Deepening our understanding of meiotic prophase I events in C. elegans by the study of mutants in PROM-1, SMC-3, RRF-3 and live imaging of chromosome end movement

Meiose ist eine spezialisierte Zellteilung, bei der der Chromosomengehalt halbiert wird. Sie ist für die Bildung von haploiden Gameten erforderlich. Während der Prophase der ersten Teilung müssen sich homologe Chromosomenpaare finden, weiters mittels des synaptonemalen Komplexes verbinden und programmierte Doppelstrangbrüche über „Cross-over“ Rekombination repariert werden, um Homologe zu verknüpfen. C. elegans Gonaden rekapitulieren die meiotische Prophase I in zeitlicher und räumlicher Anordnung. Das innere Kernhüllenprotein SUN-1 ist einer der wichtigsten Akteure im Prozess der meiotischen Chromosomen-Paarung. Wir verwendeten SUN-1-Aggregate, um die Bewegung der Chromosomenenden in vivo zu studieren. SUN-1-Aggregate sind sehr dynamisch. Sie kommen zusammen, vereinigen und zerstreuen sich wieder. Anstatt einzelner Chromosomen-Enden, die ihre „Partner“ suchen, haben wir beobachtet, dass Dupletts/ Multipletts und einzelne Chromosomenenden zusammen gebracht werden. Ich analysierte die Anforderungen für die SUN-1 Aggregatbildung, Wildtyp Kinetik der Bewegung und dokumentierte den Einfluss der richtigen Orchestrierung der Meiose und Reparatur von Doppelstrangbrüchen auf das Verhalten der Aggregate. rrf-3 kodiert für eine RNA-Polymerase, beteiligt in endogener RNA Interferenz (RNAi). Genetische Analysen zeigten, dass defekte Spermatogenese für die Unfruchtbarkeit in der rrf-3 Mutante verantwortlich sein könnte. Ich zeigte, dass Paarung der homologen Chromosomen, meiotische Chromosomenachsenmorphogenese und Reperatur von DNA Doppelstrangbrüchen normal waren; nichtsdestotrotz wurden Chromosomenfragmentierung und DNA-Brückenbildung beobachtet während der Spermatogenese. Stattdessen sind Kerne in Spermien von einem Kranz von Tubulin umgeben. „Deep Sequenzierung“ von kleinen RNAs identifizierte Kandidaten Zielgene (Wildtyp und rrf-3 vergleichend), welche von RRF-3 reguliert sein könnten (eine Zusammenarbeit mit Jonathan Jesaja Gent). Cohesin spielt eine wichtige Rolle im Zusammenhalt der Schwesterchromatiden in der Meiose und Mitose. Ich isolierte, klonierte und charakterisierte ein temperaturempfindliches Allel des smc-3 Gens. Ich zeigte, dass die Reparatur der meiotischen Doppelstrangbrüche in dieser Mutante gestört ist, währendessen Kohäsion normal ist. Der primäre Defekt dieser Mutante ist die partielle Ladung von Kohäsinmolekülen, dies führt zu partieller Ladung der Komponenten der Chromosomenachsen und weiters zu defekter Synapse. In meiner Arbeit wurde erstmals ein mutiertes C. elegans smc-3 Allel beschrieben. PROM-1 spielt bei der Orchestrierung von frühen meiotischen Prophase I Ereignissen eine Rolle. Das Fehlen von prom-1 führt zu einem meiotischen Arrest gleich nach dem Eintreten in die Meiose. Ich habe einen Suppressor von prom-1 isoliert mittels eines EMS Mutagenesescreens. Der Suppressor unterdrückt den meiotischen Arrest und führt zu normaler Ladung der Komponenten des synaptonemalen Komplexes. Wiederfunktionierende Paarung im Suppressor resultiert in Ausformung von Bivalenten. Ich habe den Suppressor auf den linken Arme von Chromosome III kartiert. Die hier vorgestellte Arbeit umfasst Einblicke in verschiedene Aspekte der meiotischen Prophase I in C. elegans unter Ausnutzung besonderer Vorteile dieses Modellsystems: der Zugänglichkeit zu genetischer Analyse und Transparenz für in vivo Zeitraffer-Zell Aufnahmen.Meiosis is the specialized cell division that allows halving of the chromosome content and is required for the formation of haploid gametes. During prophase I homologous chromosomes have to pair, synapse via the synaptonemal complex (SC) and repair programmed double strand breaks (DSBs) by cross over recombination to link homologues. C. elegans gonads recapitulate the prophase I of meiosis in a temporal and spatial order. The inner nuclear protein SUN-1 is one of the key players in the process of meiotic chromosome pairing. We used SUN-1 aggregates to monitor chromosome end movement in vivo. SUN-1 aggregates are highly dynamic. They come together, coalesce and disperse. Instead of single chromosome ends looking for their homologous partner, duplets/multiplets and single chromosome ends are brought to meet. I analyzed the requirements for SUN-1 aggregate formation, wild type kinetics and documented the influence of proper orchestration of meiosis and repair of DSBs on aggregate behavior. rrf-3 encodes an RNA-directed RNA polymerase involved in endogenous RNAi. Genetic analysis revealed that defective spermatogenesis accounts for the infertility observed in rrf-3 mutant. I showed that pairing of homologues, meiotic chromosome axes morphogenesis and repair of double strand breaks were normal despite chromosome fragmentation and DNA bridge formation during spermatogenesis. Instead sperm nuclei are surrounded by wreath of tubulin. Deep sequencing analysis of small RNAs comparing wild type and rrf-3 identified candidate targets regulated by RRF-3 (a collaboration with Jonathan Isaiah Gent). Cohesin plays an important role by holding sister chromatids together both in meiosis and mitosis. I isolated, cloned and characterized a temperature sensitive allele of smc-3. I showed that repair of meiotic DSBs is impaired in this mutant whereas cohesion is normal. The primary defect of this mutant is partial loading of the cohesion complex leading to partial loading of chromosome axis components and furthermore defective synapsis. This is the first analysis of an smc-3 disruption in meiosis in C. elegans. PROM-1 is involved in the orchestration of early prophase events. Its absence results in arrest right after meiotic entry. I isolated a suppressor of PROM-1 by EMS mutagenesis. The suppressor allele in the prom-1 deletion mutant suppresses the meiotic arrest, entails the proper loading of SC proteins and successful pairing resulting in formation of bivalents. I mapped the suppressor mutation by single nucleotide polymorphism to the left arm of chromosome III. The work presented here covers insight into various aspects of meiotic prophase I in C. elegans taking advantage of its amenability to forward genetics and its transparency for in vivo time-lapse cell imaging

Multiplexed gene control reveals rapid mRNA turnover

The rates of mRNA synthesis and decay determine the mRNA expression level. The two processes are under coordinated control, which makes the measurements of these rates challenging, as evidenced by the low correlation among the methods of measurement of RNA half-lives. We developed a minimally invasive method, multiplexed gene control, to shut off expression of genes with controllable synthetic promoters. The method was validated by measuring the ratios of the nascent to mature mRNA molecules and by measuring the half-life with endogenous promoters that can be controlled naturally or through inserting short sequences that impart repressibility. The measured mRNA half-lives correlated highly with those obtained with the metabolic pulse-labeling method in yeast. However, mRNA degradation was considerably faster in comparison to previous estimates, with a median half-life of around 2 min. The half-life permits the estimation of promoter-dependent and promoter-independent transcription rates. The dynamical range of the promoter-independent transcription rates was larger than that of the mRNA half-lives. The rapid mRNA turnover and the broad adjustability of promoter-independent transcription rates are expected to have a major impact on stochastic gene expression and gene network behavior

A New Thermosensitive smc-3 Allele Reveals Involvement of Cohesin in Homologous Recombination in C. elegans

The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11–dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation

Release of CHK-2 from PPM-1.D anchorage schedules meiotic entry

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways i

Coxiella burnetii Infection in Livestock, Pets, Wildlife, and Ticks in Latin America and the Caribbean: a Comprehensive Review of the Literature

Purpose of the Review Q fever , a bacterial zoonosis caused by Coxiella burnetii, is reported very heterogeneously in humans in Latin America. The objective of this study was to review the data on Coxiella burnetii Infection in animals in Latin America and the Caribbean. Recent Findings A comprehensive literature review was carried out in the 47 countries and territories of Latin America on various search engines and grouped into four groups: livestock, pets, wildlife, and ticks. Summary Thus, 113 studies were selected between 1950 and 2022. Among the 47 countries, only 25 (53%) had at least one publication related to C. burnetii infection in animals. The most productive country was Brazil (N = 51), followed by French Guiana (N = 21), and Colombia (N = 16). Studies in livestock from 20 countries have shown widely varying country-to-country rates of seroprevalence, ranging from 0 to 67%. Some studies from seven countries, especially French Guiana and Brazil, found antibodies and sometimes positive PCR in dogs and cats, generally in the context of investigations around human clustered cases. Knowledge remained fragmented about infection in wildlife from only five countries (Chile, Colombia, Brazil, French Guiana, and Uruguay). C. burnetii infection was identified by PCR in Chiroptera (7 species), Rodentia (6 species), Suina (2 species), Xenartha (1 species), Cingulata (1 species), and Perissodactyla (1 species). Studies on Coxiella sp. in ticks have been performed in 11 countries, mostly in Brazil, and mainly found Coxiella-like endosymbionts. Thus, data on C. burnetii infection in animals are sparse and incomplete in Latin America and the Caribbean, and more research is warranted

Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2–dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates

Quantification of pre-mRNA escape rate and synergy in splicing

Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks

European fluxes of medical gadolinium to the ocean: A model based on healthcare databases

Marine ecosystems are exposed to a multitude of stresses, including emerging metals as Rare Earth Elements. The management of these emerging contaminants represents a significant environmental issue. For the past three decades, the increasing medical use of gadolinium-based contrast agents (GBCAs) has contributed to their widespread dispersion in hydrosystems, raising concerns for ocean conservation. In order to control GBCA contamination pathways, a better understanding of the cycle of these elements is needed, based on the reliable characterization of fluxes from watersheds.Our study proposes an unprecedented annual flux model for anthropogenic gadolinium (Gdanth) based on GBCA consumption, demographics and medical uses. This model enabled the mapping of Gdanth fluxes for 48 European countries. The results show that 43 % of Gdanth is exported to the Atlantic Ocean, 24 % to the Black Sea, 23 % to the Mediterranean Sea and 9 % to the Baltic Sea. Together, Germany, France and Italy contribute 40 % of Europe’s annual flux. Our study was therefore able to identify the current and future major contributors to Gdanth flux in Europe and identify abrupt changes related to the COVID-19 pandemic

Contribution of RNA Degradation to Intrinsic and Extrinsic Noise in Gene Expression

Summary: Genetically identical cells contain variable numbers of molecules, even if the cells share the same environment. This stochastic variability is prominent when molecules have low abundance, which is the case for mRNA noise. Most studies focused on how transcription affects mRNA noise, and little is known about the role of RNA degradation. To discriminate the fluctuations in these processes during the decay of a pair of reporter mRNAs, we quantified the uncorrelated intrinsic and the correlated extrinsic noise using single-molecule RNA FISH. Intrinsic noise converges to the Poisson level during the decay. mRNAs that have a short half-life are more susceptible to extrinsic noise than stable mRNAs. However, the Xrn1 exonuclease and the NMD pathways, which degrade mRNAs rapidly, were found to have lower fluctuation, which mitigates the noise of the short-lived mRNAs. This permits low variability across the entire range of mRNA half-lives. : Baudrimont et al. show that fluctuations in transcription and RNA degradation can be distinguished by assessing whether extrinsic noise increases during decay. Unstable RNAs are more susceptible to noise, but the activity of the enzymes that degrade them fluctuates less in comparison to those that target stable mRNAs. Keywords: Saccharomyces cerevisiae, gene expression noise, single-molecule RNA FISH, surveillance pathway, birth-death model, exosome, RNA stability, galactose, stochastic deviatio