Human nucleolar protein SURF6/RRP14 participates in early steps of pre-rRNA processing.

The biogenesis of ribosomes requires tightly controlled transcription and processing of pre-rRNA which comprises ribosomal RNAs forming the core of large and small ribosomal subunits. Early steps of the pre-rRNA processing and assembly of the ribosomal subunits require a large set of proteins that perform folding and nucleolytic cleavage of pre-rRNAs in the nucleoli. Structure and functions of proteins involved in the pre-rRNA processing have been extensively studied in the budding yeast S. cerevisiae. Functional characterization of their human homologues is complicated by the complexity of mammalian ribosomes and increased number of protein factors involved in the ribosomal biogenesis. Homologues of human nucleolar protein SURF6 from yeast and mouse, Rrp14 and Surf6, respectively, had been shown to be involved in the early steps of pre-rRNA processing. Rrp14 works as RNA chaperone in complex with proteins Ssf1 and Rrp15. Human SURF6 knockdown and overexpression were used to clarify a role of SURF6 in the early steps of pre-rRNA processing in human cell lines HeLa and HTC116. By analyzing the abundance of the rRNA precursors in cells with decreased level or overexpression of SURF6, we demonstrated that human SURF6 is involved in the maturation of rRNAs from both small and large ribosomal subunits. Changes in the SURF6 level caused by knockdown or overexpression of the protein do not result in the death of HeLa cells in contrast to murine embryonic fibroblasts, but significantly alter the distribution of cells among the phases of the cell cycle. SURF6 knockdown in both p53 sufficient and p53 deficient HCT116 human cancer cells results in elongation of G0/G1 and shortening of G2/M phase. This surprising result suggests p53 independence of SURF6 effects on the cell cycle and possible multiple functions of SURF6. Our data point to the shift from pathway 1 to pathway 2 of the rRNA biogenesis caused by the SURF6 knockdown and its likely association with p53 pathway.

Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development.

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction.

Nuclear Ccr4-Not mediates the degradation of telomeric and transposon transcripts at chromatin in the Drosophila germline.

Ccr4-Not is a highly conserved complex involved in cotranscriptional RNA surveillance pathways in yeast. In Drosophila, Ccr4-Not is linked to the translational repression of miRNA targets and the posttranscriptional control of maternal mRNAs during oogenesis and embryonic development. Here, we describe a new role for the Ccr4-Not complex in nuclear RNA metabolism in the Drosophila germline. Ccr4 depletion results in the accumulation of transposable and telomeric repeat transcripts in the fraction of chromatin-associated RNA; however, it does not affect small RNA levels or the heterochromatin state of the target loci. Nuclear targets of Ccr4 mainly comprise active full-length transposable elements (TEs) and telomeric and subtelomeric repeats. Moreover, Ccr4-Not foci localize at telomeres in a Piwi-dependent manner, suggesting a functional relationship between these pathways. Indeed, we detected interactions between the components of the Ccr4-Not complex and piRNA machinery, which indicates that these pathways cooperate in the nucleus to recognize and degrade TE transcripts at transcription sites. These data reveal a new layer of transposon control in the germline, which is critical for the maintenance of genome integrity.

Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis.

The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.

Transposon control mechanisms in telomere biology.

The ends of linear eukaryotic chromosomes, telomeres, are elongated by reverse transcriptase activity provided by the enzyme telomerase, or by specialized telomeric retrotransposons. Telomerase and telomeric retrotransposons represent unique examples of structurally different, but evolutionary and functionally related machineries that generate essential chromosome structures, namely telomeres. In fact, the telomere is an example of the taming of retroelements for the maintenance of essential genome function. Many features of telomere homeostasis are conserved between telomerase and retrotransposon maintained telomeres. The retrotransposon origin of telomeres suggests that mechanisms of transposon control could be adopted for telomere regulation. The discovery of the role of Drosophila telomeric piRNAs in telomere length control and the influence of LINE-1 retroelements on telomere regulation in human cells strongly support this idea and allow us to look at telomere regulation from a new angle.

Natural variation of piRNA expression affects immunity to transposable elements.

In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain.

An acid precipitation technique: A strip assay for the large-scale DNA polymerase activity screening.

A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.

Development of novel mouse hybridomas producing monoclonal antibodies specific to human and mouse nucleolar protein SURF-6.

SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells.