Ludmila V. Olenina

Lysine methylation of nonhistone proteins is a way to regulate their stability and function.

This review is devoted to the dramatically expanding investigations of lysine methylation on nonhistone proteins and its functional importance. Posttranslational covalent modifications of proteins provide living organisms with ability to rapidly change protein activity and function in response to various stimuli. Enzymatic protein methylation at different lysine residues was evaluated in histones as a part of the “histone code”. Histone methyltransferases methylate not only histones, but also many nuclear and cytoplasmic proteins. Recent data show that the regulatory role of lysine methylation on proteins is not restricted to the “histone code”. This modification modulates activation, stabilization, and degradation of nonhistone proteins, thus influencing numerous cell processes. In this review we particularly focused on methylation of transcription factors and other nuclear nonhistone proteins. The methylated lysine residues serve as markers attracting nuclear “reader” proteins that possess different chromatin-modifying activities.

Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster

Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation.

A novel organelle, the piNG-body, in the nuage of Drosophila male germ cells is associated with piRNA-mediated gene silencing

A novel perinuclear nuage organelle, the piNG-body, is associated with piRNA silencing in testes of Drosophila. This body contains the known ovarian nuage proteins Vasa, Aub, AGO3, Tud, Spn-E, Bel, Squ, and Cuff, as well as AGO1.

Telomeric repeat silencing in germ cells is essential for early development in Drosophila

The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. We analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. Our data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

Multicolor fluorescence imaging of whole-mount Drosophila testes for studying spermatogenesis

Drosophila testes are generally considered a useful model for studying the fundamental developmental processes of heterogametic organisms. However, immunostaining of the whole Drosophila testis is often associated with insufficient resolution at the subcellular level, poor reproducibility, and incomplete staining of fixed preparations. The main problem for adequate staining is poor permeability of the organs for antibodies and antibody-coupled fluorophores. To overcome this problem we developed a protocol for whole-mount testis immunostaining yielding high-quality preparations for confocal microscopy. Many subcellular structures can be successfully resolved, such as the spectrosome, fusome, nuage granules, apoptotic bodies, and protein crystals. This method preserves the inner architecture of the testes, enabling 3D image reconstruction from a set of confocal sections. It allows one to combine the simultaneous detection of fluorescently tagged and immunostained proteins as well as TUNEL analysis for apoptosis detection.

Genetically Derepressed Nucleoplasmic Stellate Protein in Spermatocytes of D. melanogaster Interacts with the Catalytic Subunit of Protein Kinase 2 and Carries Histone-Like Lysine-Methylated Mark

SUMMARY The X-chromosome-linked clusters of the tandemly repeated testis-specific Stellate genes of Drosophila melanogaster, encoding proteins homologous to the regulatory beta-subunit of the protein kinase casein kinase 2 (CK2), are repressed in wild-type males. Derepression of Stellate genes in the absence of the Y chromosome or Y-linked crystal locus (crystal line) causes accumulation of abundant protein crystals in testes and different meiotic abnormalities, which lead to partial or complete male sterility. To understand the cause of abnormalities in chromosome behavior owing to Stellate overexpression, we studied subcellular localization of Stellate proteins by biochemical fractionation and immunostaining of whole testes. We showed that, apart from the known accumulation of Stellate in crystalline form, soluble Stellate was located exclusively in the nucleoplasm, whereas Stellate crystals were located mainly in the cytoplasm. Coimmunoprecipitation experiments revealed that the alpha-subunit of the protein kinase CK2 (CK2alpha) was associated with soluble Stellate. Interaction between soluble Stellate and CK2alpha in the nucleus could lead to modulations in the phosphorylation of nuclear targets of CK2 and abnormalities in the meiotic segregation of chromosomes. We also observed that Stellate underwent lysine methylation and mimicked trimethyl-H3K9 epigenetic modification of histone H3 tail.

Germ granules in spermatogenesis of Drosophila: Evidences of contribution to the piRNA silencing

Ribonucleoprotein-containing granules in the cytoplasm of germinal cells are known to be a common attribute of eukaryotic organisms. Germ granules appear to ensure the posttranscriptional regulation of germline mRNAs. Recent studies specify the participation of the germ granules in genome integrity maintenance by mechanisms involving short piRNAs. PIWI clade proteins and associated piRNAs are considered as key participants of the germline-specific piRNA pathway. Proteins of the PIWI clade, Aub and AGO3, concentrated in the germline-specific perinuclear granules called nuage, are involved in silencing of retrotransposons and other selfish repetitive elements in the Drosophila genome. In Drosophila testes, two types of perinuclear nuage granules are found: a large amount of small particles around the nuclei and significantly larger structures, the piNG-bodies. In this mini-review, we analyze the recent published data about structure and functions of Drosophila male germ granules, and especially their involvement in the piRNA silencing pathway.

DEAD-Box RNA helicases in animal gametogenesis

The review summarizes a current knowledge about a role of RNA helicases in the development and maintenance of gamenogenesis in eukaryotes. We focused on three RNA helicase family members—Vasa/DDX4, Belle/DDX3, and Spindle-E/TDRD9—that contain characteristic amino acid sequence motifs (DEAD box) and perform substantial conserved functions in the germinal tissues of various species from Drosophila to human. These enzymes are involved in a broad range of activities associated with the regulation of transcription, splicing, nuclear export and, especially, with translation initiation. Expression of genes required for gametogenesis is regulated mainly at the transcriptional level. RNA helicases are involved in the formation of cytoplasmic ribonucleoprotein (RNP) granules and RNA silencing. A highly conserved central domain is characteristic of DEAD-box RNA helicases and determines their basic biological activity in ATP-dependent unwinding of short RNA duplexes.

RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila

The present study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. We found that deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. We found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins.

RNA helicase Spn-E is required to maintain Aub and AGO3 protein levels for piRNA silencing in the germline of Drosophila

Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.