 One of the NSF graduate fellowship which only 10% of the graduate students get at Wisconsin or at Harvard And she was also an MBTG fellow and after she was done with using up all that money. She told me I don't pay her Enough, so I have to let her graduate With that I'll stop and let her tell you about the beautiful work. She's been doing that have not highlighted Oh, I have I have a mic That is not true you could have turned that on Okay, thank you a seam for that lovely introduction I was worried about what you would say but that was that was thank you for those kind words and Thank you to you in the audience for coming to hear about my research Especially those of you who traveled from far away to be here and those of you who might be streaming So I really appreciate it. Thanks What I'm showing you here in my title slide is an artistic rendition of what I've been working on For my thesis research and that is this molecular machine. You can think of it as a machine that makes RNA It's RNA polymerase 2 and the focus of my work has been studying this interesting part of the machine that we call the CTD or C terminal domain and My work has revolved around understanding how this domain Regulates the RNA making properties of this machine We all know that DNA stores information of life in the case of more complex eukaryotes like ourselves and mice That's billions of bases and in the case of my model organism Saccharomyces cerevisiae not shown to scale It's millions of bases. So despite their many differences They are all very conserved in their transcriptional machinery and that includes RNA polymerase 2 which has this CTD and pole 2 compared to pole 1 and pole 3 is uniquely tasked with transcribing of a very diverse Number of different types of genes because it transcribes protein coding genes What I'm showing you here is a cartoon of a typical messenger RNA mRNA Eventually gets translated. It's capped with a methylated guanosine and polyadenylated And so many Steps to get from this information to life, but one of the first points where regulation can occur is transcription and So what does the CTD look like so here is the amino acid sequence of the CTD in yeast And this is the amino acid sequence in humans. So If you take a look at it Do you find anything unusual about this? I hope what you can see is that it's highly repetitive In fact, this heptapeptide sequence is repeated 26 times with little Deviation in yeast and about 52 times in humans and it's very conserved deletion of the CTD is embryonic lethal and So the reason why the C terminal domain is so important for life is because it's required for Transcriptional events and co-transcriptional events to occur correctly. So not so much It's not so much important for the cataclytic activity of RNA polymerase. It's that RNA Proteins that are required for steps like capping so I'm showing you an example of The SEG1 capping enzyme bound to a portion of the CTD SEG1 comes in early during transcription to place this methyl guanosine cap and protect nascent RNAs from degradation and so The SEG1 enzyme for example will bind the CTD that's been phosphorylated on the fifth and seventh serine residues and in fact The CTD is highly phosphorylated at a number of residues not the proliens of course and it's the the phosphorylation is carried out by kinases which utilize ATP as a substrate and It's this Coordinated and sequential modification of the CTD that recruits the enzymes required for transcription to occur that that give it its its regulatory power and And so now that I've given you CTD 101 I want to introduce to you The CTD kinase that I've been studying And that is kin-28 in budding yeast and it's very homologous to this kinase in humans CDK7 and so this kinase phosphorylate serine 5 and 7 of the hepatad and And I mentioned already that the capping enzyme has high affinity to this combination of phosphomarks It's also known that So what I'm showing you here is a cryo-EM structure of the pre-initiation complex So before RNA polymerase 2 takes off from the promoter. It's trapped by this pre-initiation complex with mediator here in aqua and in yellow here is a portion of the CTD and kin-28 Is known to phosphorylate these residues releasing polymerase for mediator allowing initiation to occur so kin-28 is very essential Because of these functions, so it might come to you as a surprise that there was a question about whether kin-28 was required for RNAs to be produced and Just to summarize some of the confusion that existed early on about this requirement of kin-28 for RNA synthesis I have the slide here and so depending on the loss of function study used to study kin-28 There were contradictory or conflicting results So when a temperature-sensitive mutant was used where you heat shock kin-28 that's been randomly mutagenized That seemed like kin-28 was very essential for RNA production Whereas if you use the constitutively catalytic mutant of kin-28 with a tenuated kinase activity, it seemed like kin-28 function was not required and so Before I joined the lab another graduate student Elenita Cannon thought to use a different strategy to study kin-28 One that would have less indirect effects from inhibiting the kinase and so what I'm showing you here is a structure of CDK7 kin-28 and the ATP binding pocket is zoomed in and in this chemical genetic approach What she did was she modified a very conserved residue known as the gatekeeper And it's called the gatekeeper because it's kind of in the opening Where all the action happens and it tends to be a bulky amino acid So it's modified to a smaller amino acid that creates what you can think of as a pocket such that a chemical can bind with greater affinity and this chemical is an ATP analog and It has this bulky substituent that can only dock in if this mutation is made. She called this strain kin-28 AS and What she found Using this approach to inhibit kin-28 was that kin-28 was not required for RNA synthesis generally speaking And so this would be fine and you know, I wouldn't be standing here telling you about this But a couple years later Using the same exact strain from our lab the same strategy same inhibitor the complete opposite was found and The discrepancy the reason for the discrepancy that they cited was in accuracy with normalization Techniques and so there there's some confusion and in our in our community. There was also a Concern raised that this analog sensitive strain might be a leaky form of inhibition and that some modicum of ATP could still be binding and therefore that's why RNA synthesis was still going on so in the first half of my talk I'll show you how I answered this question and Put to rest I think Kin-28's role in promoting RNA synthesis so to address the concern about leaky inhibition in Collaboration with Jack Taunton who's at UCSF we developed an irreversible Kin-28 inhibition strategy So I say kin-28 inhibition strategy But really this approach is generally applicable to any kinase that you wish to study And it works off the same idea the same scaffold of having this gatekeeper mutation But to allow for irreversible inhibition. We also introduce Assisting to a conserved residue of the side of the ATP binding pocket And this allowed reactivity with an electrophilic inhibitor such as that a covalent bond was formed is formed and we call this inhibitor cmk for the reactive moiety chloromethyl ketone and We call this strain kin-28 is for irreversibly sensitive One of the the first assays we did to compare our strategy to the reversible inhibition strategy was to measure the growth of mutant yeast that have been inhibited and This is on solid media. So YPD does not have inhibitor These are the reversible inhibitor and the irreversible inhibitor What you can see is that in this analog sensitive strain the reversible inhibitor still allows growth Although there's a slight growth defect But when we use our irreversible strategy, you see a complete flat lining of growth Which is what you would expect when you inhibit an essential kinase and this is also reflected in growth in liquid media and At this point I also want to show or point out that the wild type yeast are unaffected By these inhibitors because wild type yeast Their kinases do not contain those mutations that allow for inhibition with these inhibitors Okay, so now armed with this more potent way of turning off kin-28 Now I wanted to get to the question about how do RNA levels? How are they affected by kin-28 function and so I really wanted to be careful about how we quantified RNA and I was really inspired by this approach from the Cromer lab. So We call the CDTA Which is comparative dynamic transcriptome analysis? I don't think a lot of information comes from the name But there are two key points that I want to share with you about this technique The first is that an exogenous spike in control is used to normalize between samples Because oftentimes and this is an issue with any science any any experiment really and it's difficult to Identify an internal Standard especially when you think your perturbation might affect everything which is what we Imagine might be the case when you inhibit kin-28 and so Having no internal control. We use an exogenous RNA, but instead of just one RNA We spiked an entire species of different a different yeast which had a whole transcriptome of its own and Downstream we could later separate out Pombi RNA from Theravithia RNA Okay, and I saw I said there are two key features the second feature is that we wanted to study RNA synthesis Directly and so if you just purify RNAs what you're getting is a steady-state level picture of what RNAs exist in a system Even before you perturb the system and so you can label RNAs after adding an inhibitor You can label them with a uracil analog so as you know uracil is incorporated into RNAs and Rather than uracil you can add thio uracil and this thio group allows biotinylation and therefore Separation from the total pool of RNAs and so at the end of the day we get two fractions a nascent RNA fraction which represents RNAs that were made after an ambition in the time of labeling and You also have this total fraction that is a combination of what was made after inhibition as well as what existed before Okay, so what did we find finally? What I'm plotting here is a scatter plot showing the change in expression of the About six to seven thousand yeast mRNAs each spot represents a different mRNA species and The value on the y-axis is a change in that species when you inhibit kin-28 and this is on a log 2 scale So values below zero indicate a decrease in nascent expression and what you see is that most mRNAs are decreased in nascent expression however, if you were to only look at the steady-state levels of mRNA which is plotted as A value on the x-axis it is relatively unchanged or hovering near zero and so What I found was that We I found this group of RNAs which is the majority Of RNAs we call this RNA buffering or a buffered set of RNAs so one way to think about this is Kind of like like this analogy with fountain with you you have RNAs constantly being made they're also constantly being degraded and The total RNA level is a consequence of both of these processes And so what I found was RNAs aren't being made right the synthesis of most RNAs is stopped Yet if the total level remains the same this would indicate that decay is also stopped and so we Also looked at pole-to chip to understand the mechanism by which RNAs might be decreased in their synthesis without kin-28 function and To the left here are some examples of our chip data across these various low side Serine 5 and 7 phosphorylation are Reduced as expected in fill filled in profiles Those are the inhibited samples the outline our control samples But when we look at polymerase we see that for the most part polymerase can make it to the end of a gene Even when kin-28 was inhibited although there were certain examples where there was a defect In making it to the end of the gene and so when we look at a subset of genes The ones which had the most decreased levels of mRNA expression We find this interesting phenomena what I'm showing you here is a heat map where the full change in polymerase occupancy is represented by color blue being an increase red a decrease we see that a hundred approximately 150 base pairs past the transcription start site there is an increase in pole-to density and Here's a single gene example of this so again filled in is a profile for inhibited kin-28 cells and There seems to be an increase which we think is RNA polymerase to pausing and so why do we think this? We can align our pole-to Profiles with Nucleosome positions and we see that this Pausing or increase in polymerase density lines up with the plus 2 nucleosome Which is known to be where elongation factors bind chromatin and pole-toe itself So some of these are spt 5 path 1 burr 1 ctk 1 and so what we think is that for a subset of genes Kint 28 is important for recruiting these factors and allowing for entry into elongation so without Kint 28's phosphorylation of the CTD these factors don't bind and elongation doesn't occur as normal So in summary of the first part of my talk I showed Showed you how we developed this strategy to irreversibly inhibit a kinase that we applied to Kint 28 to understand whether it was required for RNA synthesis and we show that Yes for the majority of RNAs Kint 28 is absolutely required for RNA synthesis but that this could have been missed if you only look at steady-state levels of RNA due to RNA buffering and We also discover that Kint 28 is important for entry into productive elongation Before moving on I just want to take a moment to thank Laura who is our resident artist in biochemistry She made this beautiful illustration to summarize my work and a lot of the figures have been beautified by her and So maybe this is more easy to understand Basically Kint 28 which is part of the TF2H complex acts like a conductor It phosphorylates this polymerase to train allowing for Elongation machinery to associate such that polymerase can go over nucleosomal barriers into elongation Whereas when you inhibit inhibit the kinase polymerase has difficulties in traversing this nucleosomal barrier okay So moving on to the second part of my talk And why I was motivated to do what I'm going to show you I was really intrigued by this group of genes that was buffered I was wondering why how does the self-sense that synthesis is defective and Respond by stopping decay. How is this all happening even more curious? Was this maybe 25 or 20 percent of genes that was not buffered didn't seem to be protected What what are these things? Why aren't they protected even weirder? Is this group of genes that is it doesn't even require Kint 28? And seem to be induced when Kint 28 is inhibited how does how do how do these group of genes escape regulation? So in the second part of my talk, I'm going to discuss my investigations into the post transcriptional Consequences of inhibiting Kint 28. So by post post transcriptional. I mean decay and translation and and one of the first analyses I did was a cross-correlation analysis between my Expression data set and other data sets and so what I'm going to show you in the time that I have maybe I won't do it justice So if this analysis interests you, I'd be happy to tell you how I did it after the talk So what I hypothesized was that a transcriptome that matched my inhibited Kint 28 IS Transcriptome correlated with mine might indicate or identify which factors are involved in RNA buffering and so In this this data set from the Cromer lab. They had deleted 43 different Decay factors and they also perform CDTA seek so what I was doing here was calculating a correlation coefficient between my transcriptomic changes and theirs and so This is a heat map of the r values. I found that are clustered by Their similar r values. So it's a lot of data I will just point out some key features of this so in the diagonal You see this really dark blue color Corresponds to a correlation of one and the reason for this is you have one deletion transcriptome puff one let's say Versus puff one it should be one It's exact same data set if you delete puff one and you compare it to puff two So this this second box here It's pretty blue and which makes sense because puff one and puff two are known to act in the same pathway So if you delete one its transcriptome will appear like the transcriptome of the other and so a lot of these Associations are already known and published so I want to focus on what correlates with the inhibited Kint 28 transcriptome and and so We found a number of factors that were Correlated when they were deleted from east to an inhibited Kint 28 transcriptome But what we were most surprised to see was the most correlated Transcriptome was actually cells that had been inhibited of their translation with cyclohexamide and I want to point out that a general transcription inhibitor Was actually number 30 on the list of about 40 to 50 and so this Correlation to defective translation was determined bioinformatically So we wanted to see it is this defect in translation actually happening in inhibited Kint 28 cells So one way to look at trans Translation is with polysum profiling so polysum profiling. I'm showing you here a typical profile What polysum profiling is is essentially a spectra or a profile of absorbance readings at 260 nanometers of cellular lysate that's been separated out by density and So as you might know one of the most a 260 absorbing species is our RNA and our RNA is what forms is part of ribosomal Complexes and so they can get quite heavy and therefore you get these characteristic peaks So you can see a peak for 40 s and 60 s a small and large subunit together. They are ADS the monosome and When there are multiple Ribosomes together that's known as a polysum and you get a peak for two polysums three so on so forth Okay, so what what do we see when we inhibit Kint 28? as predicted bioinformatically we see that there seems to be a defect in polysum association when you inhibit Kint 28 the profile shown here in red and So polysum profiling kind of presents a static picture of translation So we wanted to look at if proteins were actually still being made or not and to do this We utilized another metabolic labeling approach, but instead of RNAs with protein and You can add this methionine analog HPG has this alkyne group here and you can label proteins as they're being made and You can fluorescently label these nascent peptides with click chemistry reaction of this A side with this alkyne and You can run extracts on a gel and quantify or observe them fluorescently and And so this is what I did and I'm showing you a representative psi three excited gel Where I labeled with this analog for 30 minutes or an hour to the left here It is the translation or the peptide synthesis for uninhibited cells to the right inhibited This is a quantification of this gel here and what you can see is that there are some peptides still being made but the the the the level is greatly reduced and So at this point you might wonder just as we wondered that maybe this just reflects a stress response that it's a general stress response and This is might seem like a complex pathway, but it's not essentially Trust me Essentially the cell stops translation into in response to a number of stresses the stresses can be very varied but what happens is phosphorylation of this Translation initiation factor EIF to alpha so they all converge on the same thing translation initiation stops When this subunit is phosphorylated in yeast the kinase that is responsible for this is GCN 2 and And so to see if the general stress response was being activated when I inhibited kin-28 I did Western blotting to detect phosphorylated EIF to alpha I also blotted for EIF to alpha irrespective of phosphorylation and What what you can see here is that when you inhibit kin-28 with cmk. There is no Enhanced phosphorylation of this subunit So most likely the general stress response was not being activated in this way and the in translation was not being inhibited in this way Just as a positive control. We also have a rapamycin treatment Which is known to activate GCN 2 and of course a GCN 2 delete strain of yeast doesn't show phosphorylation of EIF to alpha We also checked the cap levels of mRNAs and so to do this we used an RNA IP with an antibody that can detect cap and we also used the same normalization strategy with Pombi and What I'm showing you here is RTQ PCR results full change in cap levels for these RNAs and so these RNAs come from different groups the induced group buffered group unstable group and Really, there is no change. So what I'm trying to show here is that it seems like the RNAs that were in cells they're still being there they're capped and so Which mRNAs then are still being translated? Can can we understand? Why some RNAs are translated and some are not is there and so to look at this transcriptome wide We we utilize this technique 5p seek It's essentially an RNA seek technique a special flavor of RNA seek where you can selectively capture RNAs that have a five prime monophosphate at their end. So these are degradation intermediates things that have been uncapped decapped And so but the interesting thing and that was convenient for us was that allowed us to also study translation because the ribosome protects RNAs and These five prime ends can be captured with the three nucleotide periodicity because it as it turns out the ribosome moves in three nucleotide codon units and so these libraries were prepared in collaboration with the Stein mitz group at embell and I just want to Kind of slow down here and show you what these profiles look like they don't So those of you who might be used to looking at RNA seek plots this might look kind of spiky to you and That's because I'm not plotting the entire read over the mRNA. It's only the five prime most nucleotide that's being plotted and Oftentimes you'll see a peak at the transcription start site that corresponds to an uncapped RNA You might see peaks in the open reading frame that correspond to a ribosome Sitting and waiting for a rare cognate tRNA to come in and you'll also see peaks 20 nucleotides from the stop codon where the ribosome is known to pause waiting for disassembly and As expected there is no significant difference between a wild type yeast 5p signal when treated with inhibitor Okay, so what do we see with kin-28 is We see some instances where 5p as well as RNA itself is increased in expression And we see instances where translation is clearly defective most apparent by the decrease in this 20 nucleotide from the stop codon peak and We can look at this transcriptome wide now And to the left. I'm just showing you the wild type case where there isn't much change for comparison on the top here I'm showing you the average profile of 5p at the stop codon and The bottom here is a heat map of the full change in 5p across about 3700 mRNAs with with robust 5p signal Okay, so I'll point out two things here and the first is that Irregardless of whether an mRNA is buffered or unstable the 5p seek signal is Significantly decreased indicating that Translation was severely diminished for these RNAs And the second thing I want to point out is this interesting group of genes that are induced that don't require kin-28 for their transcription There seems to be no change or even an increase in 5p And this is more apparent if you look at 5p seek signal at the start site you see this increase and You might think that this increase is just because there's more RNA in general And so what we can do is look at the three nucleotide periodicity and the strength of the three nucleotide periodicity to understand translation and So what you can do is you can take the data the 5p signal either at the start or the stop you can Fourier transform it and Identify the contribution of the periodicity to the signal. So what I'm going to show you in the next slide is a quantification of this intensity and What I want to point out here is that in this induced group of genes the three nucleotide periodicity is increased and maintained Indicating that these RNAs are still being translated But for these buffered and unstable group of mRNAs. This is not the case and so as An example of how some of these things look like these are Example genes or mRNAs that are induced when kin-28 it's inhibited These top ones are uninhibited this bottom row is inhibited and When we took a closer look at this we noticed this increase in 5p It's not just an increase but potentially a difference in start site usage So if you look here, it seems like this start site was being utilized But when cells are inhibited of their kin-28 they utilize this site instead and So we were we think that this difference in 5 prime RNA sequence might allow these RNAs to escape regulation by normal machinery when kin-28 it's inhibited and So this is kind of akin to what happens with a lot of viral RNAs that can escape host machinery They have strange secondary structure at their 5 prime that allows them to continue to be translated and not decayed And for more evidence of this is that in this induced group of genes Their 5 prime untranslated region tends to be longer. There are three primed untranslated region tends to be shorter they tend to be lowly expressed under normal conditions and this got us to thinking about RNA binding proteins and Trying to see if we could identify RNA binding proteins that would dictate stability or instability during times of RNA buffering and I compared the binding of a lot of RNA binding proteins from the taller v cross-linking data set and the two that I want to show you here where we found the most interesting pattern of binding is Nab-2 and ski-2 so these are RNA binding proteins that are associated with decay and What I'm doing here is ranking or clustering the mRNAs by whether they're induced all the way down to unstable mRNAs and We see that ski-2 Seems to correlate with stability whereas Nab-2 binding seems to correlate with instability And so that these two factors and their relative binding They could predict whether a mRNA is unstable or not when kin-28 was inhibited and so To summarize the second part of my talk I Show the post transcriptional consequences of inhibiting a transcriptionally important kinase and I identify mRNAs that don't require its Regulation or escapes its regulation. I show defects in translation that stem from RNA synthesis stopping and That this stopping of translation is not through normal stress related mechanisms and is a unique form of translation inhibition and I also show that the relative binding of these RNA binding proteins can predict whether a mRNA is stable or unstable when transcription is inhibited and So This is an ongoing area of research in our lab How exactly do these RNA binding proteins dictate stability and so what do we know about these things? We know that Nab-2 is nuclear whereas ski-2 is cytoplasmic So one of our ideas or my ideas is that it's not so much that these proteins bind and Protect the RNA, but it's more about them being an indicator of the relative distribution of the mRNAs So perhaps nuclear mRNAs are subject to decay when kin-28 shut off Because nuclear exonucleases and decay factors are still active whereas in the cytoplasm where buffering seems to happen The the decay factors are not there so one area of research that we're pursuing is to understand the relative localization of decay factors and mRNAs and another Question that we want to understand in the future is how exactly is translation stopped how do cells sense that transcription is defective and respond by stopping translation and So in conclusion I Just want to summarize my findings and Using the strategy we developed that we can apply to any kinase of interest We were able to clarify that RNA synthesis does require kin-28 except for the weird group of genes I showed you and We discovered that this could have been missed because of RNA buffering We also discovered that kin-28 required for entry into the productive phase of elongation for a number of genes I defined which mRNAs are translated and which are transcribed when kin-28 inhibited or unstable and I also discovered that nab 2 and ski 2 binding can predict stability of RNAs and And as I mentioned with regards to future directions of research that my research has opened really understanding this RNA buffering phenomena apart from just kin-28 it happens when RNA decay and synthesis is perturbed in general and one point that we want to follow is the relative distribution of decay factors and Cellular distribution of decay factors And I didn't think I'd have enough time so I didn't talk too much about it But all what I learned about kin-28 and budding yeast can potentially be applied to CDK 7 in humans In fact, there is a new line of therapeutics targeting transcription specifically CDK 7 and To understand why this even works It's important to understand which transcripts are being affected when this essential kinase is being inhibited and Before I take questions, I Have a lot of people to thank The first group of people I want to thank are my committee members Thank you for taking the time out of your busy schedule to evaluate my work and meet with me throughout the years And give your scientific input. I really appreciate it. So thank you Aaron Betty Dave Rupa and And I want to thank my collaborators Jack Taunton helped in the development of the kin-28 is strategy of inhibiting kinases I Want to thank the Steinmetz group for preparing those five PCC libraries and sequencing? I want to thank I didn't show the data here, but I want to thank Megan for help with microscopy experiments And in our own department, I have a lot of people to thank but I really want to point out the IT and media lab Thank you for all your AV and computer and network related help Kate Ryan for all your help with ipib anything ipib and I want to thank Ruchika and Amy for helpful discussions about polysyn profiling and various RNA assays so thanks for that and So Of course I would be Of course, I'd be remiss if I didn't think my PhD advisor a seam and say some choice words about a seam so So yeah, you might think a seam is really it's nice fellow, you know hello kitty so nice And we constantly try to find this picture of him with a purple mohawk Apparently he attended his own defense with a purple mohawk So try to we're trying to look out for that, but these are some of his key catchphrases QC 25 Mer Bees chugging along and So I just want to share a story with you which some of you might have already heard about And I think it's also a really good analogy or it parallels my journey in a seams lab and in graduate school So When I first joined the lab like right after I started I seem had this idea that we crash the Cold Spring Harbor meeting for eukaryotic gene regulation and that we drive there and so I don't remember why I went along with this and Yeah, so We set out at one or two in the morning and I think the reason was because we wanted to avoid traffic in Chicago and So Google Maps predicts it takes about 16 or 17 hours depending on which route you took So we didn't really stop and I also want to point out here that we could have flown only takes two hours And a few hundred dollars, but we didn't do that right Right, right. So, um, you know again, I want to remind you this is like my graduate school experience and So you might have heard of the story, but I don't think anyone's ever seen how perilous it was And so I actually have video evidence that I dug up Some part of this. I don't know. Do you remember this a seam this? Okay, yeah, so I just want to play a little bit of our journey here I don't know if we'll play though Look at this a seam. Yeah, so that was grad school for me in your lab And here's some still frames we have a quarry in the fetal position we have Juan dosing away Here's a seam gripping the steering wheel and fear and I was sitting by the side and I didn't want to go to sleep because I wanted to die awake and But okay, I'm just I'm just joking a little bit here This is a picture of the road after a while it eventually cleared right and So I think we took the trip we drove there because we learned a lot from driving there versus just taking a short flight and As Devesh always says all's well that ends well. So thank you for that interesting experience and and you know, I said this perilous graduate school, so thank you for the entire journey and That unique experience, so thank you and really also. Thank you for assembling this group of crazy characters I want to thank all the past and current members of the Ansari lab that made it such a joy to come into work and keep on working I especially want to thank Natalie for managing the lab as well as managing a seam and These particular CTD group members Cori and Juan my elders who are my original CTD mentors Juan and Shane worked closely with me on the Kintuni IS the first part of my talk Rajesh, I want to thank for keeping me company in the CTD group for the past couple of years and Oscar and Laura are honorary CTD members Oscar is my bay mate for many years and she's always been an inspiration how persevering she is with her research as well with things like ultra-marathonning and You mentioned the list of mentees that I've had the honor of working with I want to thank these guys because they really kept me motivated to come into lab because I had to take care of them Like a mother has to take care of her kids But also they provided me with a fresh perspective on what I'm doing and kept me really curious with their fresh eyes And I've told you all about the scientific support And you guys probably seen this picture a million times now at any defense from any one of us but this is a picture of us in the end of week one and I talked about scientific support I want to mention the emotional support that I received you guys have been really Keeping me going and with your own stories really inspiring me and a lot of you guys have moved on to bigger and better things some of you are moving and I'm really devastated by that, but that's okay. And So 2012 is a long time ago These are some friends. These are lab members that became friends my family and This are I think paintball trip here And it really felt like no time at all that we are here today With your support. So lastly, thank you for my funding sources and Yeah, thanks again Do you want the microphone back? Okay It was a bonding exercise Shane No, I didn't sure so the second part of your question Nabto's interaction with and ski to the interaction. So there is really nothing known about that interaction About nab to and ski to themselves nab two doesn't really have a catalytic function. It serves more as Enhancing the efficiency of decay factors in the nucleus ski to is a helicase and it's associated with decay factors in the cytoplasm and So how does kin-28 relate to them unclear? It would be interesting We've talked about this if well, we could see if so there are phosphocytes on nab to Interesting if kin-28 can phosphorylate nab to that would be a direct connection But I think that would be too to fortuitous Too easy, but maybe this is what we know about these or at least what I know Yeah, that's a good question we did some gene ontology analysis where you can kind of pick out key Key or similar terms that are related to a set of genes And we see that some of them are meiosis related Which is because I use I use a haploid strain of yeast. They shouldn't be undergoing meiosis a lot of them are meiosis related and We see also some With relations to the unfolded protein response Yeah, and so the there are some people Sandra and Connor in our lab are looking at characteristics of these proteins or these mRNAs and seeing if they're related to the stress response what exactly is their Codon usage. Maybe there's something special On your second question You know, I think I think it was it was right that people pointed out there is a Reversibility to the inhibition is a competitive kind of inhibition where a little bit of ATP could still bind and that was enough to get the machine going I think that was I think that was why Yes Yeah, they all evaluated total Yeah, good point. So for instance like the temperature sensitive strategy when if they use nascent First of all, I'll point out that that Heat shock alone activates a whole slew of different mRNAs and that would totally cloud things. So for that one I think that's what would have happened and I think for the catalytically in active mutant You would have saw the same thing that I did with IS that There there'd be less RNA being made