Identification of three candidate genes involved in N6-Methyladenosine (m6A) RNA modification in Arabidopsis

Emma O'Shaughnessy Author

07/28/2021 Added

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Identification of three candidate genes involved in N6-Methyladenosine (m6A) RNA modification in Arabidopsis Poster Presentation

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[00:00:00.384]Hello my name is Emma O’Shaughnessy
[00:00:04.384]and I have spent my summer working in Dr. Bin Yu’s lab
[00:00:07.564]on the Identification of three candidate genes involved in
[00:00:10.908]N6-Methyladenosine (m6A) RNA modification in Arabidopsis.
[00:00:16.178]in order to understand this work it is essentialto recognize
[00:00:21.663]the importance of RNA Methylation.
[00:00:25.680]The Methylation of RNA is a type of post-transcriptional modification
[00:00:29.840]that plays a vital role in the regulation
[00:00:32.425]splicing, maintenance, and translation
[00:00:35.467]of the genetic code.
[00:00:36.487]My area of focus specifically has been
[00:00:41.253]M6a-methylation
[00:00:42.404]M6a-methylation is an incredibly abundant
[00:00:45.427]form of RNA modification and has been
[00:00:48.089]shown to be responsible for
[00:00:50.519]growth, development, and
[00:00:52.053]flowering time in plants In animals,
[00:00:55.175]m6a modification regulates the development
[00:00:57.840]of the nervous and reproductive systems,
[00:00:59.877]along with certain cancers.
[00:01:01.887]For example people with the mutated METTLE genes,
[00:01:04.910]homologs of the genes responsible for m6a-
[00:01:07.754]modification in plants, often develop
[00:01:10.373]lung cancer.
[00:01:11.278]M6a modification, in plants, is
[00:01:14.597]added by a protein known by mRNA
[00:01:17.458]adenosine methylase (MTA), and the close
[00:01:21.956]homolog MTB. The modification is
[00:01:26.833]removed by the ALKBH protein family.
[00:01:31.060]While we know some of the enzymatic players
[00:01:33.756]in M6a modification, there is an
[00:01:36.877]elaborate protein complex and the
[00:01:38.998]genetic origins of all downstream proteins
[00:01:41.517]are not known.
[00:01:42.633]This is where the Bin Yu lab comes in…
[00:01:45.213]Using Mass Spectrometry
[00:01:49.213]the Bin Yu lab
[00:01:50.336]identified protein that interact with MTA
[00:01:52.706]and MTB. The Mass spectrometry first
[00:01:55.451]must use a protein of interest
[00:01:56.972]in this case EMB1706 and EMB1691.
[00:02:03.611]This protein was isolated using
[00:02:06.509]immunoprecipitation in which specialized
[00:02:08.569]antibodies are used to mark and gather
[00:02:11.800]flagged MTA and MTB along with any
[00:02:14.859]proteins that interact with MTA/MTB.
[00:02:17.539]The proteins that were attached to the
[00:02:20.365]flag-MTA and flag-MTB were then
[00:02:22.021]digested using Trypsin.
[00:02:23.172]This frees up the peptides that made
[00:02:24.954]up the proteins, allowing their chemical
[00:02:26.937]composition to be analyzed by the
[00:02:29.006]mass spectrometer
[00:02:29.906]Simply put, Mass spectrometry
[00:02:32.083]uses an electron beam to excite
[00:02:34.022]the electrons in the sample of material.
[00:02:35.986]The excited molecules move rapidly and
[00:02:38.936]bounce off of a detector. The speed at
[00:02:41.480]which they bounce off of the detector
[00:02:43.429]is recorded and used to identify the
[00:02:45.212]mass, charge, and composition of .
[00:02:47.668]the molecules.
[00:02:48.530]The sequences of the amino acids were
[00:02:52.372]determined by this mass spectrometry
[00:02:54.198]method and can now, mapped to the
[00:02:57.880]reference genome. These assigned genes
[00:03:00.115]then became candidates for contributing
[00:03:03.385]to the m6a modification.
[00:03:04.676]To investigate these candidates, we
[00:03:09.086]examined the genotypes and phenotypes
[00:03:10.906]of three T-DNA insertion mutants of the
[00:03:14.264]model organism, Arabidopsis thaliana.
[00:03:16.709]T-DNA mutations are powerful tools for understanding
[00:03:20.337]the purpose of genes by disrupting their function.
[00:03:24.337]Short segment of foreign DNA areinserted into the gene of interest, which disrupts
[00:03:28.730]function, but also acts as a marker for a mutation
[00:03:31.873]We ordered Arabidopis seeds from ABRC with mutations at the locations
[00:03:38.668]of our genes of interest. We prepared and planted these seeds and they grew
[00:03:45.648]in 16 light- 8 dark light cycle and 22 C conditions in a growth chamber.
[00:03:51.690]As the arabidopsis grew, we observed them and noted any possible phenotypic
[00:03:58.017]abnormalities.
[00:03:58.754]Once the arabidopsis had grown to a sufficient
[00:04:01.424]size samples of the leaves were taken ground
[00:04:04.431]and their DNA was extracted using isopropanol DNA extraction methods.
[00:04:09.030]Using specially designed primer we ran PCR to amplify the genomic regions and th
[00:04:17.421]T-DNA insertions. Gel electrophoresis was used to determine if the T-DNA
[00:04:22.534]insertion mutations were present in the growing arabidopsis.
[00:04:26.659]We discovered that two of the three genes we examined were homozygous,
[00:04:33.181]one for the SALK_151028 mutation and the other for CS907108 mutation, but the
[00:04:43.699]plants experienced no major differences in phenotypic expression compared
[00:04:48.700]to the wildtype.
[00:04:49.700]These results are not nearly the end of the work on this project, instead we can
[00:04:57.743]look ahead to future directions for the research. Searching for more gene
[00:05:02.335]candidates and mutations to examine is essential to developing a fuller
[00:05:06.077]understanding of the M6A methylation protein complex.
[00:05:10.351]These candidate genes could include homologs for the genes we have just
[00:05:14.202]examined, which could explain the lack of phenotypic change in our mutated
[00:05:19.853]arabidopsis or they could include possible heterozygous mutations.
[00:05:23.145]In the future, using crispr to examine the effects of gene knockouts in
[00:05:27.715]embryonic growth and development or to examine the purpose of entire gene
[00:05:32.589]families would be extremely helpful in gaining more information about
[00:05:35.954]m6A methylation.
[00:05:37.315]Thank you to Mu Li, Lu Gan, and Dr. Bin Yu
[00:05:43.947]for their help and support this summer. This research is funded by a grant from
[00:05:47.754]the National Science Foundation.
[00:05:50.420]Thank you!

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Identification of three candidate genes involved in N6-Methyladenosine (m6A) RNA modification in Arabidopsis

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