Binding of Xenopus oocyte masking proteins to mRNA sequences.

It has been shown previously that maternal mRNA, synthesized and stored in growing oocytes, is stabilized and blocked from translation through various mechanisms including restricted polyadenylation and the binding of proteins to 3' regulatory elements. In addition to binding sequence-specific proteins, the bulk of stored mRNA is packaged with a set of 'masking' proteins, the most abundant of which are the phosphoproteins pp56 and pp60. In this report these proteins are shown to be bound to heterogeneous mRNA sequences and not to the 3' poly(A) tract. Crosslinking studies demonstrate that all of the pp56/60 present makes direct contact with the RNA. In vitro binding studies confirm that pp56/60 interact with single-stranded RNA of heterogeneous sequence, such as occurring in the maternal mRNA encoding cyclin B1. However, binding is equally effective to capped and polyadenylated cyclin mRNA, to truncated mRNA lacking 5' and 3' non-coding regions and even to the antisense sequence. Lengths of 70-80 nucleotides are protected from ribonuclease digestion after protein binding. Although no extended binding motif could be detected, binding does appear to have some specificity in that it is not competed out by 100-fold excess of double-stranded RNA, transfer RNA, poly(A) and various other homopolymers and heteropolymers. The sequence which competes most efficiently is the mixed polypyrimidine, poly(C,U). Crosslinking of RNA-protein complexes, followed by ribonuclease digestion, suggests that the arrangement of proteins on RNA is as dimers. Dimerization appears to be stabilized by phosphorylation of pp56/60. These results are discussed in terms of the known structures of pp56/60.     

The DAZL family proteins are PABP-binding proteins that regulate translation in germ cells

DAZL proteins are germ-cell-specific RNA-binding proteins essential for gametogenesis. The precise molecular role of these proteins in germ-cell development remains enigmatic; however, they appear to function in the cytoplasm. In order to directly address the function of vertebrate DAZL proteins, we have used Xenopus laevis oocytes as a model system. Here we demonstrate that members of this family, including Xdazl, mouse Dazl, human DAZL, human DAZ and human BOULE, have the ability to stimulate translation and function at the level of translation initiation. We show that DAZL proteins interact with poly(A)-binding proteins (PABPs), which are critical for the initiation of translation. Mapping and tethered function experiments suggest that these interactions are physiologically important. This leads to an attractive hypothesis whereby DAZL proteins activate translationally silent mRNAs during germ cell development through the direct recruitment of PABPs.     

Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.     

Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA

In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasa and nanos 1 (nos1) mRNA. It has been shown that the 3'untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos 1 3'UTR is conserved between teleost species, we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos 1 3'UTR (GFP-nos 1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas ) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos 1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.     

In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.     

Spectrin, fodrin and protein 4.1-like proteins in differentiating rat germ cells

The presence and the distribution of proteins of the membrane skeleton in differentiating germ cells of the rat has been investigated. Immunofluorescence and immunoblotting analysis, performed using monoclonal and polyclonal antibodies to human erythroid alpha-spectrin and protein 4.1 and to brain spectrin (fodrin), demonstrated the presence of analogues of spectrin and fodrin in spermatocytes and round spermatids and of protein 4.1-like molecules in spermatocytes, spermatids and spermatozoa. Spectrin and fodrin showed molecular weights comparable to those of their analogues in erythrocytes but a distinct intracellular distribution. Fodrin was localized along the plasma membrane while spectrin appeared associated with the regions of the Golgi apparatus and of the developing acrosome. Antibodies to protein 4.1 recognized molecules with a molecular weight not comparable with that in erythrocytes, and their presence in spermatozoa was confined to specific regions of the head and of the tail.     

In vivo cross-linking of proteins to mRNA in human cells

Human KB cells were irradiated with ultraviolet light to cross-link mRNA to its associated proteins. More than 75% of both the poly(A)-containing and the poly(A)-lacking mRNAs were cross-linked to proteins after 3 min irradiation. Glycerol gradient analysis showed that no significant RNA chain breakage occurred during this treatment. Cross-linked poly(A)-containing mRNA-protein complexes were purified by oligo(dT)cellulose chromatography in the presence of sodium dodecylsulphate. CsCl gradient analysis revealed that the low salt eluted particles had a buoyant density of about 1.47 g/cm³. To determine which proteins were cross-linked to mRNA, covalent mRNA-protein complexes, labeled in their RNA moiety, were exhaustively treated with nucleases. Polyacrylamide gel analysis showed that most of the residual RNA-radioactivity was covalently bound to proteins of 73000, 69000 and 52000 molecular weight.     

Hydrogen bonds and masking in the sulfhydryl group in proteins

Szent-Györgi has shown that the relative amounts of readily titratable (“free”) SH groups vs. those readily titratable only following denaturation (“masked”) varies significantly from normal to cancerous organ tissues. It is therefore important to inquire into the nature of the two forms of protein-borne SH. Of the four suggested mechanisms for the “masking” of protein SH groups toward hydrophilic reagents, namely: (i) sequestration in hydrophobic regions—whether between chain-folds or between agglomerated protein sub-units, (ii) local steric hindrance, (iii) cyclic hydrogen-bonding to local peptide amino acid residues, or (iv) covalent bonding as in thiazoldines; the first mechanism, that of sequestration in hydrophobic regions appears from present evidence to be the most likely cause. Various spectroscopic, reaction rate and entropy arguments are presented and compared which lead to this conclusion.In addition we have calculated the binding energy of the SH group to various sets of lone pair electrons appropriate to N, O, F, P, S, and Cl atoms in molecules. The calculation was made in the configuration interaction of valence orbitals (CIVO) scheme and gave binding energies and interatomic distances in reasonable agreement with available experimental data.