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Application of the RiboTag model to define translation regulation by two testis-specific RNA binding proteins, ADAD1 and ADAD2
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Badrinath, Aditi. Application of the RiboTag model to define translation regulation by two testis-specific RNA binding proteins, ADAD1 and ADAD2. Retrieved from https://doi.org/doi:10.7282/t3-vz0j-ft21

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TitleApplication of the RiboTag model to define translation regulation by two testis-specific RNA binding proteins, ADAD1 and ADAD2
NameBadrinath, Aditi (author); Snyder, Elizabeth (chair); Belden, William J (member); Uzumcu, Mehmet (member); Rutgers University; School of Graduate Studies
Date Created2022
Other Date2022-01 (degree)
SubjectBiology, Molecular biology, Genetics, RiboTag, RNA binding protein, RNA sequencing, Spermatogenesis
Extent107 pages : illustrations
DescriptionMeiotic and post-meiotic male germ cell differentiation depends heavily on translation regulation, as male germ cells slow and then halt transcription prior to final differentiation into spermatozoa. Translation regulation is mediated in large part by RNA binding proteins (RBPs) which primarily act by binding to specific regions of target mRNAs to mediate changes in stability, ribosome association, and localization. As a result of male germ cell dependence on translation regulation throughout development, many RBPs are enriched or exclusively expressed in distinct populations of differentiating germ cells. This research focuses on two testis-specific RBPs: Adenosine Deaminase containing 1 and 2 (Adad1 and Adad2). ADAD1 is expressed predominately in the post-meiotic round spermatid and is required for post-meiotic germ cell differentiation and male fertility although the mechanism by which it acts has been unclear. ADAD2 is expressed predominately in spermatocytes and is also required for fertility. This study aimed to examine the impact of ADAD1 and ADAD2 loss on translation in their respective germ cell populations by combining Adad1 and Adad2 mutant alleles with a transgenic mouse model (RiboTag) that allows direct quantification of ribosome bound mRNAs. Using this system, mRNAs associated with an intact ribosome can be enriched via immunoprecipitation (IP) of a genetically tagged protein incorporated into the large ribosomal subunit. IPed RNAs then undergo RNA-sequencing in parallel with total RNA from the same sample. Comparison of precipitated versus total RNA thus allows quantification of ribosome association (RA) per transcript across samples, even in cases where total transcript abundance is altered.

The first part of the thesis describes the development of the analysis pipeline for RiboTag RNA-sequencing reads, utilizing the Adad2 mutant model. Initial analysis confirmed robust detection of ribosome association. However, careful examination of multiple data parameters demonstrated quality control and principal component analysis (PCA) are essential for accurate preparation of the sequencing reads. From a biological perspective, the analyses found that mutation of Adad2 resulted in reduction of spermatocyte transcript abundance concurrent with an increase of ribosome association in transcripts related to DNA damage repair and regulation of the cell cycle. These findings led to additional molecular work presented in (Chukrallah et al., in review). Overall, these results demonstrate that ADAD2 is required for proper translation in the meiotic spermatid population and may participate in the DNA damage repair pathway.

Next, the RiboTag model was applied to Adad1 mutants to determine whether ADAD1 influences translation in post-meiotic germ cells. These analyses found that while mutation of Adad1 results in minimal total transcript abundance changes, genes important for spermatogenesis showed significant reductions in ribosome-association. Among these genes were Tssk2, Akap4, Tnp1, Prm1, and Prm2. This phenomenon was not a function of overall transcript abundance or abnormal testis cellular composition. Motif analysis of transcripts with abnormal ribosome-association identified several motifs bound by RBPs such as PUM1, SAMD4A, and all three YBX proteins. These RBPs are all well characterized translation repressors in spermatids and ADAD1 loss resulted in reduced ribosome association of transcripts targeted by them. Thus, these results suggest ADAD1 may act as a novel translation activator of genes important for spermiogenesis. Future studies will aim to define the exact molecular mechanism by which ADAD1 influences post-meiotic germ cell translation.
NoteM.S.
NoteIncludes bibliographical references
Genretheses
Persistent URLhttps://doi.org/doi:10.7282/t3-vz0j-ft21
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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