A phylogenetic analysis of m6A RNA modification in land plants to elucidate protein functions

R. Witkowski Author

07/28/2020 Added

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This poster represents the work that I have completed over the course of the Nebraska SRP. I would like to thank Dr. Bin Yu and Mu Li for their guidance in conducting this research.

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[00:00:00.790]Hello. My name is Robert Witkowski,
[00:00:04.021]and I'm a plant biology student here at UNL.
[00:00:06.284]My poster presentation will summarize my summer
[00:00:09.004]conducting a phylogenetic analysis
[00:00:11.217]of m6A RNA modification in land plants
[00:00:14.297]to elucidate protein functions.
[00:00:16.776]To begin, let's focus on RNA.
[00:00:18.965]In eukaryotes, RNA is modified after being translated;
[00:00:23.350]this phenomenon is an important part of
[00:00:25.344]many gene regulatory pathways.
[00:00:27.878]There are more than 160 RNA modifications
[00:00:30.732]that we have observed, and research into these modifications
[00:00:33.888]has engendered the transcriptome,
[00:00:35.978]a comprehensive catalog of RNA transcripts similar to the genome or proteome.
[00:00:41.463]One of the most abundant of these modifications is m6A,
[00:00:44.902]or N6-methyladenosine.
[00:00:48.700]In eukaryotes, a so-called "writer" complex of proteins
[00:00:52.846]is responsible for methylating adenosine nucleotides in RNA molecules.
[00:00:56.902]In plants, this complex is composed of the methytransferase MTA,
[00:01:01.527]and the noncatalytic subunit MTB.
[00:01:05.230]These are orthologs of the mammalian METTL3 and METTL14.
[00:01:10.404]The m6A writer complex methylates the 6th postition of the adenosine base,
[00:01:15.440]hence the abbreviation m6A.
[00:01:20.478]Over evolutionary time, these molecules may have undergone substantial changes
[00:01:24.828]as a result of frequent gene and genome duplications;
[00:01:28.263]such genetic changes may have engendered a loss or gain of function
[00:01:32.263]in these writer proteins.
[00:01:34.677]The focus of this investigation has been to comparatively assess
[00:01:37.922]the rise of functional domains in MTA and MTB.
[00:01:44.204]To begin, MTA and MTB amino acid sequences were collected
[00:01:47.924]from comprehensive proteomic databases including NCBI, Phytozome,
[00:01:51.924]TAIR, and TreeGenes.
[00:01:55.025]350 collective sequences were found from 200 unique taxa
[00:01:58.664]in the Viridiplantae, which broadly includes
[00:02:01.581]mosses, ferns, conifers, and flowering plants.
[00:02:05.563]Algae were not included in this study.
[00:02:08.643]After collection, the protein sequences were aligned in the MEGA software
[00:02:12.302]using the MUSCLE alignment algorithm.
[00:02:15.542]Then, these sequences were searched in the HMMER online protein domain
[00:02:21.062]homology tool.
[00:02:22.392]Additionally, these aligned protein sequences were analyzed
[00:02:25.367]using the SPEER-SERVER in order to find specificity-determining sites;
[00:02:29.891]these sites are amino acid residues that can uniquely determine
[00:02:33.891]which substrate a protein will bind to.
[00:02:38.426]Once the sequences were aligned, phylogenetic trees were constructed
[00:02:42.047]to determine divergence of these proteins by species,
[00:02:44.603]based on amino acid substitutions.
[00:02:49.640]Letter (A) of Figure 2 shows the MTA phylogeny of 39 plant species.
[00:02:54.378]Based on similarity, proteins generally were grouped together,
[00:02:57.664]for example the Brassicaceae, shown in yellow,
[00:03:00.944]which contains the model organism Arabidopsis thaliana;
[00:03:04.784]the Fabaceae, or legumes, in green; and the Poaceae, or grasses, in blue.
[00:03:10.814]The scale bar on these phylogenetic trees represents similarity,
[00:03:14.379]where 0.1 means two species are 10% different.
[00:03:18.701]Using this scale, we can see that Selaginella moellendorfii,
[00:03:22.701]a simple, seedless spikemoss, is approximately 20% different from
[00:03:26.701]its common ancestor with Physcomitrium patens, a moss,
[00:03:30.701]and 40% different from Physcomitrium patens itself.
[00:03:37.137]Letter (B) of Figure 2 shows a similar phylogeny of MTB found in
[00:03:41.137]the same 39 species, where applicable.
[00:03:44.799]Again, the MTB of the Brassicaceae, Fabaceae, and Poaceae
[00:03:49.672]are grouped similarly.
[00:03:55.698]The HMMER protein domain search yielded hits for an MT-A70 domain
[00:04:00.269]in all proteins that were collected; the MT-A70 domain is the catalytically active
[00:04:07.344]methylation domain found in eukaryotic RNA methyltransferase proteins.
[00:04:14.057]In addition, the specificity-determining site search resulted in the identification
[00:04:18.057]of 9 highly conserved amino acid residues within the MTB or MTA
[00:04:24.007]subfamilies that are predicted to have co-evolved.
[00:04:27.812]Table 1 shoes the numbered positions of these amino acids,
[00:04:31.272]out of 72 positions total.
[00:04:35.709]In conclusion, this investigation made headway into determining
[00:04:39.121]how the m6A writer complex proteins arose and changed over time.
[00:04:43.980]Because this project was conducted completely online,
[00:04:47.433]the availability of sequence data was a limiting factor
[00:04:50.418]that made determining gene duplication
[00:04:52.269]difficult due to redundant data publications.
[00:04:55.716]Although the MT-A70 domain is conserved across eukaryotes,
[00:04:59.716]plants may have evolved unique mechanisms for RNA methylation due to the long
[00:05:04.276]and volatile evolutionary history of plant genomes.
[00:05:08.312]And finally, this investigation identified specificity-determining sites
[00:05:12.312]that may be attractive targets for further research into the structure
[00:05:15.785]of RNA-modifying enzymes.
[00:05:19.647]This work was made possible by Nebraska EPSCoR
[00:05:22.297]and the National Science Foundation.
[00:05:23.160]Thank you for your attention!

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A phylogenetic analysis of m6A RNA modification in land plants to elucidate protein functions

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