Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes
Introduction
In most animals, germ cells contain a presumptive determinant of germ cell fate called the germ (or pole) plasm. One of the constant components of the germ plasm is germinal (or polar or P) granules, which invariably originate from the perinuclear electron-dense material termed vaguely the “nuage” (for review, see [1], [2], [3]). On the basis of ultrastructural observations of germ cells in various animal species, it is commonly believed that the nuage originates in the nucleus and thus is the source of the ribonucleoproteins (RNPs) necessary for germinal granule assembly [1], [4], [5], [6], [7]. Until recently, the molecular composition of the nuage and its function remained a complete mystery. Recent studies showed that Vasa, a DEAD-box RNA helicase possibly involved in translational control of other elusive nuage RNAs and possibly indirectly involved in the microRNA pathway, is a consistent component of nuage in animals ranging from nematodes to mammals (reviewed by Raz [8]). Surprisingly, it was recently found that the nuage in germ cells of Caenorhabditis elegans and mouse spermatocytes contains splicing complex components such as Sm proteins and/or small nuclear ribonucleoproteins (snRNPs), which are necessary for the integrity and proper assembly of germinal granules and germ cell function [9], [10]. The presence of these components in nuage suggests that highly conserved splicing factors may have been adapted to a role independent of pre-mRNA splicing and that the nuage represents a juncture for the proteins shuttling between nucleus and cytoplasm and for the assembly of cytoplasmic RNPs necessary for the correct localization of various mRNAs to the germinal granules [9], [10], [11].
The biogenesis of spliceosomal snRNPs is a complex, stepwise process. Small nuclear RNA (snRNA) molecules are transcribed in the nucleus and exported through the nuclear pores into the cytoplasm. Within the cytoplasm, each snRNA molecule associates with a set of seven Sm proteins, also called the core proteins. This process is mediated by another group of proteins, the SMN complex, which consists of SMN protein and Gemins. Together, the snRNAs, Sm proteins, and SMN complex form snRNPs. Subsequently, the 5′-monomethylguanosine cap of the snRNAs is hypermethylated and transformed into a 2,2,7-trimethylguanosine (TMG) cap. Following this modification, the snRNPs are imported back into the nucleus and targeted to Cajal bodies (specialized spherical nuclear domains usually associated with the periphery of the nucleolus) where the SMN complex dissociates and the snRNPs undergo further maturation. Finally, the modified snRNPs are transferred into speckles or other nuclear destinations where as subunits of spliceosomes, they are involved in pre-mRNA splicing [12], [13], [14].
Cajal bodies contain a large number of invariable components such as their signature protein coilin, small Cajal body-specific RNAs (scaRNAs), snRNPs, SMN protein, polymerases, general transcription factors, fibrillarin, and Nopp 140. In addition, some macromolecules (e.g., small nucleolar ribonucleoproteins, snoRNPs) move transiently through this organelle (reviewed in [15], [16], [17], [18]). Among the functions ascribed to Cajal bodies are posttranscriptional modifications of snRNAs and certain snoRNAs, maturation of the 3′ end of histone mRNAs, and formation of transcriptosomes (large RNP complexes involved in different aspects of RNA transcription and processing) [15], [19]. Cajal bodies present in the germinal vesicles (oocyte nuclei) of Xenopus and some insects are exceptionally large and often numerous [15], [20], [21]. It has been hypothesized that in female gametes, the snRNPs present in Cajal bodies not only process nascent transcripts but also are a source of processing factors for the future embryo [21], [22], [23]. All this information prompted us to investigate whether the molecular components of nuage are evolutionarily conserved and whether similar to the case in C. elegans, Drosophila and mouse, the nuage and/or mitochondrial cement present in Xenopus oocytes contains the components of Cajal body or splicing machinery, such as snRNAs and associated proteins (Sm, SMN, coilin) and the DEAD-box RNA helicases.
The abundance of nuage and mitochondrial cement in female germ cells of Xenopus makes them particularly well suited for the study of the ultrastructure and molecular composition of these structures. In early oogonia of Xenopus, the nuage is visible within the nuclear pore complexes and in large quantities at the cytoplasmic face of the nuclear envelope (reviewed by Kloc et al. [2], [24]). In late oogonia and early stage I oocytes, the nuage becomes intimately associated with mitochondria and, at this stage is called the mitochondrial or intermitochondrial cement. In Xenopus, the mitochondrial cement is believed to be a direct precursor of the granulo-fibrillar material (GFM) and eventually of the mature germinal granules that reside within the METRO region of the Balbiani body also called the mitochondrial cloud [2], [5], [24], [25], [26]. Although the permanent or transient presence of several mRNAs (e.g., Xcat2, Xpat, DEADSouth, Xdazl, and XFACS [C10]) has been described in GFM and mature germinal granules in Xenopus oocytes and embryos [26], the composition of nuage and mitochondrial cement remains completely unknown. Using the combination of electron microscopy in situ hybridization and immunostaining, we studied the molecular composition of nuage and mitochondrial cement in pre-stage I and stage I Xenopus oocytes and recapitulated pathway leading from the nuage to the formation of germinal granules.
Section snippets
Standard electron microscopy
Xenopus laevis oocytes were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h. After being washed, the material was postfixed for 1 h in 1% OsO4 in the same buffer, dehydrated with a graded series of ethanol and acetone and embedded in Epon 812 (Fullam, Latham, NY). Ultrathin sections were cut on an Ultracut E microtome (Reichert-Jung). After contrasting the sections with uranyl acetate and lead citrate, we examined them with a JEOL 100SX transmission electron microscope
Origin of nuage material and formation of mitochondrial cement and germinal granules
The detailed analysis of early Xenopus oogenesis, the development of the Balbiani body (mitochondrial cloud), and oocyte polarity have been described in Kloc et al. [27]. Pre-stage I oocytes in Xenopus already have very well-defined animal–vegetal polarity, with the germinal vesicle situated at the future animal pole and the developing Balbiani body facing future vegetal pole [25], [26]. We found that the germinal vesicle of pre-stage I and stage I Xenopus oocytes had a slightly undulated
Discussion
Xenopus oocytes contain germ cell determinant, the germ plasm in a conspicuous cytoplasmic organelle called the Balbiani body (or mitochondrial cloud). The germ plasm is situated in the METRO region at the vegetal apex of the Balbiani body and contains two major components: germinal granules and mitochondria. We previously showed that various coding and noncoding RNAs are present within the germ plasm and/or germinal granules in Xenopus oocytes and embryos [26]. Ultrastructural and molecular
Acknowledgements
This work was supported in part by NSF grant to L.D.E. and core grant CA16672. We thank Prof. E. Pyza (Laboratory of Electron Microscopy, Jagiellonian University, Krakow) for use of the EM facilities and W. Jankowska, E. Kisiel and Kenneth Dunner, Jr., for their excellent technical assistance.
References (42)
- et al.
The Balbiani body and germ cell determinants-150 years later
Curr. Top. Dev. Biol.
(2004) - et al.
Mitochondrial ribosomal RNA in the germinal granules in Xenopus embryos–revisited
Differentiation
(2001) Assembly of the Drosophila germ plasm
Int. Rev. Cytol.
(2001)- et al.
P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA
Dev. Biol.
(2000) - et al.
A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis
Curr. Biol.
(2002) - et al.
Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs
Mech. Dev.
(2003) - et al.
Transport of proteins and RNAs in and out of the nucleus
Cell
(1999) - et al.
Spliceosomal UsnRNP biogenesis, structure and function
Curr. Opin. Cell Biol.
(2001) A role for Cajal bodies in assembly of the nuclear transcription machinery
FEBS Lett.
(2001)Nuclear bodies in the oocyte nucleus of ground beetles are enriched in snRNPs
Tissue Cell
(2001)
Nuclear bodies of stage 6 oocytes of Rana temporaria contain nucleolar and coiled body proteins
Exp. Cell Res.
RNA localization and germ cell determination in Xenopus
Int. Rev. Cytol.
Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus
Dev. Biol.
Formation, architecture and polarity of female germline cyst in Xenopus
Dev. Biol.
The mitochondrial cloud of Xenopus oocytes: the source of germinal granule material
Dev. Biol.
The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies
J. Biol. Chem.
DEADSouth is a germ plasm specific DEAD-box RNA helicase in Xenopus related to eIF4A
Mech. Dev.
VASA mediates translation through interaction with a Drosophila yIF2 homolog
Mol. Cell
DexD/H box RNA helicases: from generic motors to specific dissociation functions
Mol. Cell.
Germ plasm and the differentiation of the germ cell line
Int. Rev. Cytol.
Germline development in vertebrates and invertebrates
Cell. Mol. Life Sci.
Cited by (51)
Morphology and ultrastructure of the Balbiani body in the oocytes of closely related bush cricket species. Shared features reveal important aspect of functioning
2022, ZoologyCitation Excerpt :It is of note to add here that similar (50 nm in diameter) granules have been found to take part in the formation of nuage in the oocytes of a model organism, Xenopus laevis (Bilinski et al., 2004).
Convergent evolution of germ granule nucleators: A hypothesis
2017, Stem Cell ResearchKnock-out mutations of Arabidopsis SmD3-b induce pleotropic phenotypes through altered transcript splicing
2011, Plant ScienceCitation Excerpt :These characteristics resulted in the smd3-b null mutants completely inhibiting the splicing of some genes, partially inhibiting other genes, and not inhibiting the rest of the genes. The Sm/Lsm proteins of UsnRNP have diverse functions in animal systems besides their splicing functions, which are replicating chromosome telomeres, degrading mRNA, processing rRNA and tRNA, controlling germ granule integrity, and regulating germ cell fate in embryogenesis [43–52]. These diverse functions indicate the additional functions of the SmD3 proteins in plant development and differentiation.
Germ plasm dynamics during oogenesis and early embryonic development in Xenopus and zebrafish
2023, Molecular Reproduction and DevelopmentBalbiani body formation and cytoplasmic zonation during early oocyte development in two Clupeiform fishes
2022, Journal of Fish Biology