astop codon positioned in upstream positions (with respect to EJCs). Because of NMD, there is noselective pressure to maintain protein-coding genes with an upstream stop.

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3. The figure to the right hints that histone proteins areregulated at the level of mRNA stability, while the SLBP proteinis regulated at the level of protein stability.

a) How are histone mRNAs regulated? b) What is the nature of the 3' end of these histone mRNAs?

c) How is the 3' end of these histone mRNAs generated?

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a) The special class of Histone proteins, known as replicativehistones, are upregulated at the beginning of S phase and arerapidly degraded at the end of S phase. The mRNAs have astem‐loop, and when the SLBP is bound to this structure in Sphase, the mRNA is protected from degradation and the histonegets translated.

b) The 3' end does not have a poly A tail. The stem/loop is gene encoded.

c)The nuclear polyA polymerase does not add untemplated A residues. The mRNA has a U7 snRNA bindingsite. The U7 snRNA, the SLBP, and other proteins help position and recruit an endonuclease, whichcleaves the transcribed histone mRNA. Later, a TUT enzyme adds U residues to the end of the mRNA,facilitating degradation post-S phase.

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4. a) What are CUTs?

b) Compare and contrast how the 3' end of a CUT is made versus a regular mRNA

c) What role do CUTs play in cells?

d) Where in the genome might you find a CUT?

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a) cryptic unstable transcripts. Long (200‐800bp) capped and polyadenylated non-coding RNAs foundin eukaryotes. They are often transcribed in the opposite direction from close-by mRNAs

b) Regular mRNA: RNA Pol2CTD directs the activity of nuclear polyA polymerase to add many As to the 3'end of a cleaved mRNACUTs have Nrd binding sites, which recruit TRAMP, which adds a small number of A residues, but moreimportantly, recruit the exosome. The exosome does not degrade the CUT completely, but provides it witha 3' end.

c) They have been observed to downregulate gene expression by virtue of their ability to recruit SETenzymes, which have histone methylation activity. (observed in yeast retrotransposons). CUTs have alsobeen observed to help establish gene expression boundaries in chromatin.

d) look up

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In class, we discussed the proteins and domains that are found in a prokaryotic exosome-like complexknown as the “degradosome”. We also saw some slides of the predicted structures of archaeael exosomeswith each domain depicted as a different color. The slides were taken from a review paper published in2006, and since then, we have learned much about eukaryotic exosomes. Data from the archaeal exosomewas obtained first, and as the information on eukaryotic exosomes became available, the similarity inprotein domains and overall structure between eukaryotic and archaeal exosomes caused many scientiststo anticipate additional similarities in function. Such assumptions regarding function were not bourne out.Adding to the confusion, are data that turned out to be artefactual (spurious). (Careful what you read outthere!) For example, like the archaeal exosome, the human exosome is also a ring-like structure. Thehuman exosome contains six proteins that have a single domain with homology to RNase PH. In addition tothe six RNase PH-like proteins, an additional 3 or 4 proteins also comprise the “exosome ring”, and theseproteins have S1 and KH domains that you heard about in class. However, unlike the archaeal exosome, theRNase PH-like proteins in eukaryotic exosomes are not catalytically active. (Some early experimentssuggested they were. Oops.) The key amino acids that are required for catalytic RNase PH activity are notpresent in the eukaryotic exosome. In eukaryotic exosomes, the exosome ring associates with additionalproteins that supply the catalytic activity to the complex. The exoribonuclease catalytic activity explainswhy some RNAs (aberrant mRNAs) are degraded while other RNAs (normal mRNA) are not—one is abetter substrate for the exoribonuclease (although modifications to the aberrant mRNA may be required inorder for it to be a good substrate). Now for the question. Exosomes from archae and some plants are phosphorolytic, whereas humanexosomes are hydrolytic enzymes. Assume that you do not have this information regarding humanexosomes. Assume that you can purify a functional plant exosome and a functional human exosome, andassume you have a target mRNA with an AU-rich element and some AU-rich element binding proteins(AUBPs).

a) How would you design a biochemical assay to determine whether the human exosome isphosphorolytic versus hydrolytic??

b) Why are the mRNAs with an ARE and the AUBPs included in this experiment?

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a) ARE-containing mRNA (radiolabeled) + AUBPs + Pi + human exosomeàIs mRNA degraded?ARE-containing mRNA (radiolabeled) + AUBPs + H20+ human exosomeàIs mRNA degraded?ARE-containing mRNA (radiolabeled) + AUBPs + Pi + plant exosomeàmRNA is degraded? ARE-containing mRNA (radiolabeled) + AUBPs + H20 + plant exosomeàmRNA is not degraded? You can run a gel to see if the mRNA is degraded.The human enzyme is phosphoryolytic if degradation occurs in the presence of Pi, and not in assayswithout Pi. If it is hydrolytic, the presence of the Pi probably will not make much difference.

b) This allows for mRNA destruction by the exosome.

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Premature termination codons can cause dominant mutations. It is often the case that a truncatedprotein can affect the function of a wild-type, full length protein. Around 30% of mutations in humangenes are caused by Premature Termination Codons, yet most of these mutations are recessive. Whatmechanisms prevent these mutations from being dominant?

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Nonsense mediated decay

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7. In the mid-1800s, Louis Pasteur first discovered a "killer" strain of yeast which has the ability to secrete toxicproteins that are lethal to non-killer strains. Killer yeast also harbor mechanisms that make them "immune" to the toxic effects of their own secreted protein. Due to his expertise in microorganisms, Pasteur frequently advisedproducers of beer, wine, and milk products. Killer yeast strains caught the attention of Louis Pasteur, as they wereobviously undesirable strains to have around when your goal is to make bread. It turns out, one of the more common toxin systems inyeast is linked to the presence of a double-stranded RNAvirus, the L-A virus. The protein toxins are produced by L-A virus genes, not the yeast genes. However, when someyeast genes are mutated in strains harboring this L-A virus,the mutant yeast become "superkillers". Superkillers areable to kill a larger number of susceptible cells incomparison to the regular killers (wild type cells infectedwith L-A virus). In our studies of mRNA surveillance, we read about Ski7and other Ski proteins. Yes, these are the yeast genes that,when mutated in L-A infected strains, give rise to aSuperkiller phenotype. Superkiller mutants are betterkillers because they secrete an increased amount of toxic protein. This increased secretion is directly due to anincrease in the level of production of the toxic protein in Superkiller mutants.

How do yeast ski mutants give rise to extra toxic protein? What is the normal role of yeast ski genes in L-Ainfected killer strains?

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Ski proteins are required for the exosome activity in the cytoplasm. Ski proteins help to recognize astalled ribosome, a situation that might occur when the mRNA is aberrant, or when translation isstalled for other reasons (like amino acid starvation or rare codon usage). Some important viralmRNAs may have features that make them look “aberrant” to the yeast cell. These features wouldbe predicted to trigger mRNA surveillance mechanisms, leading to degradation of the viral mRNA,including the viral mRNAs that encode the toxic protein. The fact that the virus is more toxic in a skimutant indicates that the normal role of the SKI protein on the viral mRNA is, indeed, an mRNAsurveillance role. The SKI protein facilitates degradation of some viral mRNAs; without SKIfunction, more toxic protein is translated because the corresponding mRNA is not degraded.

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8. List five different types of errors or features in mRNA that can cause that mRNA to be degraded by theexosome.

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Premature stop codon

No stop codon

Short polyA tail/recognition by TRAMP in nucleus

ARE sequence in 3’ UTR

Presence of other sequences that recruit Nrd1 (like the CUTs)

Recognition by Ski7

Modification at 3’ end by TUTase (like the histone mRNA)

Targeted degradation by siRNA or miRNA (plants)

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What experimental data provides evidence that translation is required for nonsense-mediated decay?(List evidence from TWO different kinds of experiments)

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NMD is disabled by insertion of hairpin or IRE in 5’ UTR (prevents translation)

NMD is inhibited by cyclohexamide

NMD is inhibited by Suppressor tRNAs

Mutation of the AUG start codon (prevents translation)

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10. Hemophilia is a genetic blood clotting disorder. It is often caused by mutations in genes in the blood clottingproteolysis cascade. These enzymes eventually lead to the controlled conversion of fibrinogen to fibrin, which is cross-linked at the site of a wound. Hemophilia patients are given replacement factor treatments of the blood clottingprotein that they lack. The proteins are injected directly into the patient’s blood stream. The replacement proteinscan be isolated from donated human blood, and this can be dangerous when you consider the potential to co-introduce viruses. Alternatively “recombinant factor” can be isolated from human tissue culture cells that have beenengineered to overexpress the blood clotting factor protein. Recombinant factor is safer but can be expensive, as thisis a chronic condition. You have the brilliant idea to overexpress the human blood clotting factor gene in dairy cowssuch that the corresponding protein accumulates in the milk. Milk is a biological fluid that normally accumulates lotsof protein, and milk is a safer biological fluid in comparison to human blood with respect to viruses. You plancarefully, using a bovine mammary gland-specific promoter, a human blood clotting factor cDNA, and bovine 5’ and 3’UTR regions that are normally present in bovine mammary gland-expressing mRNAs. Your DNA is designed perfectlyto allow for robust accumulation of human blood clotting factor protein in cow’s milk and you make sure that thereare no errors in the sequence; however, your transgenic cows do not produce very much human clotting factor. Youhave evidence that the mRNA is transcribed and transported to the cytoplasm of mammary gland tissue, yet themRNA eventually becomes degraded before much protein is translated. What other modifications to the codingregion could you make that might help to increase the yield of protein? Why?

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Optimize the codons in the human cDNA for expression in cows. Look for codons that are usedinfrequently in cows. Replace those rare codons with those that are more frequently used in cows.(The codons should code for the same amino acid.)

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11. In the reading material, you read about the TRAMP complex. What is the enzymatic function of thiscomplex of proteins?

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TRAMP is a multi-protein complex that has RNA helicase and poly(A) polymerase activity. The PAPadds a small number of As to the 3’ end of some “imperfect” mRNAs, and is involved in the normalprocessing of rRNAs and snoRNAs and tRNAs. [TRAMP is not your husband’s girlfriend, nor is it a spaghetti-eating dog.]

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What about mitochondria? Mitochondria also have mRNAs, as well as their own translationmachinery. Mitochondria also have their own PolyA Polymerase (mtPAP), which is involved in the polyadenylation of mitochondrial mRNAs. mtPAP actually impacts mitochondrial gene expression onmultiple levels. In human mitochondria, the polyA tail appears to stabilize mRNAs. (This is not the case formitochondrial polyA tails in other organisms.) Removal of poly(A) before mRNA degradation isaccomplished by a PNPase enzyme, as in prokaryotes. Also, many mitochondrial mRNAs lack a stop codon,and instead have a U at the ultimate 3’ end. Thus, when these mitochondrial mRNAs are polyadenylated, astop codon, required for translation termination, is generated.

Questions:a) Isn’t this cool?

b) Which stop codon do you think is generated by polyAdenylation of these human mitochondrial mRNAs?

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A)yes, yes

b) UAA

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13. The exosome is crucial for mRNA surveillance of defective or imperfect RNAs as well as in normalprocessing of different classes of RNAs. We now know a lot about the structure of the exosome. Below,provide an itemized list with the names of each of the exosome-associated proteins, their functions anddescribe any enzyme activities associated with the proteins.

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Each of the monomers of the exosome has proteins with interesting domains or enzyme activities. The S1 domain is an RNA binding domain originally observed in a ribosomal protein, but now found in alot of proteins that bind RNA. Likewise there is a protein with a K-Homology domain (KH domain). This is a domain commonly observedin RNA binding proteins. The interesting enzyme is the RNase PH domain protein. Together, these proteins contribute to thepolynucleotide phosphorylase activity of the exosome. These enzymes use inorganic phosphate, not water,to cleave the phosphodiester bond. Curiously, this reaction is reversible, but the biological significance ofthe reverse reaction is unknown.

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14. RNAs acquire polyA tails in the nucleus in two very different ways. In column A below, name twodifferent classes of enzymes that add A residues to the 3' end of RNAs. In column B, describe the nature of the A tail produced by each of these enzymes. In column C, list another important enzyme or protein that istypically associated with the polyA-producing enzyme.

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A name of enzyme nature of tail (think length) associated enzyme or protein Poly A Polymerase Longer stretch of A residues(hundreds) RNA polymerase CTD, forexample TRAMP Short stretch of A residues (tens) (Look at table question 14)

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15. There is an abundance of experimental data that supports the model that translation is required fornonsense-mediated decay, NMD. Some of this data is summarized below; however, one item in the list justdoesn't make sense or doesn't support the model. Circle the data summarization that does NOT providedirect evidence that translation is required for NMD. a) NMD of an mRNA with a Premature Termination Codon is inhibited by insertion of hairpin or IronResponse Element in the 5' UTR of this mRNA. b) NMD of an mRNA with a Premature Termination Codon is inhibited by cyclohexamide c) NMD of an mRNA with a Premature Termination Codon is inhibited when splicing is inhibited. d) NMD of an mRNA with a Premature Termination Codon is inhibited by Suppressor tRNAs.e) NMD of an mRNA with a Premature Termination Codon is inhibited by mutation of the AUG start codon in this mRNA.

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c) NMD of an mRNA with a Premature Termination Codon is inhibited when splicing is inhibited.

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16. 5-15% of human mRNAs contain AREs in their 3' UTRs.a) What is the consequence of having ARE sequences in the 3' UTR?

b) What protein directly binds to the ARE sequences, thereby providing further instructions?

c) What kind of genes have these ARE sequences? (what do these genes do?)

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a) The mRNA will be less stable/have a short half‐life in the cell

b) KSRP TTP for example

c) 5-10% of human genes have these sequences. They are associated with immune system function, cell cycleand other regulatory activities. These are natural sequences in the gene, not aberrant sequences ormutations.

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17. miRNAs are made by cells and used to (mostly) negatively regulate target mRNA. However, miRNAsdo not last forever. What is the first step that triggers miRNA degradation? (List the name of the enzymeand what it does.)

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TUT (terminal Uridinyl transferase) Adds un-templated Us to the 3’ end. This targets the miRNA fordegradation.

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18. a) What is no-go mRNA decay? (What kind of mRNA does this refer to?)

b) List at least two different cellular conditions that would lead to a no-go condition for an mRNA?

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a) mRNA surveillance mechanisms that act on mRNAs that are stalled in the elongation phase of translation

b) structural impediments in the mRNA; amino acid starvation or other conditions that limit charged tRNAs;less‐frequently‐used codons in the reading frame CAN lead to translational stalling, expecially undernutrient‐deprivation conditions.

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19. a) What is a no-body?

b) What kind of events and proteins are associated with no-bodys?

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a) A specialized region in the nucleolus that is associated with RNA surveillance, especially for rRNAs

b) surveillance or exosome activity, NRD1

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20. What kinds of aberrant mRNAs trigger non-sense mediated decay? (be specific)

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mRNAs undergoing translation with IN-FRAME, premature stop codons that are typically more than50nt upstream from the last splice junction

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21. What kinds of aberrant mRNAs trigger no-go decay? (be specific)

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mRNAs undergoing translation that have structural impediments (hairpins) or damage, mRNAswith infrequently used codons, mRNAs with other kinds of damage

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22. What kinds of aberrant mRNAs trigger non-stop decay? (be specific)

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mRNAs undergoing translation that lack a stop codon

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23. In the mRNA surveillance mechanisms listed above, ribonucleases act on the 5’ end and the 3’ end ofthe aberrant mRNA. List the names of the nucleases:

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5’->3’ exonucleaseXRN3’->5’ exonucleaseEXOSOME

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24. What is a TUT enzyme and describe a role does it plays in RNA surveillance:

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Terminal Uridylyl Transferase or RNA uridylyltransferase belong to the polymerase Beta family ofnucleotidyl transferases and catalyze the addition of multiple U residues to the 3’ end of RNAs. Thismodification occurs on some RNAs, such as histone mRNAs and pre-microRNAs, and thismodification leads to the degradation of the modified RNA.

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25. (1pt) TRUE or FALSE: mRNA surveillance mechanisms are specific to RNA Polymerase II transcripts.

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Flase

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26. List TWO proteins that are required to activate the ribonuclease (exosome) that is involved in aparticular mRNA surveillance mechanism

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Ski7

Rrp47

TRAMP4 or TRAMP5