Lecture #11 (Translation #2) Flashcards

Question

What happens to GCN4 expressiion if ORF4 AUG is mutated in media?

Answer 1

Answer - GCN4 epxression should go up - In non-starvation conditions ORF 4 distracts the ribosome form getting to GCN4

Answer 2

Answeer - GCN4 expression goes down (because ORF4 will be more distracting - Will get translation of ORF4 = will stop before GCN4 = no GCN4 expression

Answer 3

To study – make a reproter and get readouts --> then do ribosomal profiling Test ribosomal profiling using starvation (good model because know a lot about starvation) - mRNA seq = don in all ribosomal profling experiment

Answer 4

Chart: - mRNA seqeuncde in green – shows mRNA is there - Bottom chart = starvation ; Top chart = rich media In Rich media – see ORF1 being translated (have big peak of ribosomes in ORF1) + Less ribsomes in ORF2 (less tranlsation) + have a small amount of ribsomes in ORF3 + Few ribsomes in GCN4 - Rich media = not tranlsating GCN4 IN starvation – Have more ribosomes in GCN4 (Means GCN4 is expressed and being translated) - ALSO Have many ribosomes before ORF1 (unexpected)

Answer 5

Looked at Upstream of the ORFs for near cognates of AUG that might result in ribosomes starting there --> THEY found non-conical start sites such as UUG and AUA upstram of the ORF = maybe those are start site = have ribosomes upstream of ORF After looking for conical starts sits – Made reporters for GCN4 expression (linked to lac z) where they mutated 1 of the non-conical starts sites or the other non-conical start sites or both of the non-conical start sites --> Asks how that affects regulation Results: Two bars for each condition in chart – Untreated and Starved - WT = no GCN4 in rich media ; have GCN4 in starvation - When Muatant of 1 uORF + Mutant of other uORF + mutant both you maintain regulation END - Don't know why have ribosomal reads upstream of ORF1 BUT the reads don’t seem to be important for the regulation

Answer 6

Uses ATF4 (similar to GCN4) Mammals = have 4 different eIF2a kinases --> affects eIF2 association with eIF2b ATF4 = Transcrtion factor ATF4 = has two upstream ORF - ORF1 = always translated - ORF2 = overlaps with ATF4 (when make ORF2 then NOT making ATF4) ; ORF2 in ATF4 = similar to ORF4 in GCN2 Normal = translate ORF 1 and ORF2 Starving = no eIF2a = make ORF1 BUT fail to make ORF2 --> get ATF4 --> ATF4 is a transcription factor that goes to the nucleus and turns on genes to save the cell from whichever stress it was

Answer 7

Integrated stress response refers to the shut down of translation AND a transcription response that is needed for cell survival The integrated stress response is a major mechanism of translational control downstream of four related kinases (GCN2, PERK, PKR and HRI)

Answer 8

At the end = need to turn off the stress signal at some point - IF ISR is not turned off = leads to apoptosis = needs to be regulated Solution – have phosphatases that turn off the phosphorylation

Answer 9

Many genes have uORFs and don’t use 95% rule of kozak (dones't use first AUG of the main instad uses AUG in uORFs) - uORFs are regulatory (know they are regulatory because mutate the upstream AUG and see if make more of downsream gene) = central to gene specific regulation

Answer 10

Chart shows the Level of phosphorylated eIF2a vs. Level of translation Overall: High phosphorylated eIF2a = low translation Low Phosphorylated eIF2a = have high translation Have zone in middle where fix things

Answer 11

ATF4 is such an important pathway that companies tried to make drugs that target the pathway --> did a drug screen How do you do a drug screen - Use reporter + cell line (NOT done in mice) --> need readout - Make a reporter with ATF4 (ATF4 is hooked up to firefly Leusiferase --> can look at cell to see if have leciferase) - In reporter whether or not you have luecerferase is a function of whether or not uORF1 or uORF2 blocks translation of ATF4 - Reporter is looking at expression of ATF4

Answer 12

Promotes the misfoloding of proteins in ER = actiavtes the ATF4 pathway through PERK Thapsagardin = activates PERK kinase --> PERK phosphorylates eIF2a --> Phosphorylation allows AFT4 to be expressed (because ribosomes can ignore ORF2) No Thamsargardin = not making ATF4 Have Thapsagardin = make a lot of ATF4 Shows reporter works

Answer 13

1. Once show reporter works--> Put cels in wells in dish and add durgs to each well and ask if there us anything that blocks production of ATF4 Chart - Thapsagardin signal – Want inhibition of ATF4 even when add Thapsagardin - Found 400 drugs that passed some threshold --> then narrowed to 28 --> then chose ISRIB 2. Once found ISRIB drug = need to titrate and show the binding constant (found trans vs. Cis compounds have different binding constants)

Answer 14

Drug that blocks expression of ATF4 in cells that are exposed to Thapsagardin How does ISRIB work: ISRIB binds to eIF2b

Answer 15

Because ISRIB binds to eIF2b = functions at the bottom of the pathway EIF2b = guanosine exchange factrs --> means ISRIB functions at the bottom of the pathway (means it should be effective on every feed in pathway (should work on PERK and HIR etc) ; should work for all GCN proteins)

Answer 16

ISRIB woks in certain titration range and then truns off EIF2b is active as a tetrome and less active as an octomer --> ISBRIB = activates the tetromeric form of eIF2b and inhibits the octomer ISRIB = Function like a Reohstat for eIF2b activity - Low amounts staples together eIF2b and enhances activity - Too much stabilizes tetramer rather than the octomer

Answer 17

ISRIB = used to target aging + head injury - Exapmple - given to rats after they have head injuries ---> Rats need to turn on ISR response BUT will need to turn it off and the wasn’t always happening - Because of the Rheostat behavior of ISRIB you can manipulate the pwathway in just the right way at the right concetration ATF4 pathway = dangerous pathway – IF not turned off it is bad

Answer 18

There are multiple ways to target the ISR (effects on health are not always predictable) - Many companies want to target this pathway - Pathway is an example of how to globally shut down translation and specifically express genes Example to target ISR = ISRIB (targets the last step of ISR)

Answer 19

Global Translation control #2 - CAP dependent regulations (connected to TOR)

Answer 20

TOR = growth regulator in Eukryotes (growing cells need TOR) - TOR = drives translation TOR function – regulates 4E - Level at which 4E is available dictates how much translation takes place (4E is a critical regulator)

Answer 21

4E binding proteins bind to 4E --> regulate 4E (when 4E binding proteins bind to 4E they block 4E from recognizing CAP) 4E binding proteins are regulated by phosphorylation --> Phosphorylate 4E and 4E binding proteins = 4E and 4E bidning proteins can’t intercat with one another + Phosphorylation of 4E allows 4E to bind to CAP = get tranlsation - Phosphorylation of 4E and 4E binding proteins promotes translation

Answer 22

TOR – affects translation by regulating 4E When amino acids are high cell growth is high --> TOR phosphorylates 4E binding proteins = stops the 4E binding proteins from binding to 4E = 4E is able to function – get translation When nutrients are low = TOR is off = 4E bnding proteins are not phosphorylation = have no translation

Answer 23

TOR sits on a lysosomes and responds to amino acid levels by interacting with key proteins that read out Amino Acid levels - Exmample - 1 Proteins looks at leucine + 1 proteins looks at methionin --> key amino acids that the TOR system is informed by to turn on/off

Answer 24

Rapa and Torin inhibit Tor

Answer 25

Western blot – shows phosphorylation states for TOR targets - Treatment groups = Vehicle (normal cells) + Rapa (inhibits TOR) + Torin (inhibits TOR) + No amino acid group - eIF2a – has two bands (top is the phoporylated state ; bottom is the protein) --> shows the eIF2a doesn’t talk to TOR (because no change in any of the treatment groups) - S6Kinase and 4EBP = major target of TOR --> in all three conditions (inhibiting TOR and AA starvation) = have a decrease in amount of phosyrlation of 4EBP + S6Kinase (Phosrylated in vechile and then blcok phosphorlyation in the inhibitors/AA starved)

Answer 26

Healthy cells = have a lot of polysomes on mRNA When add TOR inhibitor = number of polysomes decreases and 80S increases = have a block in tranlsation - Have global shut down in tranlation when turn off TOR or starved for amino acids

Answer 27

Translation efficiency = Ribosome footprint/mRNA

Answer 28

Looking to see if yo turn off all of the genes or if there are some that are still expressed (what mRNA are still bound to ribosome ween have Torin or no Torin) - Looks at translation efficiency --> asking how effectively mRNA is being translated --> SEE some mRNA are better translated and some are less well translated Results: 1. GCN4 = expressed in TOR inhibition (in the blue on the chart) - Blue = GCN4 – gene you make in crisis 2. Some genes are not tranlsated - Green = genes that you stop making (mRNA of genes that are not tranlsated under the conditions

Answer 29

TOP mRNA = genes that are not translated when you inhibit TOR TOPs (temrinal oligio pyrimidine) = all mRNAs have a 5’ CAP THEN a C nucleotide THEN a string of pyridimidines following the C nucleotide TOPs = All proteins that encode the ribsomes and tranlation factors (region responsible for tranlation) - No Amino acids = don’t wnat to make ribomes = don't translate TOP genes when have no TOR

Answer 30

Different websites look at different start sites - Found 100 mTNA in TOPs that are well regulated in known TOPs they already knew about - See that some websites didn’t calls something a TOP or they called it a TOP like mRNA (disrupted TOP) --> sows that make sure you look at the TOPs (how you define them is importnat) - When get a list of genes that are well regulated – look if they are TOPS or to and why they were defined as TOPs and are there different ways to define TOPs or where the Transcription starts sites is

Answer 31

TOPS are regulated by gene specific regulation ((specific motifs are recognized by specific factors that affects the mRNA)

Answer 32

4E = global Effects vs. TOPs has specific effect

Answer 33

Issue with Ribosome profiling in general – taking an average (Translation effcicney is averga ribosome density per mRNA) Issue with average – Might have a population of mRNA are tranlated and some that are not (can’t know answer based on avergae density of ribosmes) Solution: Take polysome and sequence each fraction across polysome – seqence each fraction (Sequnec each mRNA) in the population and where it is

Answer 34

Left charts: See Actin in deep fraction (not in light faction) (fraction 6/7) ; actin is an example with high translation (lots of polysomes) Right Chart: DMSO Treated - 1. DMSO A260 is well translated (lloks the same as active) 2. ALL the non-TOP mrNA (chart at the bottom) = all in the deep fraction (look like actin) = well tranlsated 3. TOP gene - Every TOP is half loaded with ribosomes and half not loaded (averge Ribosomal profile would not be able to show the biphasic natre) Add Torin (inhibit TOR) - get global movement in if the mRNA is translated 1. In the non-TOP with TOR (bottom right chart) = see shift in many mRNA = have global movement in whether or not the mRNA is being translated 2. In TOP gene = fully turn off the TOPs (TOPs are specifically truned off)

Answer 35

There are multiple inputs into the CAP recognition complex TOR = feeds to S6K + Feeds to 4E Binding proteins (when not 4E avaible) + Feeds to MINK1/2 (phosphorylates 4E binding proteins) SHOWS that many signaling pathways are important for cell growth – signals feed at the cell membrane and mTOR is a major regulatory node

Answer 36

TOR = improtant pathway for cancer + cell growth + development Hyperactivity if pathway = found in majority of cancer

Answer 37

Regulation is most common at the stage of translation initiation both in normal conditions and in cells that are stressed (TOPs and uORFs)

Answer 38

Viruses routinley target CAP dependent tranlsation When virus enters the cell they do not have enough to survive on their own = need to use things in Eukaryotes host cell BUT they also need to fight their way out - Need to use reagents in the host cell for their own virus homeostasis BUT they can’t use everything in the host cell because the host cell needs to stay alive Solution = Irises

Answer 39

Iris = internal ribosome entry site Iris = mechanism in Eukaryotes that allow for evading general shut down (mechanism of specific gene regulation)

Answer 40

Viruses – make their own mRNA BUT doesn’t want to compete with the Eukryotic system because need the host cell to survive Viruses will cleave PolA binding protein + cleave 4G complex + decaps host mRNA --> ALL disrupts the elements for CAP dependent translation --> once CAP dependent mechansim is detsroyed cells/viruses still need to have a way to translate important things for host cell to survive Solution = Iris --> iris = dones’t need CAP – just needs ribsomes and tRNA and syntehtases

Answer 41

How to look for Iris function – use bicystronic reporter assay Bicytronic reporter has 2 different reproters (Example Renella and Firefly – have different colors so you can measure both indepentely How does bicytronic work: - Normal cell – scanning happens = reacignize the first ORF BUT should not recgiizie the 2nd ORF (Because the first ORF would lead to normal termination and recycling and would not get tranlation of the second ORF) - In Bicyrtonic - Put in an iris seqeunce ) to see if can get expression of the second reporter --> if get expression from second ORF then there must something sepcial about the sequence that does NOT depend on CAP

Answer 42

Reporter is danergous because need to be certain that there is 1 peice of RNA in cell that inlcudes both reproters (IF insertion encodes a promoter or splicing feature and get exprression of the 2nd ORF in normal CAP dependent tranlation)

Answer 43

Iris has complex 3D structure Example - Cricket --> escapes the need for all Initiation factors in the cell because it will bind to the ribosome between large and small SU - Cricket = Evades eveyrthing the cell normally needs for RNA folding

Answer 44

Overall - Cricket places the Ala codon in the A site of ribosome --> doesn't need to recgizne CAP or eiF2a to load initiator tRNA - Doesn’t need Met tRNA because Cricket has secondary structure that looks like tRNA/mRNA decoding event (looks liek start site BUT it is not start site it is a peice of mRNA) How does this happen - Cricket will bind to the P site using tRNA like structure then Ala can come in --> translation can start

Answer 45

Irises = like the shine delgarno sequence - Because shine delgarno alows you initiate internally on mRNA and places the codon of interstest where you wnat to start things

Answer 46

In bacteria – the main way to regulate expression of specific mRNA is to target the Shine delgarno sequence (regulate the availibity of the SD seqence) Done using RNA binding proteins to that bind to the SD and block SD under certain conditions - Small RNAs regulate access to SD and tRNA binding proteins can binds to SD

Answer 47

Bacteria = have riboswitches

Answer 48

Riboswitches = small elements in 5’ UTR of bacterial genes - Riboswitches = fold to a 3D structure in cis to gene of interest Ex. can bind thymine pyrophsphate --> block the thymine pyrophasphate gene from being expressed - Lots of thymine pyrophasophate = don’t need to make the gene that makes thymine pyrophate (auto feedback loop) What happens: 1. High Thymine - thymine binds = SD is sequestered in some RNA secondary structure 2. Low Thymine - the riboswitch structure is different = SD is available = get translation

Answer 49

Can be done through: 1. 5’ UTR binding porteins 2. 3’ UTR elements that regulate intiation through CAP dependent mechansims (MORE common) - 3’ UTR regulation = very common (very common during development)

Answer 50

Have example 5’ UTR binding porteins that is iron regulated Protein does’t bind or binds Iron based on level of iron = regulates mRNA involved in iron biology - Proteins binds in absence of Iron and blocks translation (physically blocks scanning)

Answer 51

Overall - Elements binds to 3’ UTR and affect if the ribosome intiated in 5’ UTR

Answer 52

Have different proteins and mRNA in the worm development Example (image) - oocytes are moving down a track and are released for fertilization --> Can see that there are different patterns of where the proteins are found

Answer 53

To show that the proteins are not regulated by transcription – put the genes under a constitutive promoter + put GFP fused on the 3’ UTR of the the different mRNA --> ask where GFP is expressed - Constitutive promoter means the protein would be made everywhere/all of the time) Results – show that GFP expression a the begining or the middle or the end in a manner that depends on which 3’ UTR they have (shows 3’ UTR drives protein expression in a localized manner)

Answer 54

miRNAs binds to the 3’ UTR for regulation (common regulating feature in mammalian genes) - There are 100s of miRNA and each miRNA has multiple targets = many genes are regulated by miRNAs - miRNA is not turning genes fully on/off more like changes regulation by 20% miRNA = Usually binds to the 3’ UTR- miRNA binds to 5’ seed sequences in context of AGO protein --> targets the mRNA for degredation

Answer 55

Translation and decay are tightly controlled – no CAP = not translated and the mRNA will be degraded (shows a close relationship between translation and decay)

Answer 56

Experiment using Xenopus eggs – Xenopus oocytes are full of mRNA waiting for fertilization occurs (transcription already occurred but waiting in the oocytes for translation) As an oocyte that is waiting to be fertilzied there is no polyA tail on the mRNA BUT instead it is bound by a collection of proteins

Answer 57

Overall - based on whether or not 4E is compenent and can recruit 4G - Shows how feature in 3’UTR can regulate specific mRNA rather than the whole cell How does this work - In the tranlsatonally inactive mRNA AAUAAA that is normally recognized by CPSF in the nuclues is not recognized + 4E is bound to CAP BUT 4E is not bound to 4G instead 4E is bound to Maskin (Maskin is bound to CPEB) CPEB binds to the CPE element in the 3’ UTR - Maskin has 4G like element THEN a kinase becomes active when sperm enters teh cell = get phosphyration = phosphrylatiion leads to the recuritemnt if CPSF that will clevae AUAAAA seq --> get PAP -> PAP puts on the polyA tail -> PolyA binding proteins is recruied (recuites eiF4G--> eIF4G have affinity to displace the maskin - Example of a sequence in 3’ UTR that does what Torin did genrally (Torin played with how likley will you make active complex based on 4E binding proteins that sequester the protein)

Answer 58

Bacteria - Much regulation happens through the 5’ UTR of bacterial mRNAs Eukryotes - Much regulation happens through the 3’ UTR of eukaryotic mRNAs

Answer 59

Nonsense mediated decay = quality control pathway - RNA polymerase is busy making lot of things = it ends up making a lot of nonsense --> have nuclear for qualty control BUT many things can escapae

Answer 60

When there is an early stop codon nonsense mediated decay heppens - The cell knows if there is a mutation that gives a premature stop codon --> mRNA is recognized as weird and targeted for decay

Answer 61

Overall - Cells knows based on the fact that stop codons are almost always in the terminal exon because of NMD + based on the fact that during splicing the splicing machinery makes an EJC - By contrast the start codon can be in any exon Process: Normally - Normal stop codon = clear all EJC then hit Stop codn (no EJC downstream) When have premature stop codon - ribosome sees a stop codon upstream of the EJC they know is is a bad mRNA = recruits UPFs (UPFs = proteins that are important for nonsense mediaed decay) --> degraded the mRNA

Answer 62

Collection of proteins that clamps on the RNA - EJC marks all of the splice sites - EJC = deposited on the exon:exon boundary after each splicing event - If have 20 exons there are 19 EJCs Function - EJC helps pull the mRNA out of the nucleus Important protein in EJC = 4A3 (clamps on RNA with complex around it)

Answer 63

Nonsense mediated decay modulates phenotypes of genetic diseases Chart = shows diseases that are associated with an early stop codon - Early stop codon is bad because in the middle of protein you want to make = now get short protein = bad Example – defects in beta globulin - if have an earlier stop codon you are the disease is not as sever as if you have a late stop codon - Earlier stop codons = recognized by NMD - Late stop codon = less recognized by NDM (disease is more challemging)

Answer 64

It is better not to make a truncated protein (because the protein can have dominant effects and disrupt things) - If mRNA transcript is targets for decay it is better - Phenotypes associated with late stop codons that escape NMD = more detrimental than ones that are early Overall: Recognize early stop codon --> target mAN for decay = won’t keep making bad protein IF have early stop codon (but later in the transcript) = make toxic protein that doesn’t function

Answer 65

Rachel thinks that many lnc RNAs are transcriptional noise that came out of nucleus that failed to be recognized by nuclear exosome as nonsense Say lncRNA is RNA junk - on average there will be an AUG at some point in RNA+ on averae will encounteer a stop codon within 3 out of 20 codons (random RNA 1 in 20 codons ins a stop codons) = on averge will make a short protein --> get long tail that wasn’t translated = on averge these are targeted by NMD When do experiments and sequence lncRNA in a cell and put in NMD inhibitor = get more of them because the lncRNA are targeted by NMD

Answer 66

mRNA damage Example mRNA damage = premature polA in ORF (have poly A tail before the stop codon) --> end up geting Lys, Lys, Lys residues in a row (get many AAAAAAA = get Lys amino acids n a row) - Cell knows this string is a mistake and has systems to recognize this issue)

Answer 67

Goal for dealing with problematic mRNA = get rich of the mRNA + degrade the protein + Get ribosome off the mRNA Bectria = use tmRNA to fix mRNA damage

Answer 68

Group in japan working in the RNA --> notice the end looks like Ala tRNA (saw GU wobble in the stem --> thought if the RNA gets charged with alanine) fount that tmRNA does get charged with Alanine Group in Australian – trying to express a mammalial protein --> couldn't express the protein --> Sequence the protein --> Find all of the mamamlial protein they are looking for have a C terminal tag of 11 AMino acids (11 Amino Acids were not part of the vector) --> Do BLAST --> find that the only thing that encodes that 11 amino acids seqence in bacteria is the tmRNA - When trying to epxress the protein they Ffund that there are a lot of degredation products on teh gel

Answer 69

Ala tRNA is amazing – only thing synthetase cares about GU wobble bp at position 3 in the stem (if put a GU wobble BP into any tRNA alanine sythetase will add an Alanine to the tRNA)

Answer 70

tmRNA has an end with Alanine AND sequence that is ORF that endocdes 11 Amino acid sequence What does tmRNA do - fixes stuck ribsomes - When have a ribosome that is stuck need the ribosome back + protein is bad because never reached the stop cond so we don’t wnat that protein How does tmRNA fix probelm - tmRNA = feeds into the A site of the ribsome with help of protein --> brings Alanine to A site using part of tmRNA that looks like tRNA) --> makes peptdie bond --> feeds in ORF on the tMRNA and ribsome tranlsates that ORF --> at the end of the ORF have a stop codon = then ribosome can leave

Answer 71

1. Had a ribosome stuck on mRNA that is now come to a termination codon so it can be freed 2. 11 Amino acids sequence is a degron tag (targets that protein for decay in bacteria by CLIPXP) = not accumulating bad protein)

Answer 72

Uses ubiqtuitin + E3 liages + endonucleases + recyling machinery Uses system when have truncated messages or polyA sequences in the middle or damaged mRNA that ribosome can’t get past Process – recruits E3 ligases that target the nascent peptide of decay + bring in endonucleases that lead to decay of mRNA - Need to decay mRNA because you don't want to keep translating a bad mRNA - Have factors that will do that help dissocate the ribsome

Answer 73

At the heart of BOTH the Eukyotic and bacteria systems for dealing with problematic mRNA = a ribosome collision How does the cell know that a ribosome is in trouble -- Because when a ribsome is slow because it is stuck --> THEN another ribosome will collide with the stuck ribosome --> when ribosomes collides it acts as a recruitment surface that everything comes in on - After collision the downstream events are distinct