Exploring iCLIP-data analysis with 7SK RNA and RBM7

Luke Buettner Author

04/05/2021 Added

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Spring 2021 Research Presentation

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[00:00:01.260]Hello. I am Luke Buttner and today I'm going to talk about exploring.
[00:00:04.050]iCLIP data analysis while looking at 7SK RNA and the Protein RBM7.
[00:00:09.840]To start. When you ask a few questions to understand what's going on, such as,
[00:00:13.380]why is 7SK RNA important? Why do we care
[00:00:16.350]how 7SK RNA and proteins. specifically RBM7, interact?
[00:00:20.940]And what is iCLIP data analysis? after these questions have been answered,
[00:00:24.900]we can try to replicate the iCLIP process used in some published papers.
[00:00:30.860]Starting with the importance of
[00:00:32.270]7SK RNA. We look at how it functions. In this figure.
[00:00:35.540]We can see a few key pieces of information.
[00:00:37.550]First, normally the 7SK RNA
[00:00:39.460]complex sits in a sequestered state and the positive transcription
[00:00:43.520]elongation factor P-TEFb is held by the RNA complex.
[00:00:47.810]This results in a paused state of elongation and often cell defects, though
[00:00:52.250]once receiving specific signals,
[00:00:54.080]the 7SK complex moves to a released state and PTEFB is able to move
[00:00:59.030]freely.
[00:00:59.990]Once released P TEFb is able to attach a phosphate to the
[00:01:04.250]polymerase 2, elongation enzyme,
[00:01:07.460]and it shifts to processive elongation.
[00:01:12.230]Next, we look at how
[00:01:13.380]7SK RNA interacts with proteins, specifically with the RNA binding
[00:01:18.060]motif protein seven RBM7,
[00:01:20.750]as a result of induced genotoxic stress.
[00:01:24.020]The reason we were looking at RBM7 specifically is due to its property to
[00:01:27.560]promote cell survival after damage resulting from UV radiation.
[00:01:31.940]In this case,
[00:01:32.840]UV radiation is being replicated by the chemical four nitroquinoline 1 oxide,
[00:01:37.730]four NQO, which will be shown later, this signal,
[00:01:41.840]caused by the induction of genotoxic stress leads to change in how RBM7
[00:01:46.280]binds with 7SK.
[00:01:48.380]This change leads to the release of PTEF B followed by processive elongation and
[00:01:53.150]ultimately the survival of the effect itself.
[00:01:58.190]Now we see how iCLIP works.
[00:02:00.830]The first steps are to use ultraviolet light to weaken the RNA protein structures
[00:02:04.310]To the point they can bond covalently.
[00:02:06.740]Then insertion of a barcode to help identify the complex in the future.
[00:02:11.600]Next,
[00:02:11.960]the complex is reverse transcribed to cDNA where through adapter insertion PCR
[00:02:16.340]amplification occurs,
[00:02:17.900]and we are left with numerous copies of the CDNAs for easier computational
[00:02:21.320]analysis.
[00:02:22.940]Once we have the cDNA.
[00:02:24.020]Libraries,
[00:02:24.680]we can start the high throughput sequencing process to see how often the
[00:02:28.650]7SK and RBM7 Crosslink.
[00:02:30.680]moving on to the actual project.
[00:02:35.750]To answer the question,
[00:02:36.680]can we replicate the iCLIP process use in published papers? We
[00:02:41.360]look at the
[00:02:43.940]papers that have already completed the process. Here
[00:02:46.460]we have data from arrayexpress, a geo database that was used in this project.
[00:02:50.690]Once we get data from here, we can move on to
[00:02:55.010]purifying it, to remove the adapter and the barcode,
[00:02:57.950]as well as low quality data. Following mapping of the human human genome.
[00:03:02.380]We adjust for coverage and use a Python script to pull out the gene of interest.
[00:03:06.550]Finally, we perform statistical analysis to show the quantitative.
[00:03:09.680]Results.
[00:03:12.990]And here's a generalized workflow of the major steps needed to complete the
[00:03:16.230]analysis and the processes to achieve the steps.
[00:03:19.800]So first we can see we get the iCLIP data and then we check it to make sure
[00:03:23.910]that the data is quality and then we remove the adapter.
[00:03:27.750]And if we find data that isn't of a high enough quality,
[00:03:31.020]remove that at the same step here,
[00:03:33.900]we recheck the data quality to make sure that the trimming worked well.
[00:03:39.570]Next, we moved to the demultiplexing step stage to remove the barcode.
[00:03:44.760]After that we can align the human genome with the 7SK RNA complex
[00:03:50.070]and this step doesn't need to be completed every single time we do the process,
[00:03:54.600]as long as we're maintaining the same
[00:03:57.390]complex
[00:04:00.090]Like 7SK, we only need to do at once. Next,
[00:04:04.050]we align the
[00:04:06.300]previously trimmed data to the genome and we shifted
[00:04:10.380]one nucleotide to account for coverage, which is
[00:04:13.950]where we assume that cross-linking starts one nucleotide before where
[00:04:18.840]the data matches the genome.
[00:04:23.700]Next
[00:04:23.910]we use Python to pull out the gene of interest to
[00:04:26.460]perform statistical analysis and
[00:04:28.380]You get a graph.
[00:04:31.590]Here we compare the results of the published paper with that over our own.
[00:04:34.710]And we can see in both that there's a spike in the frequency of cross-linking
[00:04:38.310]going from the DMSO control to the four NQO treated samples.
[00:04:42.510]You can see it in both as a small spike.
[00:04:47.760]Yeah.
[00:04:47.880]Next using Varna an app to map data to sequences.
[00:04:50.970]We can take into information from the iCLIP process and see how it looks
[00:04:54.900]right on the structure of 7SK zooming into just the STEM Loop three from
[00:04:59.400]the 7SK structure.
[00:05:00.960]It is possible to see the spikes from the earlier graph translate with the
[00:05:04.320]darker colors,
[00:05:05.760]representing a higher frequency of cross-linking. looking at nucleotides 250
[00:05:10.080]and 251.
[00:05:10.950]We can better visualize their previous results and come to the conclusion that
[00:05:14.820]Four NQO treatment representing damage from UV radiation led to a higher
[00:05:19.290]amount of cross-linking between 7SK RNA and RBM7 .
[00:05:23.280]We can also see that, that we were able to successfully replicate the iclip
[00:05:27.630]Process. Going forward
[00:05:31.080]I'll be looking at other proteins related to 7SK,
[00:05:33.720]such as HEXIM1 LARP7, and DDX21.
[00:05:37.410]And repeating the iCLIP analysis process for each.
[00:05:40.200]I will also be analyzing the recent publication, the kinetic landscape of an RNA,
[00:05:44.190]binding protein in cells,
[00:05:45.990]looking to extract data related to 7SK to perform a similar
[00:05:49.980]analysis. I say thanks to the Eichhorn lab led by Dr.
[00:05:54.090]Eichorn, my peer mentor, Amr Sobeh,
[00:05:56.520]and my follow lab members also to UNL's first-year research experience,
[00:06:00.190]FYRE for funding and direction for this research.

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Exploring iCLIP-data analysis with 7SK RNA and RBM7

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