Source: UNIVERSITY OF VERMONT submitted to NRP
RNA METHYLATION AS A MECHANISTIC LINK BETWEEN GENOTYPE AND PHENOTYPE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1021717
Grant No.
2020-67016-31577
Cumulative Award Amt.
$200,000.00
Proposal No.
2019-05533
Multistate No.
(N/A)
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2023
Grant Year
2020
Program Code
[A1201]- Animal Health and Production and Animal Products: Animal Breeding, Genetics, and Genomics
Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405
Performing Department
Animal and Veterinary Sciences
Non Technical Summary
Genetic and epigenetic factors contribute towards phenotypic variation in complex traits.Epigenetic modifications can affect transcription and translation and have been shown to influence variation in economically important phenotypes in agricultural species. Methylation can be studied in the context of either DNA methylation or RNA methylation. While DNA methylation regulates gene transcription, RNA methylation affects translational fidelity. The proposed project is designed to elucidate the effect of DNA and RNA methylation on gene expression and investigate the potential contribution of epitranscriptomics towards functional annotation of genomes. To accomplish this goal, we shall utilize a comparative genomics technique involving samples from both cattle and sheep which will undergo DNA whole genome bisulfite sequencing, RNA whole genome bisulfite sequencing and RNA-Seq. Subsequently, we will interrogate similarities and differences in RNA and DNA methylation between cattle and sheep. Our long-term goal is to elucidate the role of methylation applied to phenotypic variation and to provide a foundation upon which future projects can utilize RNA-WGBS to determine the extent to which RNA methylation affects phenotypic variation in complex traits.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043399108050%
3043620108050%
Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3620 - Meat, sheep; 3399 - Beef cattle, general/other;

Field Of Science
1080 - Genetics;
Goals / Objectives
The overall goal of this proposal is to elucidate the effect of DNA and RNA methylation on gene expression and investigate the potential contribution of epitranscriptomics towards functional annotation of genomes. To achieve this goal, the following specific aims will be accomplished: 1) Combine RNA-Whole Genome Bisulfite Sequencing, DNA-Whole Genome Bisulfite Sequencing and RNA-Seq to evaluate the impact of the epitranscriptome and epigenome on gene expression in cattle and sheep. 2) Examine conservation and variation of RNA methylation and DNA methylation between cattle and sheep.
Project Methods
RNA and DNA derived from the spleen, placenta, and mammary gland tissues of 4 Hereford cows and 4 Sheep will sent for RNA Whole Genome Bisulfite Sequencing, DNA Whole Genome Bisulfite sequencing and RNA-Seq. Returned sequence reads will be quality trimmed, aligned to the latest version of the respective reference genome and methylation levels will be calculated for each cytosine in the epigenome and epitranscriptome. In addition, transcript abundance will be calculated for each transcriptome. Analysis will be undertaken to characterize the similarities and differences for the epigenome and epitranscriptome within each tissue between cattle and sheep. These analyses include the identification of differentially methylated regions and characterizing patterns of nucleotide methylation. Regions of differential methylation will be identified using the list of methylated CpG sites previously identified and a combination of calling software. DNA and RNA methylation patterns will also be compared within tissue for each animal to determine the extent of posttranscriptional modifications in the genome and identify residual DNA methylation. Previously identified differentially methylated regions will be analyzed to determine if conservation and variation of DNA and RNA methylation exist with tissue between species.

Progress 06/01/20 to 05/31/23

Outputs
Target Audience:This research is designed to investigate the potential contribution of epitranscriptomics towards functional annotation of animal genomes. Therefore, the target audience includes individuals and groups who have an interest in functional annotation of genomes as well as those interested in epitranscriptomics. This may include individuals from the scientific community, industry and/or producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We were unable to secure funding to acomplish both the scientific aims and for students or postdocs to conduct the work. Therefore, no professional development was accomplished. How have the results been disseminated to communities of interest?Results were disseminated via 2 oral presentations and a presentation at Plant and Animal Genome and the International Society for Animal Genetics. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? MeRIP-Seq, Whole Genome Bisulfite Sequencing (WGBS) and RNA-Seq have been generated forCaruncle, Mammary Gland, and Spleen from four cattle and 4 sheep. All data were aligned to the most recent version of their respective reference genomes. For MeRIP-Seq, peaks harboring N6-methyl adenosine (m6A) modifications were identified and characterized and differential RNA methylation was called between tissues. For WGBS, methylation levels were calculated for each 5-methylcytosine (5mC) and differentially methylated regions were called between tissues. For RNA-Seq data, transcript abundance was measured. Subsequently, Me-RIP and WGBS result were overlapped to identify the number of 5mC located within m6A peaks. Furthermore, the relationship between m6A and gene expression was examined as was the relationship between 5mC and gene expression. Furthermore, this data was used as preliminary data for USDA NIFA grant 2022-67016-32616entitled Establishing a Pan-Epigenome for Cattle and Sheep.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: McKay, SD and BM Murdoch. 2023. RNA Methylation as a Mechanistic Link between Genotype and Phenotype. International Plant and Animal Genome XXX, San Diego, California.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: McKay, SD, S Xie and BM Murdoch. 2023. RNA Methylation as a Mechanistic Link between Epigenotype and Phenotype. 39th International Society of Animal Genetics Conference, Cape Town, South Africa


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:This research is designed to investigate the potential contribution of epitranscriptomics towards functional annotation of animal genomes. Therefore, the target audience includes individuals and groups who have an interest in functional annotation of genomes as well as those interested in epitranscriptomics. This may include individuals from the scientific community, industry and/or producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results are being disseminated via oral and poster presentations conferences such as Plant and Animal Genome. What do you plan to do during the next reporting period to accomplish the goals?During this reporting period, all data analysis will be completed. A manuscript will be written that clearly and concisely summarizes the major findings of the analyzed data.

Impacts
What was accomplished under these goals? Caruncle, Mammary Gland, and Spleen from four cattle and 4 sheep have undergone MeRIP-Seq to identify transcriptomic regions harboring N6-methyl adenosine (m6A), total RNA-Seq for identification and quantification of mRNAs and long noncoding RNAs (lncRNAs). Finally, whole genome bisulfite sequencing was performed in the same tissues of the same animals for genomic identification and quantification of 5-methylcytosine. These data sets will facilitate evaluation of the potential impact of epitranscriptomics and epigenomic modifications on gene expression in cattle and sheep.

Publications


    Progress 06/01/20 to 05/31/21

    Outputs
    Target Audience:This research is designed to investigate the potential contribution of epitranscriptomics towards functional annotation of animal genomes. Therefore, the target audience includes individuals and groups who have an interest in functional annotation of genomes as well as those interested in epitranscriptomics. This may include individuals from the scientific community, industry and/or producers. Changes/Problems:As proposed in this project, tissues from four adult sheep were collected at the University of Idaho in the first part of 2021. However, COVID-19 inhibited our ability to perform laboratory work and send out samples. Therefore, no sequencing was performed. Additionally, recently published manuscripts question the reliability of WGBS for RNA. Therefore, we have decided to perform Me-RIP instead of WGBS on RNA. While Me-RIP does not have a single base resolution, the reliability and accuracy of the method are not in question. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?When COVID permits, samples will be sent out for sequencing and analysis.

    Impacts
    What was accomplished under these goals? Samples were collected from sheep but sending out samples for sequencing was inhibited by COVID-19.

    Publications