I am a third year cellular biology major at University of California, Davis (UCD), planning to become a research scientist in cellular or computational biology. My previous research experiences have focused on understanding stem cell expression in zebrafish (a tropical freshwater fish) and on host reproductive responses induced by infecting bacteria that support vertical transmission in Drosophila melanogaster (fruit flies).
In the Draper stem cell lab at UCD, we studied germline regulation in the model organism zebrafish to reveal common regulatory mechanisms that may provide insight as to how the germline is stably established in humans. We identified a gene required for germ line stem cell specification in zebrafish by producing loss-of-function mutations in embryos and studying the associated phenotypic result in mutant adults. We found that such mutants were sterile; the stem cell population in these mutants enters meiosis prematurely and is quickly depleted, thereby precluding continuous production of gametes throughout the life of the fish. I used fluorescent in situ hybridization (FISH) to determine how early in development the stem cell population of mutants and wild-type siblings begin to differentiate and when the stem cell population in mutants is fully depleted.
The identified gene has an orthologous gene in humans that carries out a parallel function. Thus, understanding the role and regulation of the zebrafish gene and applying this knowledge to the human ortholog may reveal novel therapies for human cancers that can be attributed to defective germline stem cell regulation, such as many human childhood tumors and adult testicular cancers1.
At the Turelli Evolution and Ecology lab at UCD, I studied bacteria-host interactions between Wolbachia pipientis, a maternally transmitted intracellular bacteria, and D. melanogaster. Wolbachia affect their hosts in a myriad of ways to increase their prevalence in host populations. For example, infection by some strains of Wolbachia protects the host from viral infection. This trait is being exploited for human health benefits. Bites from the common Dengue fever vector mosquito, Aedes aegypti, no longer transmit the Dengue virus to humans if the mosquito is infected with some strains of Wolbachia. Thus, wild mosquito populations are being infected with Wolbachia in areas where the disease is prevalent to prevent human infections2.
In the Turelli lab, I focused on a different host manipulation by Wolbachia. Cytoplasmic incompatibility (CI) alters host reproduction by reducing egg hatching in crosses unfavorable to Wolbachia infection spread. Working with another undergraduate, I characterized bacterial transmission of Wolbachia as a function of paternal age and of infection titer, or Wolbachia “concentration.” We confirmed that CI decreases with male age, and we discovered that CI is triggered in crosses that reduce titer. Understanding the complex interplay between Wolbachia and its host may result in more effective measures for controlling pathogenic infections in the human population.
I am excited to be working in the Brem lab at the Buck Institute for Research on Aging this summer because I am interested in participating in leading-edge scientific research that may lead to real-world biomedical treatments that could enhance the quality of life for a multitude of people.
Validating and Investigating the Regulatory Impact of 3’UTR Length Changes in Lung Adenocarcinoma Tumors
To be expressed as protein, genes must first be transcribed into pre-mRNA and be edited to make a mature mRNA, which includes a 3’ stretch of nucleotides at the end of the transcript. These regions are downstream of the terminal amino acid stop codon and, thus, are not translated into the protein polypeptide chain; hence, they are referred to as 3’ Untranslated Regions (3’UTRs). 3’ UTR length is determined by cleavage site selection by the cleavage and polyadenylation complex after which the poly-adenine tail is added3. For a given gene, the length of its 3 ‘UTR may vary based on tissue, cell cycle stage, and genetic background. Changes in 3’ UTR length result in the inclusion or exclusion of RNA binding motifs recognized by regulatory factors. The presence or absence of these regulatory motifs can impact mRNA stability, turnover, and translation. One largely unexplained hallmark of cancer cells is the use of transcript forms with short 3’ UTRs4. In cancer, the mechanisms of 3′ UTR length changes, their regulatory effects, and their global impact on tumorigenesis are almost completely unknown.
Figure 1. Processing of RNA to prepare it for translation.
Multiple Polyadenylation Sites (PASs) are present on the RNA, which can be used by cell machinery to create alternatively polyadenylated mRNA. Picture Credit: Kristina A. Clemens, Brem Lab.
Ongoing work in the Brem lab has focused on developing computational tools to identify novel, tumor-specific 3’ UTR length changes from mRNAseq data and devising wet-lab experimental strategies to understand the regulatory impact of these changes during tumorigenesis. Computational analysis carried out on a cohort of tumor-normal lung transcriptomes has shown that the 3’ UTRs of mRNA transcripts are consistently altered in tumors compared to normal control lung tissues from the same patients.
While interning in the Brem lab, I will use a combination of bioinformatic and experimental biology techniques to accomplish the following aims: 1. Devise quantitative PCR assays to measure the abundance of long and short transcripts of top candidate genes predicted by the lab’s computational surveys. 2. Develop expression constructs that will be introduced into cells to test the regulatory impact of short and long transcripts forms of top candidate genes. 3. Identify RNA binding motifs enriched in the transcripts undergoing tumor-specific 3’ UTR length changes using the Meme Suite5. 4. Apply computational strategies developed in the Brem lab to interrogate additional cohorts of tumor-normal sequencing data from The Cancer Genome Atlas6 to investigate the causal, genetic basis for 3’ UTR length changes.
The work of this project—identifying 3’ UTR length changes in cancer and understanding their mechanism and impact—is important for two reasons. First, many such changes could be detected in tumors and used as diagnostic markers. Second, many tumor-specific 3’ UTR forms could ultimately prove to be of use as novel targets for RNA-based and small-molecule drug treatments.
Future Plans:
Next year, during my fourth and final year at UC Davis, I will return to the Turelli Lab to work on localizing the distribution of Wolbachia within fly cells throughout their lifetime using FISH microscopy and apply some of the computational skills from my SRF internship to conduct genomic analyses of the bacteria. After I receive my bachelor’s degree, I hope to enroll in a cellular or computational biology Ph.D. program to gain the skills and experience for a career as a research scientist.
References:
1. Malkin, David, et al. “Germline mutations of the p53 tumor-suppressor gene in children and young adults with second malignant neoplasms.” New England Journal of Medicine 326.20 (1992): 1309-1315.
2. 2. Jeffery, J. A., Yen, N. T., Nam, V. S., Hoffmann, A. A., Kay, B. H., & Ryan, P. A. “Characterizing the Aedes aegypti population in a Vietnamese village in preparation for a Wolbachia-based mosquito control strategy to eliminate dengue.” (2009): e552.
3. D. C. Di Giammartino, K. Nishida, and J. L. Manley. Mechanisms and consequences of alternative polyadenylation. Mol Cell, 43(6):853–66, Sep 2011. PMCID: PMC3194005.
4. C. Mayr and D. P. Bartel. Widespread shortening of 3’ UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell, 138(4):673–84, Aug 2009. PMCID: PMC2819821.
5. Robert C. McLeay, Timothy L. Bailey, “Motif Enrichment Analysis: a unified framework and an evaluation on ChIP data”, BMC Bioinformatics, 11:165, 2010.
6. The Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543-550, July 2014. PMCID: PMC4231481.
