Chemical Mapping 7SK snRNA

Jacob Sorensen Author

04/05/2021 Added

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Enjoy my research on chemical mapping 7SK!

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[00:00:01.050]Hi, my name is Jacob Sorenson,
[00:00:02.850]and I'm going to be talking to you about my research on chemical mapping,
[00:00:05.970]the secondary structures of seven S K S and RNA
[00:00:10.350]organisms contain coding RNA, which is also known as M RNA,
[00:00:14.340]which can be translated into proteins as well as non-coding RNA,
[00:00:17.850]which can not be translated.
[00:00:20.040]Non-coding RNA is a diverse class of RNA with many different functions.
[00:00:25.080]An example of non-coding RNA is small nuclear RNA,
[00:00:28.260]which function and the regulation of gene expression,
[00:00:31.380]seven S K S and RNA makes up the core of the seven S K ribonucleoprotein
[00:00:36.270]complex and is abundant in the human body.
[00:00:39.330]Seven SK functions to regulate the activity of positive transcription,
[00:00:43.770]elongation factor B or P T E F B,
[00:00:47.160]which in turn regulates RNA polymerase two,
[00:00:49.890]which promotes the efficiency of M RNA in the cell.
[00:00:54.420]A big part of my research prior to conducting experiments on my own was
[00:00:58.290]analyzing literature pertaining to 700 K probing.
[00:01:01.680]I did a lot of research on methods to get a good understanding on the how and
[00:01:05.790]why of chemical mapping as well as obtain raw data that I could
[00:01:10.470]input into spreadsheets for later. Comparison,
[00:01:14.040]figure one on the right is a structure from a paper published by the Howard
[00:01:17.550]Chang lab.
[00:01:18.540]Here I see shape was used to probe seven SK when bound to different proteins.
[00:01:23.040]Hexham one in bath to see how it affected the structure.
[00:01:27.090]This paper was one of the biggest inspirations for my project and gave me a lot
[00:01:30.720]of ideas on the direction I want my project to go.
[00:01:34.770]The purpose of my study is to use chemical mapping methods and computational
[00:01:38.490]modeling to determine the seven SK RNA secondary structure of STEM loop three
[00:01:42.900]specifically in various buffer conditions by determining the structure of seven
[00:01:47.160]SK in various conditions,
[00:01:49.320]the function can later be hypothesized by understanding how the RNA interacts in
[00:01:53.430]certain circumstances.
[00:01:55.410]Further structural analysis can help determine how PTE F B binds and releases in
[00:02:00.360]the seven S K R and P complex.
[00:02:03.780]Here's a little overview on chemical mapping.
[00:02:06.390]It works by modifying bases that are solvent exposed or accessible to the
[00:02:10.200]probing agent. Also known as single stranded regions, depending on the probe.
[00:02:14.910]Different positions on different bases can be modified DMS.
[00:02:19.140]Methylates the end seven position of adenine and N three position of cytosine
[00:02:24.210]shape isolates the two primary Doxil group of solvent exposed spaces
[00:02:29.310]because of this,
[00:02:30.240]it is not based specific and can modify all four bases.
[00:02:35.490]CMCT is a carbo Diomede,
[00:02:38.010]which covalently a ducks gone in and [inaudible].
[00:02:41.130]And finally [inaudible] forms five member rings on single-stranded guanine,
[00:02:45.960]different chemical probes provide complimentary data on seven S K secondary
[00:02:50.640]structure and can be used together to form a more comprehensive structure.
[00:02:55.620]Finally, magnesium is a crucial part to this process because it allows for the
[00:03:00.130]stabilization of the secondary structures in buffer additions,
[00:03:04.620]before chemical mapping,
[00:03:06.150]I had to first optimize my transcription reactions of STEM loop three.
[00:03:11.520]The reactions were run for three to four hours at 37 degrees Celsius.
[00:03:15.540]And as you can see in table one, the conditions that were optimized,
[00:03:19.470]where the Ribo nucleoside triphosphate levels, the magnesium concentration,
[00:03:24.330]the DNA template concentration, as well as the enzyme conscience concentration.
[00:03:29.460]After the transcription reactions,
[00:03:31.650]the yield was analyzed on Agros gels
[00:03:36.600]in table two,
[00:03:37.530]you can see the final optimized values for seven S K STEM loop through
[00:03:40.950]transcription, which I used in my case, chemical mapping.
[00:03:44.940]The first step in chemical mapping is to fold the RNA.
[00:03:48.870]You need to heat and cool it to destabilize any preexisting secondary
[00:03:52.770]structures. After that, you can prepare the folding buffers.
[00:03:56.460]The three I use were number one, sodium could catalase magnesium,
[00:04:01.260]number two, sodium Kolkatta late, only in number three,
[00:04:05.010]magnesium and heaps. Yeah.
[00:04:07.410]Folding buffers were then added to my RNA samples for 30 minutes to induce RNA
[00:04:11.970]folding into form secondary structures.
[00:04:15.240]After folding modification took place.
[00:04:18.660]Michael probe I chose to use was DMS.
[00:04:21.390]The DMS was added each RNA tube. In addition,
[00:04:26.250]water was added to separate tubes to serve as a background subtraction later on
[00:04:31.110]in the process,
[00:04:32.730]the modification reaction occurred for exactly six minutes when it was quenched
[00:04:36.630]the BME. And after that it was purified
[00:04:42.150]and the concentration was taken on a cubit, right? Figure three.
[00:04:46.470]You can see the sites on adenine inside ezine that DMS modifies
[00:04:52.050]after modification reverse transcription took place to the modified RNA.
[00:04:56.490]I added Tieger,
[00:04:57.510]which shows the reverse transcriptase enzyme as well as a barcode slash reverse
[00:05:01.800]transcription primer. After the reverse transcription,
[00:05:05.610]the reaction was quenched with quench acid to stop the enzymatic activity.
[00:05:11.340]The newly synthesized cDNA DNA fragments were then purified.
[00:05:15.720]Lastly,
[00:05:16.680]the samples were prepped for sequencing [inaudible] master mix was added to the
[00:05:20.610]cDNA along with forward and reverse primers for PCs.
[00:05:24.870]After the PCs,
[00:05:27.060]the nude double stranded DNA was run on 2% precast Agros
[00:05:31.830]Eagles for 10 minutes,
[00:05:33.510]where it was extracted purified in quantified on a cubit.
[00:05:38.190]Again, lastly, it was input yeah, into the sequence.
[00:05:42.330]The sequencer spit out data,
[00:05:44.220]and that data was input into Varna to predict secondary structures
[00:05:49.350]now onto my data and results. Seven S K STEM loop,
[00:05:52.560]three can adopt two different secondary structures. Number one on the left.
[00:05:56.340]And number two, the right here are the results for buffer condition.
[00:06:00.740]Number one,
[00:06:02.050]as you can see a lot of the solvent exposed a and C basis have
[00:06:06.520]reactivity values.
[00:06:09.250]The scale is set so that blue represents zero or lower activity in
[00:06:14.050]anything above 0.4 is red,
[00:06:17.590]which signifies high reactivity values. As you can see,
[00:06:21.430]there are some reactive double stranded base pairs in
[00:06:26.020]both confirmations,
[00:06:28.780]which suggests that both structures are formed buffer
[00:06:33.460]condition, number two, produced pretty similar results. However,
[00:06:38.350]the basis that our solvent exposed seem to have lower reactivity values.
[00:06:43.210]This could be due to the absence of magnesium,
[00:06:47.050]not stabilizing the structure. And lastly, buffer condition. Number three,
[00:06:51.970]produced really similar results to buffer condition. Number two,
[00:06:56.110]in conclusion,
[00:06:57.250]both secondary structure variations have data that suggests they form.
[00:07:01.480]This is very preliminary data,
[00:07:03.070]but I'm really excited to be making progress during a pandemic as observed
[00:07:07.990]in literature and through data analysis,
[00:07:09.670]both [inaudible] secondary structures seem to form an all three conditions.
[00:07:14.050]The percentage is still to be determined in the future.
[00:07:17.320]I hope to incorporate protein into my buffers to determine the
[00:07:21.910]structure of the Seminis K RNP. In addition,
[00:07:25.510]I would like to use other probes to get a more comprehensive structure.
[00:07:29.890]Ultimately, my goal is to answer the question,
[00:07:32.260]how do RNA protein interactions affect secondary structures?
[00:07:36.700]Finally, I want to say a big thank you to Dr.
[00:07:39.190]Eichorn as well as the rest of the Icahn research group for their contributions
[00:07:42.670]to my project. In addition, I want to thank Dr.
[00:07:45.490]Yes-man for his assistance in the procedure,
[00:07:48.340]data processing and data interpretation.
[00:07:51.310]I would also like to thank the entire UCare fellowship program for giving me
[00:07:55.090]this opportunity.
[00:07:56.530]Stay tuned for more exciting seven SK chemical mapping research.

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Chemical Mapping 7SK snRNA

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