Amyloids are fibrous protein aggregates associated with age-related diseases, such as Alzheimer’s disease and Parkinson’s disease. The role of amyloids in the etiology of neurodegeneration is debatable, but genetic and molecular evidence supports a causative relationship between amyloidogenesis and disease. Amyloidogenic proteins are constitutively expressed throughout the lifespan of an organism, and yet only become pathogenic in certain situations. This led to a hunt to understand how amyloidogenic proteins could be modified in order to become aggregation-prone. One possibility that has garnered attention is phosphorylation, primarily because several amyloid aggregates such as tau and α-synuclein are often highly phosphorylated in disease. However, the contribution of phosphorylation to disease progression remains unclear.While amyloid aggregates are typically described as irreversible and pathogenic, some cells utilize reversible amyloid-like structures that serve important functions. One example is the RNA-binding protein Rim4 which forms amyloid-like assemblies that are essential for translational control during S. cerevisiae meiosis. If Rim4 is unable to translationally repress its mRNA targets, cells mis-segregate chromosomes during meiosis resulting in aneuploid gametes. Importantly, Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset which allows previously repressed transcripts to become translated. In Chapter 1, I describe the significance and complexity of protein phosphorylation as it relates to disease-associated amyloids and why Rim4 is an ideal model for studying this phenomenon. The objective of this thesis is to examine the mechanisms underlying clearance of Rim4 amyloid-like assemblies. The work described in Chapter 2 focuses on identifying co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. I demonstrate that yeast 14-3-3 proteins, Bmh1 and Bmh2, bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. In Chapter 3, I explore the mechanism underlying 14-3-3 contribution to Rim4 amyloid-like disassembly. I find that 14-3-3 proteins are critical for the interaction between Rim4 and its primary kinase Ime2, thus facilitating downstream multi-site phosphorylation of Rim4. In Chapter 4, I explore additional roles for 14-3-3 proteins in general protein aggregate homeostasis. I find that 14-3-3 mutants exhibit greater protein aggregate burdens. Additionally, 14-3-3 mutants accumulate ubiquitinated proteins and are sensitized to proteasome mutations, suggesting a role for 14-3-3 proteins in proteasome function. Collectively, the studies described in this thesis support a protective role for 14-3-3 proteins in protein aggregation that may have implications for amyloid biology in human disease.

<p>Conserved across metazoans, cell division depends upon the synchronous assembly and disassembly of a robust, mitotic spindle for the congression and separation of duplicated chromosomes. Composed of mostly microtubules, mitotic spindle generation depends on three different microtubule nucleation mechanisms to build its distinctive bipolar assembly. These three mechanisms are centrosomal-based, kinetochore-based, and branching microtubule nucleation. Branching microtubule nucleation occurs when microtubules nucleate from the sides of pre-existing microtubules within the mitotic spindle. Without branching microtubules, a weaker spindle apparatus can result in mitotic delay, chromosomal misalignment, multi-polar spindles, and/or aneuploidy. </p> <p>Several important complexes and proteins mediate branching microtubule nucleation. These proteins are the γ-tubulin ring complex (γ–TuRC), the homologous to augmin subunits (HAUS) complex (or simply augmin), the targeting protein for Xklp2 (TPX2), colonic and hepatic tumor overexpressed gene (chTOG), and echinoderm microtubule-associated protein-like 3 (EML3) among others. This work focused on discerning the molecular architecture of the augmin complex while also endeavoring to establish heterologous expression and purification methodologies for the γ–TuRC and TPX2. </p> <p>Augmin consists of proteins HAUS1–8 (H1–8) which bind to the sides of pre-existing microtubules and orient the γ–TuRC, the template for making microtubules, via NEDD1 to create new microtubules at shallow angles (~<20°). Despite its importance in cell division, the structure of augmin has eluded determination. This work utilized a multi-pronged approach of the baculovirus insect cell protein complex expression, cryo-EM, new protein structure prediction methodologies, and crosslinking mass spectrometry (CLMS) to elucidate the molecular architecture of the augmin complex. Further work studying the isolation, structure prediction and comparison across model organisms, and phosphorylation studies was also conducted. The results will aid the structure-assisted development of novel chemotherapeutics that target the augmin complex as well as provide deeper insights into how this complex functions in cell division. </p> <p>To help better understand the molecular mechanisms, regulation, and interactions between the different machinery involved in branching microtubule nucleation, the γ–TuRC and TPX2 also became a focus of this work. My primary effort was to overexpress and purify from the heterologous baculovirus insect cell protein complex expression system sufficient quantities of γ–TuRC for biochemical and biophysical characterization. Thus, efforts shifted to establish an expression and purification methodology for this complex. Similarly, a methodology for purification of TPX2 were also initiated. The goal of these endeavors is to establish <em>in vitro</em> biochemical reconstitution of branching microtubule nucleation utilizing the augmin complex, γ–TuRC, and TPX2 utilizing total internal reflection fluorescence microscopy (TIRF-M). </p> <p>Lastly, in unrelated work, a section on other work focuses on the roles of anti-CRISPR proteins that inhibit the Csy surveillance complex from <em>Pseudomonas aeruginosa</em> can be found. Cryo-EM studies revealed the structures of AcrIF4, AcrIF7, and AcrIF14. These anti-CRISPR proteins inhibit the Csy complex by different mechanisms. AcrIF4 prevents conformational changes necessary to recruit a Cas2/3 nuclease for degradation of invading mobile genetic elements while AcrIF7 acts as a dsDNA mimic preventing invading phage DNA recognition. Lastly, AcrIF14 functions by binding in the grove where the crRNA of Csy is and prevents hybridization between target invading MGE DNA and the crRNA. These mechanisms exemplify convergent evolution among anti-CRISPR proteins while also showing the diversity of structures produced by phages in their ongoing molecular arms race with their hosts.</p>

Meiosis is the specialized process of cell division utilized during gametogenesis in all sexually reproducing eukaryotes, which consists of one round of DNA replication followed by two rounds of chromosome segregation and results in four haploid cells. Crossovers between homologous chromosomes promote proper alignment and segregation of chromosomes during meiosis. Crossover interference is a genetic phenomenon in which crossovers are non-randomly placed along chromosomes. Crossover assurance ensures that every homologous chromosome pair obtains at least one crossover during Prophase I. Crossovers physically connect homologous pairs, allowing spindle fibers to attach and separate homologs properly. However, some organisms have shown an ability to segregate chromosomes that fail to receive at least one crossover, a phenomenon termed distributive disjunction. In Saccharomyces cerevisiae, mutation of either Tid1 or Ndj1 results in a similar defect in crossover interference. The overall number of crossovers is not substantially different from the wild type, however they are distributed more randomly with respect to each other. In this thesis, the roles of Tid1 and Ndj1 on crossover assurance and distributive disjunction have been further elucidated through use of knock-out mutants and tetrad dissection. To analyze meiotic chromosome segregation in isogenic tid1 and ndj1 strains, the spore viability of dissected tetrads was utilized as an indirect measure of nondisjunction events. An elevated number of 2- and 0- spore viable tetrads were seen in ndj1, but not tid1 yeast, confirming previous results. Elevated 2- and 0- spore viable tetrads are an indication of meiosis I (MI) nondisjunction, commonly resulting from failure of crossover formation. These results suggest crossover assurance is disrupted in njd1, but not tid1 mutants. However, MI chromosome segregation is an indirect readout of crossover formation; distributive disjunction, for example, can lead to proper segregation of achiasmate chromosomes. To determine if distributive disjunction is functional in yeast, wild type, tid1 and ndj1 versions of diploid yeast carrying a single homeologous pair of chromosomes were constructed. These strains have one chromosome (chr. III or V) replaced with one from a closely related species of yeast. The homeologous chromosome functionally replaces the homolog, however crossovers are significantly reduced between homeologs. A spore viability pattern typical of MI nondisjunction was detected in ndj1 mutants, but not in tid1 mutants. In the context of these homeologs, this pattern is suggestive of a role for Ndj1, but not Tid1, in distributive disjunction. Further, these results suggest that tid1 and ndj1 mutant yeast may not be different in their competence for crossover assurance. To directly assay competence for crossover assurance in native mutants, the incidence of E0 chromosome pairs (those lacking crossovers) was determined. To do this we assayed crossover formation along the length of chromosome III of isogenic wild type, ndj1 and tid1 mutant strains. The incidence of E0 chromosomes was comparably elevated in both tid1 and ndj1 mutant yeast, suggesting that crossover assurance is nonfunctional in both strains. We find evidence that supports the idea that interference and assurance are genetically linked. Our data also suggests that distributive disjunction may be genetically separable from some meiotic genes.

Budding yeast cells have the capacity to adopt distinct physiological states depending on environmental conditions. Vegetative cells proliferate rapidly by budding while spores can survive prolonged periods of nutrient deprivation and/or desiccation. Whether or not a yeast cell will enter meiosis and sporulate represents a critical decision which could be lethal if made in error. Most cell fate decisions, including those of yeast, are understood as being triggered by the activation of master transcription factors. However, mechanisms that enforce cell fates post-transcriptionally have been more difficult to attain. Here, we perform a forward genetic screen to determine RNA-binding proteins that affect meiotic entry at the post-transcriptional level. Our screen revealed several candidates with meiotic entry phenotypes, the most significant being RIE1 which encodes an RRM-containing protein. We demonstrate that Rie1 binds RNA, is associated with the translational machinery, and acts post-transcriptionally to enhance protein levels of the master transcription factor Ime1 in sporulation conditions. We also identified a physical binding partner of Rie1, Sgn1, which is another RRM (RNA Recognition Motif)-containing protein that plays a role in timely Ime1 expression. We demonstrate that these proteins act independently of cell size regulation pathways to promote meiotic entry. We propose a model explaining how constitutively expressed RNA-binding proteins, such as Rie1 and Sgn1, can act in cell-fate decisions both as switch-like enforcers and as repressors of spurious cell fate activation. Chapter 1 serves as a brief overview of the importance cell fate decisions and details how sporulation in the budding yeast Saccharomyces cerevisiae can be used as a model to understand the pathways and mechanisms underlying these decisions. This chapter focuses on the importance of the meiotic master regulator IME1 and the different effectors of regulation that govern its expression at the transcriptional and post-transcriptional levels. Chapter 2 describes the significance of RNA binding proteins and how they can influence cell fate decisions with a focus on cell cycle modifications that shift mitosis to meiosis. Chapter 3 explains the methodology that I used to discover two RNA-binding proteins that play key roles in meiotic entry: Rie1 and Sgn1. Chapter 4 describes my work to dissect the pathways governed by Rie1 and Sgn1. Chapter 5 discusses the potential mechanisms by which Rie1 and Sgn1 could drive entry into meiosis. Collectively, the studies described in this thesis demonstrate that Rie1 and Sgn1 affect the cell fate decision to enter meiosis in budding yeast by activating as translational activators of IME1.

Sexual reproduction requires meiosis, a specialized cell division program that halves the chromosome number of germ cells in order to generate gametes. Chromosome number reduction is achieved by two successive rounds of cell divisions after a single round of DNA replication. In meiosis I, homologous chromosomes (homologs) recombine to produce at least one reciprocal DNA exchange, called crossover (CO), in each chromosome. COs ensure proper segregation, as the resulting tetrad chromosomes are bisected during the first division to form dyads. In meiosis II, dyads split allowing segregation of single chromatids, resembling mitosis. During prophase I, the earliest stage of meiosis I, each pair of sister chromatids is arranged in series of loops tethered longitudinally by a structure called the chromosome axis, which serves as a scaffold for the machinery that promotes the formation of programmed DNA double-strand breaks (DSBs). Recombination is initiated when the DSBs are resected to produce single-stranded DNA (ssDNA) overhangs that seek out their homologs, promoting pairing. Then the synaptonemal complex (SC) forms, physically linking homolog axes through transverse filaments. In the context of the SC, meiotic recombination repairs the DSBs and turns a small fraction of them into COs. DSB formation will continue on unsynapsed axes and it is only terminated by the complete synapsis of all homolog pairs in pachytene stage. Meiocytes that fail to complete recombination and/or synapsis are eliminated (spermatocytes by mid-pachytene; oocytes from late prophase I, but at or before follicle formation), as these defects can cause chromosomal abnormalities that will be passed down to the offspring, causing severe diseases or death. Our group and others suggest that there are two simultaneous pachytene checkpoints, one that is activated by persistent DSBs and the other by asynapsis. This issue has proven very difficult to approach, since defective DSB formation/repair will inevitably affect synapsis by impairing homology search and strand invasion. The meiosis-specific HORMAD2 protein binds preferentially to unsynapsed axes, where HORMAD2 promotes the recruitment of ATR kinase. The ATR-dependent accumulation of γH2AX (histone H2AX phosphorylated on Ser139) is thought to generate a checkpoint signal from unsynapsed chromosomal regions as part of the synapsis checkpoint. In the case of spermatocytes ATR activity is concentrated to the non-homologous regions of sex chromosomes, which remain unsynapsed. This leads to γH2AX accumulation and subsequent transcriptional silencing of the sex chromosomes. This mechanism is called meiotic sex chromosome inactivation (MSCI) and is essential for the survival of spermatocytes, since the expression of sex chromosome-linked genes is toxic to spermatocytes beyond mid pachytene. Despite the importance of HORMAD2, the mechanism for its interaction with the unsynapsed chromosomes was unknown. Furthermore, it remained untested if HORMAD2 binding to the axis is actually required for the synapsis checkpoint. To address whether HORMAD2 binding to the axis is required for its function, I generated a mouse strain that expresses an altered version of the constitutive chromosome axis component SYCP2. The mutant SYCP2Δex16 lacks a short peptide sequence, called closure motif, which is predicted to bind HORMAD2. Immunofluorescence (IF) in spermatocytes showed that the localization of HORMAD2 to the chromosome axis is lost in Sycp2Δex16/Δex16, while axis formation is not impaired. Consistently, immunoblotting showed that HORMAD2 was depleted from insoluble fractions (chromatin-rich) of Sycp2Δex16/Δex16 testis extracts, while present in the soluble and total fractions. These results confirmed that the closure motif of SYCP2 serves as anchor for HORMAD2 on meiotic chromosome axis. Sycp2Δex16/Δex16 males are infertile. IF staining of cryosections of testes showed a complete loss of spermatocytes beyond pachytene. The number of cells undergoing apoptosis increased relative to the wild type, most of them in stage IV of the epithelium cycle, which corresponds to mid-pachytene. Thus, the onset of arrest coincides with the activation of the checkpoint that prevents asynaptic/DSB repair defective meiocytes from progressing beyond mid-pachytene. Nonetheless, global SC formation, and patterns of DSB formation and recombination foci in Sycp2Δex16/Δex16 are similar to the wild type. By early pachytene, the last stage reached by Sycp2Δex16/Δex16 spermatocytes, most cells have completed synapsis and DSBs are repaired. Only a small percentage of pachytene cells show a degree of incomplete synapsis. Although this suggests a synapsis-enhancing role of HORMAD2, it is thought to be concomitant to the MSCI failure observed in this mutant. In Sycp2Δex16/Δex16, 83.7% of pachytene spermatocytes have an abnormal distribution of γH2AX on sex chromosomes, accompanied by impaired accumulation of ATR and BRCA1 (another MSCI-promoting protein). The phenotype of Sycp2Δex16/Δex16 suggests a misregulation of pachytene checkpoint functions due to loss of HORMAD2-dependent ATR signaling from unsynapsed axes, and I propose that this misregulation accounts for the meiotic arrest phenotype. Females do not require MSCI, as their XX chromosomes can synapse. Accordingly, Sycp2Δex16/Δex16 females are fertile. Nonetheless, previous reports indicate that HORMAD2 has a role in sensing asynapsis, but not persistent DSB, as part of a checkpoint for quality control in oocytes. As expected, Sycp2Δex16/Δex16 cannot rescue oocytes in a DSB repair and synapsis defective background. Sycp2Δex16/Δex16 females have higher oocyte numbers than wild type. This could suggest that oocytes with defective synapsis that are usually culled in wild type ovaries, are not eliminated in Sycp2Δex16/Δex16, supporting HORMAD2 role in signaling asynapsis. Further testing in a synapsis defective background is underway. Sycp2Δex16/Δex16 seems to phenocopy Hormad2-/-, supporting a model where HORMAD2 binding to the unsynapsed axis is required for enabling its checkpoint-activating functions.:List of figures I List of tables II List of abbreviations III Acknowledgments V 1. Introduction 1 1.1. Meiosis and gametogenesis in mouse 1 1.2. Prophase of the first meiotic division 3 1.2.1. Meiotic recombination 4 1.2.2. The chromosome axis and the synaptonemal complex 5 1.2.3. The interplay between DSBs and synaptonemal complex formation 8 1.3. Surveillance mechanisms in prophase I 9 1.3.1. HORMAD1 and HORMAD2 in the male pachytene checkpoint 11 1.3.2. HORMAD1 and HORMAD2 in the female prophase checkpoint 12 1.4. HORMAD1/2 interaction with the chromosome axis 14 1.5. Aim of the project 15 2. Materials and methods 16 2.1. Generation of a mouse model lacking the closure motif 16 2.1.1. Generation of a mutant using CRISPR-Cas9 system 16 2.1.2. Identification of mutant candidates by PCR and HMA 17 2.1.3. Genomic DNA sequencing for the selection of founders 19 2.1.4. cDNA sequencing 20 2.2. Mice 21 2.2.1 Mice from a homogenized genetic background 21 2.3. Methods for the fixation of tissues 22 2.3.1. Nuclear surface spread spermatocytes 22 2.3.2. No-spin sucrose spreads 23 2.3.3. Testis fixation and cryosectioning 23 2.3.4. Ovary fixation and cryosectioning 23 2.3.4. Fixed Wild Type Cells (FWTC) 24 2.4 Staging 24 2.4.1. Staging of spermatocytes from nuclear surface spreads 24 2.4.2. Staging of epithelial cycles in cross-sections of seminiferous tubules 25 2.4.3. Staging of oocytes 26 2.5. Protein extraction 27 2.5.1. Total protein extraction and western blotting 27 2.5.2. Fractionation assay and western blotting 27 2.6. Immunofluorescence microscopy 29 2.6.1. Staining conditions 29 2.6.2. Imaging 30 3. Results 31 3.1. HORMAD2 recruitment to the axis requires the closure motif of SYCP2 31 3.1.1. Generation of a mouse mutant lacking the closure motif 31 3.1.2. Axial HORMAD2 localization is lost in Sycp2Δex16/Δex16 spermatocytes 34 3.1.3. HORMAD2 is present in Sycp2Δex16/Δex16 spermatocytes but greatly decreased from the chromatin-rich fraction 36 3.2. HORMAD1 localization to the axis seems to be independent of the closure motif of SYCP2 37 3.3. Loss of HORMAD2 localization to the axis causes infertility in males 38 3.3.1. Low levels of H1t signal indicate that Sycp2Δex16/Δex16 spermatocytes do not progress beyond mid-pachytene 39 3.3.2. Defect in spermatogenesis of Sycp2Δex16/Δex16 is caused by mid-pachytene arrest 41 3.4. Axis formation is not impaired in Sycp2Δex16/Δex16 spermatocytes 44 3.5. HORMAD2 localization to the axis plays a minor role in SC formation 45 3.5.1. In Sycp2Δex16/Δex16 spermatocytes, autosomal SC formation does not seem impaired, but abnormal XY synapsis is observed 45 3.5.2. IHO1 is properly removed from axes in Sycp2Δex16/Δex16 and Hormad2-/- spermatocytes 48 3.5.3. Synapsis at early pachytene is mildly defective in Sycp2Δex16/Δex16 and Hormad2-/- spermatocytes 50 3.6. HORMAD2 localization to the axis is not essential for DSB repair 52 3.7. Loss of HORMAD2-dependent ATR signaling from unsynapsed axes causes a misregulation of pachytene checkpoint 55 3.7.1. Abnormal sex body formation in Sycp2Δex16/Δex16 is an indicator of defective ATR signaling on unsynapsed sex chromosomes 55 3.7.2. ATR localization is affected by the loss of HORMAD2 localization to the axes 58 3.8. HORMAD2 seems to be required for a checkpoint mechanism in females that is activated by persistent asynapsis 60 3.8.1. HORMAD2 is not essential for fertility in females 60 3.8.3. HORMAD2 localization to the axis is not required for the elimination of oocytes with persistent DSB 60 4. Discussion 63 4.1. The closure motif in SYCP2 is the binding site of HORMAD2 to the unsynapsed axes 63 4.1.1 The role of HORMAD1 in the recruitment of HORMAD2 to the chromosome axis 64 4.2. Loss of HORMAD2 localization to the axes causes mid-pachytene arrest 65 4.2.1 Persistent DSBs and/or asynapsis are unlikely to trigger the mid-pachytene arrest in Sycp2Δex16/Δex16 spermatocytes 66 4.3. Loss of HORMAD2 from the unsynapsed axes impairs the recruitment of ATR activity on sex chromosomes, triggering the elimination by the mid-pachytene checkpoint 67 4.3.1 Increased asynapsis of sex chromosomes is most likely an effect of defective ATR signaling 68 4.3.2 ATR-dependent silencing of sex chromosomes is required for survival of spermatocytes beyond pachytene 69 4.4. HORMAD2 localization to the axis has a limited role in the prophase checkpoint in females 69 4.4.1 HORMAD2 seems to be required for a checkpoint mechanism in females that is activated by persistent asynapsis 70 4.4.2 HORMAD2 localization to the axis is not required for the elimination of oocytes with persistent DSB 70 4.4.3 HORMAD2 might play a role in the checkpoint that monitors unrepaired DSBs during female prophase 71 4.5. Final remarks 72 5. Summary 73 5. Zusammenfassung 75 References 78

No description available.

No description available.

In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. Due to the lack of transcription in the fully-grown oocyte, critical oocyte processes including cell cycle progression, chromosome segregation, formation and maintenance of the meiotic metaphase spindles, maternal mRNA recruitment and degradation, fertilization and egg activation are all under post-transcriptional, translational, or post-translational control. Despite advances in understanding mechanisms regulating the translational control of cytoplasmic maternal mRNAs, it is unknown whether localized maternal mRNAs exist in mouse oocytes and what mechanisms are responsible for their control. Maternal mRNAs were isolated from metaphase II (MII) mouse oocytes, microsurgically-removed MII spindle-chromosome complexes, and enucleated MII oocytes and analyzed by cDNA microarray analysis. The analysis identified enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. Maternal mRNAs involved in cellular compartments and processes related to the cytoskeleton, chromatin/nucleus, and cellular signaling were enriched on the MII spindle. Using immunofluorescence and confocal microscopy, MIS18A, a protein encoded by a spindle-localized maternal mRNA, was confirmed to be associated with the MII spindle along with components of the ribosome translational machinery. The key translational regulator, EIF4EBP1, was observed to undergo a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appeared at different positions along the spindle at different stages of meiosis. Overexpression of EIF4EBP1 mutants had a profound effect on the maintenance of MII arrest. Approximately 24% of oocytes expressing a phosphodeficient (Threonine 69 to Alanine) EIF4EBP1 mutant underwent spontaneous activation, suggesting EIF4EBP1 phosphorylation is important for translation of maternal mRNAs and maintenance of MII arrest. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types. / Molecular Biology and Genetics

In the intricate realm of biology, meiosis stands as the remarkable process responsible for generating genetically diverse haploid gametes from diploid cells. In 2000, Pezzi et al., identified STAG3 as a novel meiotic-specific synaptonemal-complex associated protein belonging to the highly conserved family of stromalin nuclear proteins. Later, over the years, research groups characterised the depletion phenotype of STAG3 in mice, where deficiency of STAG3 causes severe chromosomal defects and early meiotic arrest. These studies together collectively highlighted STAG3 as the most important meiotic cohesin. Traditionally, the role of the cohesin complex was understood as maintaining cohesion between chromatids during cell division. However, over the years, this perception has evolved significantly, expanding to include the regulation of dynamic chromosomal configurations during meiosis. With the realisation of STAG3's importance in meiotic progression, the next pressing question becomes: how does STAG3 coordinate this intricate process? This study sought to address that question by examining the STAG3 interactome in male germ cells, aiming to uncover novel pathways through which STAG3 contributes to maintaining meiotic progression. Through successful purification of the STAG3-REC8 complex, its ability to form functional complexes in-vitro was demonstrated. During my PhD thesis, I discovered links between STAG3 and DNA repair mechanisms beyond the well-known homologous recombination /non-homologous end joining pathway in meiotic recombination. By looking at the meiotic-specific protein interactome bound to the STAG3-REC8 complex through Mass Spectroscopic analysis, we identified STAG3 involvement in PARP-1-mediated repair of DNA double-strand breaks occurring outside of the programmed DSB repair during the zygotene stage of prophase I. PARP-1 is an ADP-ribose polymerase which acts as a first responder that detects DNA damage and facilitates the activation of the DNA repair pathway. STAG3 shows a preferential interaction with PARP-1 when spermatocytes are challenged with extensive DNA damage. Furthermore, the interaction of SMC3, another component of the cohesin complex, with PARP-1 during DNA damage suggests that STAG3, as part of the cohesin complex, contributes to DNA damage repair in spermatocytes. To gain deeper insights into the distinctive characteristics of STAG3, an extensive analysis of spermatogenesis in mice expressing a C-terminus truncated form of STAG3 was performed. The C-terminal region of STAG3 is not conserved among the stromalin family members, and hence it was speculated that this region might have unique functions to meiosis. Removal of the C-terminal end comprising 47 amino acids led to an early meiotic arrest, mirroring the phenotype in most cohesin subunit deletion mutants. The phenotype observed mimics the complete STAG3 depletion phenotype to some extent. The truncated STAG3 resulted in an arrest at a late zygotene/early pachytene-like stage during meiotic prophase I. One of the most notable observations was the significant reduction in the length of the axial elements (AE) in this mutant. Despite stable expression of and localisation of STAG3 to the axis, the axis length decreased by over 60%. This mutation compromised synaptonemal complex formation, leading to the early meiotic arrest. Although SYCP1 loads onto the axis and initiate synapsis, the shortened axial elements could not synapse, marked by HORMAD-1, a well-known asynapsis marker. The average number of SYCP3-marked stretches was 35 in this mutant. The increased number of AE and shortened axis length did not result from chromosome fragmentation because most chromosomes/axes had intact telomere and centromeric signals, validated by RAP1 and ACA foci, respectively. Centromeric and telomeric cohesion may be partially affected as some chromosome showed aberrant telomeric and centromeric defects. C terminal truncated STAG3 impairs synapsis between homologous chromosomes, but the sister chromatid cohesion remains largely unaffected. Also, this deletion did not affect the loading of the cohesin complex subunits onto the chromosome axis. The early meiotic arrest resulted in underdeveloped gonads, leading to infertility in otherwise healthy mice. Taken together, these results suggest novel roles for STAG3 in meiosis, and the meiotic-specific C terminal region of STAG3 is critical for proper meiotic progression in mice.

The meiotic cell cycle is modified from the mitotic cell cycle by having a premeiotic S phase which leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis, and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is expressed only during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show a decreased intragenic meiotic recombination and a delay at the onset of meiosis I. When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding for two proteins with different function depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimeras show a meiotic-specific regulation of splicing, exactly as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 and Fkh2. While Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and pre-meiotic S phase. / El ciclo meiótico se diferencia del ciclo mitótico por tener una fase S pre-meiótica caracterizada por altos niveles de recombinación, dos rondas de división nuclear sin síntesis de DNA entre las dos y una segregación cromosómica reduccional. Rem1 es una ciclina que sólo se expresa en meiosis en la levadura de fisión Schizosaccharomyces pombe. Celulas con rem1 deleccionado presentan una tasa de recombinación intragénica disminuida y un retraso en el inicio de meiosis I. Cuando se expresa ectópicamente en células creciendo vegetativamente, Rem1 induce un arresto en G1 seguido de catástrofe mitótica. Este trabajo describe que la expresión de rem1 está regulada a nivel de la trascripción y el procesamiento, codificando para dos proteínas con funciones diferentes dependiendo de la retención intrónica.. Hemos determinado que la regulación del splicing de rem1 no depende de ninguna región transcrita del gen. Además, cuando el promotor se fusiona a otros genes que contienen intrones, las quimeras presentan una regulación específica de meiosis como el rem1 endógeno. Esta regulación depende de dos factores de transcripción de la familia Forkhead, Mei4 y Fkh2. Mientras Mei4 induce la transcripción y el splicing de rem1, Fkh2 es responsable de la retención intrónica del tránscrito durante crecimiento vegetativo y fase S pre-meiótica.