The roles of short ORF-derived proteins in meiosis

In contrast to all other cells in our body, which contain 46 chromosomes, germ cells (gametes), such as sperm and egg cells in humans, only contain 23 chromosomes. This ensures that an embryo originating from the fusion of gametes after fertilization ends up containing a normal set of 46 chromosomes. The number of chromosomes in the parent cells is reduced during the process of meiosis, a specialized type of cell division during which cells undergo two consecutive rounds of chromosome segregation after only a single duplication. Meiosis allows for sexual reproduction and contributes to genetic diversity, which is essential for the long-term fitness of organisms. Meiotic errors cause infertility, miscarriages, and major birth defects including Down syndrome in humans, although their causes are not well understood, likely due to our poor understanding of the cellular control of most aspects of meiosis. Gene expression is the process by which genes are turned on to produce proteins, the primary functional units of cells. It is highly regulated in time and space during meiosis, which leads to dramatic remodeling of nearly every cellular part, resulting in a full metamorphosis, producing products that do not resemble the starting cell. Budding yeast, which serves as a representative model organism in meiosis research, has been found to synthesize thousands of formerly unknown short proteins. These proteins are produced from short gene sequences, termed short open reading frames (sORFs), and are shorter in length than the historically determined threshold of 100 amino acids, which is why earlier analyses have failed to detect them. In total, 2555 sORFs are expressed in meiotic yeast, with the functions of the resulting proteins being completely unknown to date. The synthesis of these short proteins takes place in highly controlled time windows, which suggests that they play a specific and time-dependent role in meiosis. My work focuses on revealing the molecular functions of these short proteins in meiosis and aims to gain important insights into the regulation of meiosis and, more broadly, gene expression in eukaryotes. I plan to disturb short protein synthesis and investigate how these manipulations influence a yeast cells ability to progress through meiosis to identify sORF that have important meiotic functions. The proposed study will identify the function of a new class of proteins, as well as determine their roles in meiosis. These data will contribute to a better understanding of the molecular control of meiosis, with the potential to explain reasons for the many meiosis- related errors known to exist in humans, resulting in infertility and birth defects.

In this study I analyzed the function of short DNA sequences that have generally been ignored in genomic studies but recently shown to potentially play important roles in specific cellular pathways. Traditionally, a gene has been defined as a part of our DNA that, by default, contains at least 300 building blocks or base pairs. Based on this historical idea of gene configuration, DNA units shorter than that, so-called short open reading frames (sORFs), have generally been ignored. Recent studies have identified functions of individual sORFs in specific cellular processes. A large-scale study conducted in our lab in the model organism budding yeast revealed that thousands of sORFs are translated during meiosis, the specialized cell divisions of germ cells that in humans produces sperm and egg cells, likely exerting a meiotic function. This is also of medical relevance as errors in meiosis lead to birth defects including Down's syndrome and infertility. In my studies I aimed to determine the function of sORFs in meiotic yeast cells by inhibiting or increasing their expression, the process by which these are converted into a functional product, and analyzed how this affects the cells' ability to go through meiosis. To do so, I first applied a modified version of a method that has been derived from an ancient bacterial defense mechanism against foreign (and potentially harmful) DNA - the CRISPR-Cas9 technology. In the modified version (CRISPRi) I used, the DNA, in my case the one of the sORFs, is not destroyed but inhibited in its expression. I could show in initial control studies that the inhibition of known meiotic genes led to a meiotic defect in yeast, which proved the applicability of this method for my study purposes. I could subsequently identify a number of sORFs that likely exert a meiotic function as cells went through meiosis slower upon inhibiting the expression of these sORFs. We are now trying to determine the exact roles and cellular functions of these sORFs in follow-up studies. In the second part of my study I increased the expression of sORFs during meiosis by producing them from a plasmid, which is a vector that helps to introduce DNA into an organism and could identify sORFs whose over-expression caused meiotic defects. We are currently trying to determine their exact function in further experiments. In summary, my studies classified DNA sequences that have formerly been ignored as functionally important in meiosis and put sORFs into the spotlight from which they had been excluded for decades. My data show that we have to reassess our traditional idea of a "gene" in order not to label DNA sequences as redundant that in reality play important cellular roles.