Irina A. Stepanova

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well‐studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.

Modification of nucleic acids using [3 + 2]-dipolar cycloaddition of azides and alkynes

The use of azide and alkyne cycloaddition reaction in the synthesis of conjugates of nucleic acids and oligodeoxyribonucleotides is reviewed. Data on the chemical and enzymatic methods for introducing azides and alkynes into DNA are summarized.

Characterization of Tribolium castaneum oocyte nuclear structures using microinjection of a fusion nuclear protein mRNA

Nuclear architecture comprises a number of extrachromosomal structures, nuclear compartments, or domains. We use the oocytes of the red flour beetle, Tribolium castaneum, as an experimental model. This species was introduced as a novel laboratory insect to complement studies performed in Drosophila for genetic (the T. castaneum genome is fully sequenced and aligned), developmental, andcell-biology questions.Many researchers consider T. castaneum ‘‘less derived’’ and ‘‘more ancestral’’ than fruit flies. In many organisms, oocyte chromosomes join together in a limited volume of the oocyte nucleus, forming a karyosphere (karyosome) at the diplotene stage of meiotic prophase (GruzovaandParfenov, 1993). Chromosomes in the karyosphere may be further separated from the nucleoplasm by a complex, extrachromosomal karyosphere capsule (KC), whose biological significance is still unclear. We employed a gene-engineering approach to obtain a dynamic picture of the distribution of a newly synthesized protein in the T. castaneum oocyte nucleus. We microinjected an in vitro-synthesized, 5’-capped mRNA containing the coding sequence of the exon junction complex protein Y14, fused to a myc tag (Fig. S1, D) into oocytes. This approach allowed us to trace the nuclear dynamics of newly synthesized protein. This RNA was successfully translated within 3 h (Goodman et al., 2012) of microinjection, and the Y14 fusion protein is imported into the oocyte nucleus. A significant level of fluorescence was registered in the perichromatin region of the karyosphere,where loose DNA loops are located. Y14 was differentially localized at each oocyte stage. In the previtellogenesis stage (Fig. 1A), when the KC begins to form, Y14 resides in SC35 domains. By the vitellogenic stage, however, Y14 is completely redistributed to the well-formed KC rather than SC35 domains (Fig. 1B). Based on these dynamics, it could be supposed that, like in somatic cells, oocyte SC35-containing nuclear bodies represent transient domains where some proteins involved in mRNA transport and export can reside. Somatic extrachromosomal nuclear domains contain resident, slow-mobile proteins, including the proteins of the nuclear matrix. In contrast, pre-mRNAsplicing factorsarehighly dynamicand, despite their apparent residence in nuclear speckles (5 10 times higher concentration than elsewhere in the nucleus), donotdwell in thesespeckles longer than inotherpartsof the nucleoplasm. It is therefore possible that oocyte nuclear bodies contain other proteins that will contribute to theKCat the end of oocyte growth, of which the Y14-myc fusion protein is the first to be confirmed. Thus, the KC may be a site of residual transcription within the vitellogenic oocyte. Chromatin rearrangement, which is a characteristic feature of oocyte growth, is therefore clearly accompanied by a redistribution of nuclear proteins between different oocyte nuclear structures. This change in localization could affect how nuclear maturation proceeds.

Simple reagent for the synthesis of oligonucleotides labeled with 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine

Synthesis of a phosphoramidite reagent for the preparation of oligonucleotides labeled at the 5′-end with a fluorescent dye, 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine, is described. The efficiency of this reagent is confirmed by the synthesis of several labeled oligonucleotides.

Non-nucleoside phosphoramidites of xanthene dyes (FAM, JOE, and TAMRA) for oligonucleotide labeling.

This unit describes the preparation of 5‐ and 6‐carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4′,5′‐dichloro‐2′,7′‐dimethoxy‐fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non‐nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy‐FAM and carboxy‐JOE as pentafluorophenyl esters. Acylation of 3‐dimethylaminophenol with 1,2,4‐benzenetricarboxylic anhydride gave a mixture of 4‐dimethylamino‐2‐hydroxy‐2′,4′(5′)‐dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5‐ or 6‐carboxytetramethylrhodamines. The dyes were converted into 6‐aminohexanol‐ (JOE), 4‐trans‐aminocyclohexanol‐ (FAM and JOE), and hydroxyprolinol‐based (TAMRA) phosphoramidite reagents. Curr. Protoc. Nucleic Acid Chem. 52:4.55.1‐4.55.33.

Phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for the synthesis of modified oligonucleotides

The synthesis of phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for the introduction of the residues of biotin, lipoic acid, amino groups, and terminal acetylene groups at different positions of the oligonucleotide chain has been described. The efficiency of the reagents and supports has been confirmed by the synthesis of the corresponding modified oligonucleotides.