Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
RIBOSOME-BOUND TRANSCRIPTOMICS AS A LINK BETWEEN GENE EXPRESSION TO PROTEIN TRANSLATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024719
Grant No.
2021-67016-33417
Cumulative Award Amt.
$200,000.00
Proposal No.
2020-02835
Multistate No.
(N/A)
Project Start Date
Jan 1, 2021
Project End Date
Dec 31, 2023
Grant Year
2021
Program Code
[A1201]- Animal Health and Production and Animal Products: Animal Breeding, Genetics, and Genomics
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
(N/A)
Non Technical Summary
An ongoing goal of animal genomics is to identify those variants that directly cause phenotypic variation in economically important traits. Many genomic variants have been identified but phenotypic variation is often more complex than a singular cause. Large, genome-wide association studies rarely identify causal variants within gene bodies, and recent efforts to annotate non-coding regions of the genome have made progress towards improving our understanding of how genome variation leads to different phenotypes. The translatome refers to the entirety of messenger RNAs associated with ribosomes and undergoing active protein synthesis. Functional annotation of the genome is necessary in order to fully discern the effects of polymorphisms on phenotypic variation. To determine the role of translation regulation and efficiency in the functional annotation of genomes, we must first decipher the translatome. Our long-term goal to understand how genetic variation among livestock can impact phenotypic variation through altered translation efficiency and contribute this knowledge to the advancement of genetic selection for animal breeding and production.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30433991080100%
Knowledge Area
304 - Animal Genome;

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

Field Of Science
1080 - Genetics;
Goals / Objectives
The overall goal of this proposal is to elucidate the relationship between the abundance of functional RNAs and genome-wide translation efficiency and its potential role in the functional annotation of genomes.In order to achieve this goal the following specific aims will be accomplished:1)Examine the relative relationship between transcript abundance in cells or tissues to the translation of those transcripts into protein using ribosome footprinting, and2)Combine RNA sequencing, tRNA sequencing, and ribosome footprint sequencing to evaluate the impact of tRNA abundance, synonymous mutations, and optimal codon usage on translation.
Project Methods
Objective 1. Examine the relative relationship between transcript abundance in cells or tissues to the translation of those transcripts into protein using ribosome footprinting.mRNA (RNA-seq) and ribosome-bound RNA sequence (ribo-seq) data will be generated for each of the 24 samples in this study (8 fibroblast lines, 8 primary muscle lines, 8 frozen skeletal muscle tissue samples). Cell cultures will be treated with cycloheximide, a translation inhibitor, one hour prior to cell lysis. Ribosome-bound mRNAs will be treated with RNaseI and all unbound RNAs degraded. After inactivation of RNaseI and washes ribosomal RNA (rRNA) will be depleted using a commercial rRNA depletion kit. The efficiency and fidelity of ribosome footprinting methods on flash-frozen tissues will be assessed on sample libraries generated using a commercially available kit and methods previously described. In either method, frozen tissues will be mechanically pulverized in the presence of cycloheximide. Ribosome footprint assays generate short sequence read data with specific lengths such that precisely which codon occupies the A-site within the ribosome can be determined. We will compute transcript expression using RNA-seq read data and identify actively translated mRNAs by mapping ribosome-bound read fragments. Overlapping mRNA and ribo-seq datasets will be merged and normalized to estimate translation.Objective 2. Combine RNA sequencing, tRNA sequencing, and ribosome footprint sequencing to evaluate the impact of tRNA abundance, synonymous mutations, and optimal codon usage on translation.Whole-genome DNA resequencing will be carried out for each fibroblast cell line to minimum sequence coverage of 30X. All cell lines and tissues will be genotyped using the DNA-Seq data and variants called using the GATK pipeline. tRNAs will be purified and sequenced for all samples. Differential mRNA and tRNA expression between sexes will be determined in the fibroblast cell lines and between primary muscle cell cultures and flash-frozen tissues. For DNA and RNA, our analysis will result in the identification of allele-specific synonymous SNPs. Genotype and tRNA sequencing data will combine with the mRNA and ribosome footprint sequencing data generated in Objective 1 to identify the impact of synonymous SNPs on codon bias and translation efficiency. Ribosome occupancy will be calculated for each codon in transcripts identified as being heterozygous in the third base of any codon. Translational efficiency will be calculated based on the number of normalized ribosome footprint sequenced reads divided by the number of normalized RNA-Seq reads in the form of RPKM. Counts per million normalized read counts will be tabulated for all codons for which read data exists.

Progress 01/01/21 to 12/31/23

Outputs
Target Audience:The target audience for this research project were other scientists, particularly animal genomicsts. Furthermore, scientists working for or with stakeholder groups such as industry, pharma, and breed associations requiring additional information centered on genetic interactions. Changes/Problems:The primary challenge during this project was dealing with the lingering effects of the covid pandemic. This created issues in obtaining laboratory supplies, interacting with students, and particularly travels between collaborators. What opportunities for training and professional development has the project provided?A female Ph.D. student was trained during her graduate career as a result of working on this project and is now a postdoctoral fellow within the USDA-ARS system. Furthermore, two undergraduate animal science students were mentored by this Ph.D. student, assisting her on aspects of the project and are now pursuing their own PhDs in the animal genomics area. Through a new collaboration resulting from funding of this project, another female PhD student at Utah State University has received training as well. How have the results been disseminated to communities of interest?Results from this project have been published in 3 first (and corresponding) authored journal papers as well as 2 additional papers in collaboration. Multiple published abstracts and presentations at national and international conferences were products of this grant funding. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Towards Goal 1: Using a kit-based approach to sequence ribosome footprints from fibroblast cell lines and post-mortem tissues (skeletal muscle, kidney, and liver) in cattle. We developed a new collaboration with Utah State University to carry these findings to cell culture to study translational efficiency and transcript abundance on cell differentiation (myoblasts) during muscle development. We were able to extend our research project to include comparative fibroblast-based ribosome footprinting in sheep, goats, and cattle. Towards Goal 2: We sequenced mRNA, tRNA, and ribosome protected fragments from post-mortem tissues (skeletal muscle, kidney, and liver) as well as differentiating myoblast cell lines. We were able to characterize differential expression of tRNAs and mRNAs. For the first time, we created a working atlas of translational efficiency in varying tissues in livestock. Furthermore, we were able to identify novel putative open reading frames within the bovine, ovine, and caprine genomes. We especially focused on identification of micropeptides, functional polypeptides typically less than 100 amino acids in length. This improves genome annotation. From these datasets we published 3 manuscripts as first or corresponding authors, an additional two as collaborators, and contributed numerous sequence library datasets to the SRA databases. Finally, we trained two female undergraduate researchers who are now pursuing PhD training in animal genomics labs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Influence of Maternal BLV Infection on miRNA and tRF Expression in Calves. Pathogens
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Current challenges and future of agricultural genomes to phenomes in the USA. Genome Biology
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Implications of tRNA abundance on translation elongation across bovine tissues. Frontiers in Genetics
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Ribosome profiling reveals stage-specific translational regulation during muscle differentiation. Genome Biology


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The target audience for this project is other scientists, including animal scientists and genomicists. Changes/Problems:The lingering effects of thepandemic created unique and unanticipated challenges. Abattoir partners continue torestrictour access to prevent the spread of protect their plants. A protential collaboratorat another academic institutionwasunable to host visits as proposed in the grant. What opportunities for training and professional development has the project provided?A female Ph.D. student has been actively working on this project. Furthermore, two undergraduate animal science students have been mentored by this Ph.D. student, assisting her on aspects of the project. Through a new collaboration resulting from funding of this project, another female PhD student at Utah State University has received training as well. How have the results been disseminated to communities of interest?The current results have been published orsubmitted for publication (Frontiers in Genetics) and two published abstracts (ASAS-OKCand PAG) in 2022. A poster was presented by the Ph.D. student at the American Society for Animal Science meeting. The student also gave a talkatPAG. What do you plan to do during the next reporting period to accomplish the goals?Finalize data analysis and finish drafting manuscripts for publication. Continue current work on myoblast differentiation to better explain the role of tRNAs, codon usage, and transcript abundance on a specific phenoytpe (muscle development).

Impacts
What was accomplished under these goals? Towards Goal 1:Using have continued to utilize akit-based approach tosequenceribosome footprints from fibroblast cell lines and post-mortem tissues (skeletal muscle, kidney, and liver) in cattle. We have initiated a new collaboration with Utah State University to carry these findings on in cell culture to study translational efficiency and transcript abundance on cell differentiation (myoblasts) during muscle development. Towards Goal 2: We have sequenced mRNA, tRNA, and ribosome-protected fragments from post-mortem tissues (skeletal muscle, kidney, and liver) as well as differentiating myoblast cell lines. We have fullydeveloped ananalysis pipelineto merge these unique data types into informative relationships. We published one manuscript and submitted two more for publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Goldkamp A, Li Y, Rivera RM, Hagen DE. Differentially expressed tRNA-derived fragments in bovine fetuses with assisted reproduction induced congenital overgrowth syndrome, Frontiers in Genetics, 2022
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Goldkamp AK, Hagen DE. Insights into translation through tRNA sequencing and ribosome profiling. International Plant and Animal Genome Conference (PAG) XXIX, Virtual, January 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Goldkamp AK, Hagen DE. Global assessment of translational regulation within bovine tissues. Advances in Genome Biology and Technology (AGBT)-Agriculture, San Diego, California, April 2022.


Progress 01/01/21 to 12/31/21

Outputs
Target Audience:The target audience for this project is other scientists, including animal scientists and genomicists. Changes/Problems:The ongoing pandemic has created unique and unanticipated challenges. Primarily, collaborative abattoir partners have restricted our access to prevent the spread of covid within their plants. Further, collaborators at other academic institutions were unable to host visiting scientists (as proposed in the grant) and we were unable to travel during certain times of the year in 2021. Finally, ordering and receiving labware, disposables, and reagents have been incredibly challenging due to supply chain shortages and issues. What opportunities for training and professional development has the project provided?A female Ph.D. student has been actively working on this project. Furthermore, two undergraduate animal science students have been mentored by this Ph.D. student, assisting her on aspects of the project. How have the results been disseminated to communities of interest?The current results have been submitted for publication (BMC Genomics) and two published abstracts (ASAS-Louisville and ISAG-Virtual) in 2021. A poster was presented by the Ph.D. student at the American Society for Animal Science meeting. The student also gave talks at both ASAS and the International Society of Animal Genetics. The PD gave an invited seminar, presenting data from this project at the University of Idaho, Texas A&M University,and Oklahoma State University. What do you plan to do during the next reporting period to accomplish the goals?We will wrap up all sequencing and comparative analysis, fulfilling both goals.

Impacts
What was accomplished under these goals? Towards Goal 1: We used previously published methods along with two separate commercially available kits for comparative purposes. In our hands, one commercial kit outperformed other methods. Using this kit we sequenced ribosome footprints from fibroblast cell lines and post-mortem tissues (skeletal muscle, kidney, and liver). We generated correlations between transcript abundance and ribosome-protected fragments, developing probabilities for translational efficiency. Towards Goal 2: We have sequenced mRNA, tRNA, and ribosome-protected fragments frompost-mortem tissues (skeletal muscle, kidney, and liver). We have begun developing analysis pipelines to merge these unique data types into informative relationships for future publication.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Goldkamp A, Li Y, Rivera RM, Hagen DE. Characterization of tRNA Expression Profiles in Large Offspring Syndrome, BMC Genomics, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Characterization of tRNA expression profiles in large offspring syndrome. American Society of Animal Science Annual 2021 Meeting Abstracts, Louisville, Kentucky, July 15, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Insights into translation through tRNA seqeuencing and ribosome profiling. Proceedings of the 38th International Conference on Animal Genetics, Virtual, July 28, 2021.