Developmental roles and molecular mechanisms of Asterix/GTSF1

Maintenance of germline genomic integrity is critical for the survival of animal species. Consequently, many cellular and molecular processes have evolved to ensure genetic stability during the production of gametes. Here, we describe the discovery, characterization, and emerging molecular mechanisms of the protein Asterix/Gametocyte-specific factor 1 (GTSF1), an essential gametogenesis factor that is conserved from insects to humans. Beyond its broad importance for healthy germline development, Asterix/GTSF1 has more specific functions in the Piwi-interacting RNA (piRNA)-RNA interference pathway. There, it contributes to the repression of otherwise deleterious transposons, helping to ensure faithful transmission of genetic information to the next generation. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications

Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos

Purpose: Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. Methods: Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. Results: GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. Conclusions: To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos

Biomolecular NMR spectroscopy: Application to the study of the piRNA-pathway protein GTSF1, and backbone and side-chain spin relaxation methods development

The structural dynamics of proteins and other macromolecules typically serve crucial roles for their respective biological function. While rigid protein structures are used in classic “lock and key” descriptions of enzymology and receptor-ligand interactions, more and more evidence suggest that the majority of molecular interactions occur on the spectrum between induced-fit binding and conformational selection binding. This model of biomolecular interaction requires, to differing degrees, conformation plasticity and dynamics of the protein itself. To characterize the determinants and implications of protein dynamics, there exists no more suited biophysical technique than nuclear magnetic resonance (NMR) spectroscopy. This method is capable of probing the individual atomic nuclei of proteins in a site-specific manner. Furthermore, NMR spectroscopy is unique in being able to access timescales from picoseconds to seconds, providing information on events from bond vibration and libration to protein folding and ligand binding. The breadth of biophysical information accessible by NMR spectroscopy has led to its widespread use in the study of protein dynamics. The work presented herein involves i) the use of NMR for investigation of structure and dynamics in two separate biological systems that demonstrate a high degree of flexibility for folded proteins and ii) the improvement of pulse sequences and methodology for better characterizing picosecond to nanosecond backbone and side-chain dynamics. The organizing principle of this work, which is best exemplified in the structural studies of the piRNA-pathway protein Gametocyte-specific factor 1, is the unmatched capability of NMR spectroscopy to decipher molecular details within dynamic protein systems. First, the molecular structure and RNA-binding properties of gametocyte-specific factor 1 (GTSF1) of the piRNA effector pathway were investigated. A partially disordered protein with two Zn finger domains, the work presented here describes the isolation of a GTSF1 protein construct amendable to study by NMR spectroscopy. Chemical shift assignment of GTSF1 allowed site-specific observation of amide correlations, which established the basis for NMR structure calculation of GTSF1 and the evaluation of binding to candidate RNA sequences, with goal of the identification of an in vivo RNA binding partner for GTSF1. The work presents compelling data that indicate GTSF1 Zn finger 1 specifically binds a motif GGUUC(G/A) RNA, which in this study was found in the T-arm loop of transfer RNA. Zn finger 2 is affected by the interaction with RNA, but the available structural and binding data indicate that the second Zn finger is a more dynamic, breathable entity, supported by cysteine chemical shift and structural differences between the two GTSF1 Zn fingers. Although it’s currently speculative, the function of GTSF1 might first require binding of RNA to the more stable Zn finger 1, which then leaves Zn finger 2 poised for binding to another molecular species. tRNA-derived fragments that include the T-arm TC loop have been recently implicated in silencing of transposable elements in mammalian cells. GTSF1, which was identified in a genetic screen for piRNA-pathway proteins as vitally required for gene silencing, might plausibly act as a sensor of transcription of transposable elements and help initiate Piwi-piRISCs-mediated chromatin modification and heterochromatin formation. Next, NMR spectroscopy is used to investigate protein thermostability in psychrophilic (cold-loving) cytochrome c552. Isolated from the bacterium Colwellia psychrerythraea (Cp), previous work has implicated two conserved Cpcyt c552 methionine residues, which are both conserved across psychrophilic and psychrotolerant cytochromes, as acting in dynamical ligand substitution with a third methionine that is the axial heme ligand. It is proposed that elevated backbone dynamics in these methionine residues and the ability for them swap into the axial ligand position accounts for an uncharacteristically high melting temperature (Tm) compared to meso- and thermophile c-type cytochromes. Progress was made in NMR sample preparation and backbone chemical shift assignment of both redox states of Cpcyt c552, and insight from 1D 1H NMR experiments focused on the heme group bound to Cp cytochrome c552 is discussed. Additionally, chemical shifts are used to predict protein dynamics as a first test of a multiple methionine axial ligand hypothesis. Initial data analysis predicts relatively large measures of Random Coil Index for residues surrounding the native axial heme ligand, and shows the hyperfine shifts localized to the residues surrounding the heme. Future experiments will selectively record methyl group dynamics of methionine residues for elucidation of rate constants of methionine substitution and to determine the structural properties of this minor conformation. Finally, two NMR methodology studies are presented in this thesis: a novel simultaneous-acquisition TROSY pulse sequence for measurement of backbone spin relaxation rates (R1 and {1H}-15N heteronuclear NOE) and a side-chain 2H spin relaxation method for using multifield experimental datasets for better sampling of the spectral density function. Together, these pulse sequences represent significant advancements in NMR measurement of microscopic rate constants and more nuanced detail of protein dynamics

U11-48K-proteiinin toiminta ja säätely U12-tyypin intronien silmukoinnissa

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.Väitöskirjassani olen kuvannut lähetti-RNA:n silmukoimiseen osallistuvan proteiinin U11-48K toimintaa ja säätelyä. Lähetti-RNA:n silmukointi on välttämätön vaihe geenien ilmentymisessä, ja sen häiriöt voivat aiheuttaa sairauksia. Näin ollen silmukoinnin ja sen säätelymekanismien tunteminen ovat tärkeitä sekä yleisesti geenien ilmentymisen ymmärtämiselle että silmukoinnin häiriöistä aiheutuvien tautien diagnosoinnille ja hoidolle. Lisäksi väitöskirjassani kuvatut tulokset antavat viitteitä aitotumallisten eliöiden ja niiden lähetti-RNA:n muokkaukseen osallistuvien prosessien evoluutiosta. Aitotumallisia eliöitä, kuten kasveja ja eläimiä, erottaa tumattomista eliöistä, kuten bakteereista ja arkeista, mm. niiden geenien rakenne. Aitotumallisten eliöiden geenit ovat tyypillisesti jakaantuneet proteiineja koodaaviin jaksoihin eli eksoneihin, joita erottavat toisistaan introneiksi nimitetyt jaksot. Geenien ilmentymisen ensimmäinen vaihe on DNA:n geenien sisältämän informaation kopioiminen lähetti-RNA:ksi, joka toimii mallina proteiinien synteesille. Proteiinisynteesin onnistumiselle on oleellista, että lähetti-RNA:sta poistetaan proteiinia koodaamattomat intronijaksot. Tämä silmukoinniksi nimitetty prosessi on siten välttämätön välivaihe aitotumallisten eliöiden geenien ilmentymiselle. Useimmissa aitotumallisissa eliöissä introneja on kahta toisistaan selvästi eroavaa alatyyppiä, jotka tunnetaan U2- ja U12-tyypin introneina. Tässä tutkimuksessa olen selvittänyt U12-tyypin intronien silmukointia ja sen säätelyä. Tulokseni osoittavat, että proteiini U11-48K on välttämätön U12-tyypin intronien silmukoinnille, ja siten myös U12-tyypin introneja sisältävien geenien ilmentymiselle. Jotta näiden geenien toiminta ei häiriintyisi, on myös U11-48K-proteiinin määrää soluissa säädeltävä tarkasti. Väitöskirjassani osoitankin, että U11-48K:n ilmentymistä säädellään muuttamalla sitä itseään koodaavaan lähetti-RNA:n silmukointia: ylimääräiset U11-48K:ta koodaavat lähetti-RNA:t silmukoidaan poikkeavalla tavalla, mikä johtaa niiden tuhoamiseen ja proteiinisynteesin estymiseen. Sekä U12-tyypin intronit että U11-48K-proteiinin määrää säätelevä mekanismi ovat säilyneet useimmilla hyvin kaukaista sukua olevilla aitotumallisilla eliöillä, kuten eläimilla ja kasveilla. Tämä viittaa siihen, että molemmat prosessit olivat olemassa jo kaikkien aitotumallisten eliöiden kantamuodossa, todennäköisesti noin miljardi vuotta sitten. Näiden mekanismien säilyminen näin pitkiä aikoja osoittaa, että niillä on ja on ollut merkittävä rooli aitotumallisten eliöiden, myös ihmisen, elintoimintojen kannalta. Aiemmissa tutkimuksissa onkin todettu useiden perinnöllisten sairauksien johtuvan silmukointimekanismien häiriöistä. Näin ollen on mahdollista että kuvaamani säätelymekanismin häiriöt saattavat myös olla osallisena joihinkin toistaiseksi määrittämättömiin periytyviin tauteihin

The function of gametocyte specific factor 1 (GTSF1) in mammalian oocyte and ovarian follicle development

A detailed understanding of the genes and mechanisms that regulate oocyte growth and maturation underpins the development of improved methods of assisted conception. Gametocyte specific factor 1 (GTSF1)-a putative marker of gamete developmental competence, is highly conserved across species but in mammals demonstrates a sexual dimorphism in its function. In mice, male mutants for Gtsf1 have an infertile phenotype, whereas female mutants appear to have normal ovarian function. It is hypothesised that GTSF1 regulates oocyte development in monovular species such as the sheep. Initial studies characterised the expression and cellular distribution of GTSF1 across cDNA libraries spanning ovine oogenesis and embryogenesis and by using in situ hybridisation of fixed tissue. GTSF1 expression was confined to gonadal and embryonic tissues with highest expression in the ooplasm of germinal vesicle (GV)-staged secondary oocytes. The gene sequence of GTSF1 was obtained and the gene and predicted protein sequences revealed close homology across many species with two conserved CHHC zinc finger domains known to bind RNA. Functional analysis of the role of GTSF1 during sheep oocyte maturation was conducted using short interference RNA (siRNA injection) in conjunction with oocyte in vitro maturation (IVM) and oocytectomised cumulus shell co-culture. This system was validated using siRNA knockdown (kd) for a known oocyte-specific gene, Growth differentiation factor 9 (GDF9). The effect on GTSF1 kd was evaluated following the microinjection of 770 GV oocytes with siRNA target against the sixth exon of ovine GTSF1. The effects of GTSF1 kd were evaluated in 57 MII oocytes and cumulus shells. Targeted kd of GTSF1 in GV oocytes followed by IVM and cumulus shell co-culture did not affect oocyte meiotic progression or cumulus expansion. Microarray analysis using the bovine GeneChip Affymetrix array revealed that 6 down-regulated genes (TCOF1, RPS8, CACNA1D, SREK1IP1, TIMP1, MYL9) following the GTSF1 kd were associated with developmental competence, RNA storage, post-transcriptional modifications and translation. Immunofluorescent studies localized GTSF1 protein to the P-body in GV ovine oocytes. Collectively these results suggest a possible role of GTSF1 in post-transcriptional control of RNA processing, translational regulation and RNA storage which may impact on oocyte developmental competence