ISSN 0012�4966, Doklady Biological Sciences, 2012, Vol. 444, pp. 198–201. © Pleiades Publishing, Ltd., 2012.Original Russian Text © D.S. Bogolyubov, F.M. Batalova, A.M. Kiselyov, V.N. Parfenov, 2012, published in Doklady Akademii Nauk, 2012, Vol. 444, No. 6, pp. 691–694.

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In this report, we present the first data on the struc�ture of the extrachromosomal nuclear compartment inthe oocytes of Tribolium castaneum, a novel laboratoryinsect. This object was chosen due to the growinginterest of many specialists to it, because T. castaneumis likely to be brought to the leading edge as an experi�mental model; the genome of this species is fullysequenced and available to the research community [1].

The extrachromosomal part of the cell nucleus, in3�dimensional terms, is a highly�ordered area. It con�tains numerous nuclear domains (nuclear bodies).Among them, Cajal bodies (CBs) and interchromatingranule clusters (IGCs) which are present in many celltypes and suggested as universal [2, 3], are distinguished[2, 3]. In oocytes, a peculiar subnuclear structure, akaryosphere capsule, takes part in compartmentaliza�tion of the extrachromosomal nuclear space. This com�plex structure forms at the defined stages of oogenesis,around the chromosomes united into the karyospherethat occupies a limited area of the nucleus [4]. Inten�sive exploring the main functions of the universal andevolutionarily conserved nuclear domains (CBs andespecially IGCs) during the last time showed a widespectrum of their participation in regulation of differ�ent steps of gene expression, from transcription untilthe mRNA nuclear export [2, 3]. These domains playa significant role in the assembling, modificationsand/or storage of pre�mRNA splicing factors includ�ing small nuclear (sn) RNAs.

SnRNAs together with the certain proteins(snRNPs) are components of the spliceosomes, com�plicated ribonucleoprotein complexes catalyzing pre�

mRNA splicing [5]. Amongst splicing snRNAs, U1,U2, U4, and U5 snRNAs, which are parts of majorspliceosomes, are synthesized by RNA polymerase IIand have a complex post�transcriptional biogenesisthat includes nuclear and cytoplasmic phases [6]. Oneof the essential cytoplasmic steps of the processing ofU1, U2, U4, and U5 snRNPs is hypermethylation ofthe standard 7�monomethyl guanosine triphosphatecap on the 5'�end of U1, U2, U4, and U5 snRNA mol�ecules resulting in formation of the 2,2,7�trimethylguanosine (TMG) cap before these snRNAs return tothe nucleus. In the nucleus, snRNPs undergo the final

Distribution of 5'�Trimethylguanosine Capped Small Nuclear RNAs in Extrachromosomal Oocyte Nuclear Domains of the Laboratory Insect, Tribolium castaneum1

D. S. Bogolyubov, F. M. Batalova, A. M. Kiselyov, and Corresponding Member of the RAS V. N. Parfenov†

Received March 6, 2012

DOI: 10.1134/S0012496612030192

Institute of Cytology, RAS, St. Petersburg, Russia

1 The article was translated by the authors.

† Deceased.

5 µm

K

K

K

K

Fig. 1. A fragment of T. castaneum oocyte nucleus with thekaryosphere and its capsule at the ultrastructural level.Chromatin�containing area (encircled) occupies a limitedarea of the nucleus; karyosphere capsule (K) is well�devel�oped and contains morphologically different extrachro�mosomal elements; arrows indicate extrachromosomalnuclear bodies.

CELL BIOLOGY

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stages of their processing in the CBs and IGCs.Kinetic studies have shown that snRNPs importedinto the nucleus migrate first in CBs and only laterappear in IGCs and on the chromosomes [7]. In theCBs, final formation of snRNP particles occurs [8],certain nucleotides of snRNA molecules are methy�lated and pseudouridinilated [9], and the spliceosomesubunits [U4/U6 · U5] tri�snRNP are formed [10].The basic IGC function is the transient storage ofsplicing factors (snRNPs and SR�proteins) [3]. Coilinprotein is suggested as one of good immunologicalmarkers of the CBs [2], whereas SR�protein SC35 is amarker of IGCs [11]. TMG cap that characterizes themolecules of “matured” snRNAs is apparently a typi�cal additional marker for both the CBs and IGCs.

The data obtained in our study suggest that not onlythe nuclear domains specified above, but also otherextrachromosomal structures involved in the compart�mentalization of the nuclear contents, can take part inthe distribution of TMG�capped snRNAs.

MATERIAL AND METHODS

Female imagoes of the red flour beetle, T. casta�neum (Herbst.) (Coleoptera�Polyphaga: Tenebrion�idae) were used. For immunoelectron microscopy,ovaries were fixed in a solution containing 4% formal�dehyde, freshly prepared from paraformaldehyde, and0.5% glutaraldehyde in 1 × PBS for 2 h, than in 2%formaldehyde in PBS overnight at 4°С and embeddedin LR White resin. For immunofluorescent (confocal)microscopy, squashed preparations of isolated oocytenuclei were prepared, frozen in liquid nitrogen, thanfixed in 2% formaldehyde in 96% ethanol for 30 min,rinsed in 70% ethanol and PBS. Monoclonal antibod�ies anti�SC35, K121 against the TMG cap of snRNAs,a polyclonal rabbit serum against Drosophila coilin(kindly donated by J.G. Gall, Carnegie Institution ofWashington) were used as primary antibodies. Goat

anti�mouse or anti�rabbit antibodies conjugated with10 or 15 nm colloidal gold particles (for immunoelec�tron microscopy) or with FITC (for immunofluores�cent microscopy) were used as secondary antibodies; inthe latter case, the preparations were additionally coun�terstained with 1 µg/ml To�Pro�3 dye to reveal DNA.

RESULTS AND DISCUSSION

Our ultrastructural and immunocytochemicalstudy carried out on the oocyte nuclei in the laboratoryinsect, Tribolium castaneum, showed that the nucleusof growing oocyte contains the karyosphere (aka kary�osome) that is formed by the chromosomes located ina limited area of the nucleus and is separated from therest of the nucleoplasm by an extrachromosomal

0.2 µm 0.2 µm

SC35Coilin

Fig. 2. Ultrastructural identification of the Cajal body with anti�coilin antibody (on the left) and the counterpart of interchroma�tin granule clusters with anti�SC35 antibody (on the right).

0.5 µm 0.5 µm

TMG TMG

Fig. 3. The Cajal body (on the left) and the counterpart ofinterchromatin granule clusters (on the right) as viewed af�ter immunogold labeling of the ultrathin section of oocytenucleus with anti�TMG antibody. Only Cajal body is la�beled.

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BOGOLYUBOV et al.

material, the capsule of the karyosphere. Besides,numerous extrachromosomal nuclear bodies (domains)are seen both joined with the capsule and scatteredfreely in the nucleoplasm (Fig. 1).

At the ultrastructural level, T. castaneum oocytenuclear bodies are complex fibrillar structures; differ�ent parts of the bodies can be distinguished by theirelectron density and the level of the packing of thefibrils. Using antibodies against coilin and SC35 pro�teins (the markers of CBs and IGCs, respectively), weidentified CB and IGC counterparts among thesebodies (Fig. 2). At the same time, T. castaneum oocytenuclear bodies that contain the marker SC35, canoni�cal for typical IGCs, differ from typical IGCs by theirorganization: these bodies have a fibrillar rather thanfibrogranular structure and may be referred to as IGCanalogs only. SC35 domains display a similar structurein the oocytes of several other insects [13, 14].

As it was supposed, TMG�capped snRNAs wereclearly revealed immunocytochemically in the CBs ofT. castaneum oocytes. At the same time, the absence ofprominent accumulation of these RNAs in SC35domains (IGC counterparts) was a surprise for us.Instead, an antibody against the TMG cap labeledspecifically an extrachromosomal material in the kary�osphere capsule (Fig. 4), that it was also confirmed atthe ultrastructural level (data not shown). We knowonly one study [15] carried out on the weevil oocytes,that has documented localization of snRNPs, includ�ing TMG�capped snRNAs, in some elements of thekaryosphere capsule rather than in nuclear bodies.

Our study is the first that provides the data on thestructure and molecular composition of the “univer�sal” nuclear domains (CBs and IGCs) in the oocytesof a novel model object, T. castaneum. It shows thatnow has ripened the necessity of expansion of the cir�cle of model objects on which the problems of struc�

tural and functional architectonics of the cell nucleusare investigated to establish the fundamental princi�ples of the structure, molecular composition and func�tions of extrachromosomal nuclear domains. Besides,modern exploring the karyosphere and its extrachro�mosomal capsule which are the unique structures ofgerm cell (mainly oocytes) in many animals becomesrelevant. It is likely that a role of the karyosphere cap�sule is not restricted by its function as a structural ele�ment of the nuclear matrix that confines the chroma�tin from the rest of the nucleoplasm, mechanicallysupporting the chromosomes in a limited area of largeoocyte nucleus (germinal vesicle) during a long diplo�tene stage of meiotic prophase [4]. Our data on theunusual distribution of TMG�capped snRNAs in thenucleus allow assuming that the certain elements ofthe karyosphere capsule may take part in the integra�tion of different nuclear processes including thoserelated to snRNA biogenesis.

This work was supported by RFBR (grant no. 11�04�01258) and the granting program of RAS “Molec�ular and cell biology.”

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10 µm

TMG�cap DNA

ks ks

Fig. 4. Localization of TMG�capped snRNAs in the karyosphere capsule in T. castaneum oocyte nucleus (on the left); on theright, an image of the same nuclear fragment after revealing of DNA with fluorescent dye To�Pro�3; ks, karyosphere; nuclear con�tour is outlined. Confocal microscopy.

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DISTRIBUTION OF 5'�TRIMETHYLGUANOSINE 201

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