Coevolution of 7SK RNA and MePCE

Elizabeth Griggs Author

07/27/2020 Added

53 Plays

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Comparison of 7SK RNA and MePCE sequences and structures between model organisms in order to obtain information regarding coevolution and regions required for proper transcriptional regulation.

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[00:00:01.270]Hello, my name is Elizabeth Griggs,
[00:00:03.300]and I will be presenting on the coevolution
[00:00:05.340]of 7SK RNA and MePCE, an RNA stabilizing protein.
[00:00:10.730]Transcription is a highly regulated process
[00:00:13.060]as it controls the expression of RNA within the cell.
[00:00:16.320]Within the elongation phase of transcription,
[00:00:18.840]the enzyme, RNA polymerase II, undergoes a regulatory pause,
[00:00:23.010]where phosphorylation by Positive Elongation Factor b
[00:00:26.520]or P-TFEb occurs, leading to productive elongation.
[00:00:30.310]The 7SK small nuclear ribonucleoprotein
[00:00:33.860]is an inhibitory complex, composed of 7SK RNA
[00:00:38.290]and the proteins methyl phosphate capping enzyme (MePCE),
[00:00:42.680]La-related protein 7 (LARP7), HEXIM1 and P-TEFb,
[00:00:47.950]which acts as a primary regulator of this process.
[00:00:51.310]The goal of this study is to understand
[00:00:53.410]the RNA sequence, structure
[00:00:55.390]and RNA-protein interactions between 7SK RNA
[00:00:59.110]and the accessory proteins for key model organisms.
[00:01:02.170]By comparing the sequences and structures
[00:01:04.450]information regarding coevolution will be obtained.
[00:01:07.490]The highly conserved regions and interactions
[00:01:09.700]will provide insight into the regions required
[00:01:12.050]for proper transcriptional regulation.
[00:01:14.560]For this poster presentation,
[00:01:15.870]I will focus on the results pertaining to MePCE,
[00:01:18.810]which functions by binding to
[00:01:20.050]and methylating the 5' end of 7SK RNA
[00:01:23.470]in order to protect it from exonuclease degradation.
[00:01:27.810]The sequences for 7SK RNA
[00:01:30.000]and the accessory proteins
[00:01:31.320]were collected from RFAM and the NCBI database.
[00:01:34.160]The sequence and structural alignment
[00:01:35.730]for the 7SK RNA sequences of 46 mammal species
[00:01:39.760]was obtained in order to generate a consensus sequence
[00:01:42.460]and secondary structure.
[00:01:44.020]For each accessory protein,
[00:01:45.620]a sequence and structural alignment of five model organisms
[00:01:49.000]was performed to build a three dimensional structure,
[00:01:51.230]color coded by conservation score.
[00:01:53.590]Finally, using these alignment results,
[00:01:55.670]along with previously reported MePCE-7SK RNA interactions,
[00:02:00.310]the conservation of key MePCE residues that interact
[00:02:03.080]with the 5' triphosphate of 7SK RNA was identified.
[00:02:08.330]Figure three displays a two dimensional consensus sequence
[00:02:11.150]and secondary structure for 7SK RNA for 46 mammal species,
[00:02:15.860]color-coded according to percent conservation
[00:02:18.270]with the most conserved nucleotides deep red
[00:02:20.630]and the least conserved blue.
[00:02:22.350]In general, the secondary structure is as expected
[00:02:24.960]with four main stem loops
[00:02:26.370]and stem loop three being separated from stem loop four
[00:02:28.990]by a short double stranded helix
[00:02:31.000]that includes the 5' end of the RNA.
[00:02:33.870]The 5' end, where the interaction with MePCE occurs,
[00:02:36.710]is nearly perfectly conserved,
[00:02:38.400]indicating the importance of these interactions
[00:02:40.450]in stabilizing 7SK RNA.
[00:02:43.150]Within stem loop one is a nearly perfectly conserved
[00:02:45.830]GAUC-GAUC motif and a UU bulge,
[00:02:50.700]which is critical to the binding of HEXIM 1
[00:02:52.750]to 7SK RNA in humans.
[00:02:54.870]The high conservation of these motifs
[00:02:56.750]in nearly all mammals of study
[00:02:58.550]indicates that they have a similar function.
[00:03:01.150]The consensus secondary structure of stem loop two
[00:03:03.760]shows the most variance from the secondary structure
[00:03:06.010]of individual sequences due to the single stranded RNA
[00:03:09.240]within the middle of the stem loop.
[00:03:10.900]However, no accessory proteins
[00:03:12.480]bind to the RNA within this region,
[00:03:14.450]which is why insertions and substitutions can occur.
[00:03:17.730]Finally, the poly U tail is highly conserved
[00:03:20.220]as it is a binding site of LARP7,
[00:03:22.320]which aids MePCE in RNA stability.
[00:03:25.210]The 3D structure of MePCE shown in figure five,
[00:03:28.480]shows that the active site
[00:03:29.720]containing S-Adenosyl Homocysteine (SAH),
[00:03:32.950]the cofactor that remains after the transfer
[00:03:35.250]of the methyl group from S-Adenosyl Methionine (SAM)
[00:03:38.760]to the 5' guanine of 7SK RNA,
[00:03:41.600]is highly conserved between the five organisms.
[00:03:44.690]The alpha helices and beta strands
[00:03:46.470]that form the active site are highly conserved and are red,
[00:03:49.840]whereas those that are more distant
[00:03:51.170]are less conserved and are blue.
[00:03:53.100]More specifically, within secondary structures
[00:03:55.440]that are less conserved,
[00:03:56.800]the residues positioned toward the active site
[00:03:59.230]are more conserved than those that are positioned
[00:04:01.450]away from the active site.
[00:04:04.610]The MePCE-RNA binding interface is also highly conserved.
[00:04:07.920]In particular, the N- terminal helix,
[00:04:10.260]that forms on binding 7SK RNA.
[00:04:12.760]In humans, this tyrosine rich helix
[00:04:14.890]interacts with the 5' triphosphate
[00:04:17.020]for proper positioning near SAM within the active site.
[00:04:20.860]The 5' triphosphate is stabilized by multiple hydrogen bonds
[00:04:24.530]and stacking interactions with key MePCE amino acids,
[00:04:28.350]which are shown in figure six.
[00:04:30.290]Among the five model organisms, tyrosine 421
[00:04:33.300]is perfectly conserved,
[00:04:34.700]indicating this residue has the essential function
[00:04:37.260]of hydrogen bonding to the gamma phosphate.
[00:04:39.730]On the other hand, tyrosine 424
[00:04:41.890]is only 60% conserved with phenylalanine
[00:04:44.750]at this position in fruit flies
[00:04:46.320]and isoleucine in roundworms.
[00:04:48.300]However, these amino acids are both hydrophobic,
[00:04:50.840]as is tyrosine,
[00:04:52.050]allowing them to interact similarly with RNA.
[00:04:55.070]Table one details
[00:04:56.300]the conservation of these interactions described,
[00:04:59.000]while figure seven summarizes the percent conservation
[00:05:01.840]of all 12 amino acid residues
[00:05:04.010]involved in the triphosphate binding tunnel.
[00:05:06.550]Of all 12 amino acids,
[00:05:08.200]six are perfectly conserved among the five model organisms.
[00:05:12.860]The results of this study show that the 7SK RNA nucleotides
[00:05:16.580]and MePCE amino acid residues
[00:05:18.690]involved in RNA-protein binding recognition
[00:05:22.480]are highly conserved.
[00:05:24.020]Thank you.

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Coevolution of 7SK RNA and MePCE

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