Non-canonical Cajal bodies form in the nucleus of late stage avian oocytes lacking functional nucleolus

Abstract

In the somatic cell nucleus, there are several universal domains such as nucleolus, SC35-domains, Cajal bodies (CBs) and histone locus bodies (HLBs). Among them, CBs were described more than 100 years ago; however, we still do not have a final understanding of their nature and biological significance. The giant nucleus of avian and amphibian growing oocytes represents an advantageous model for analysis of functions and biogenesis of various nuclear domains. Nevertheless, in large-sized avian oocytes that contain transcriptionally active lampbrush chromosomes, CB-like organelles have not been identified yet. Here we demonstrate that in the pigeon (Columba livia) oocyte nucleus, characterized by absence of any functional nucleoli, extrachromosomal spherical bodies contain TMG-capped spliceosomal snRNAs, core proteins of Sm snRNPs and the protein coilin typical for CBs, but not splicing factor SC35 nor the histone pre-mRNA 3'-end processing factor symplekin. The results establish that coilin-rich nuclear organelles in pigeon late-stage oocyte are not the equivalents of HLBs but belong to a group of CBs. At the same time, they do not contain the snoRNP/scaRNP protein fibrillarin involved in 2'-O-methylation of snoRNAs and snRNAs. Thus, the nucleus of late-stage pigeon oocytes houses CB-like organelles that have an unusual molecular composition and are implicated in the snRNP biogenesis pathway. These data demonstrate that snRNP-rich non-canonical CBs can form in the absence of nucleolus. We argue that pigeon oocytes represent a new promising model to investigate CB modular organization, functions and formation mechanism.

Three-dimensional organisation of RNA-processing machinery in avian growing oocyte nucleus

“…2b). A detailed description of these remarkable (Khodyuchenko et al 2012). Confocal images also clearly demonstrate that in avian GVs, the previously described centromereassociated protein bodies (Gaginskaya and Gruzova 1969;Khutinaeva et al 1989;Saifitdinova et al 2003) do not contain RNA or DNA and appear as 'black holes' in nuclei stained for nucleic acids ( Fig.…”

“…After a series of washes, the oocytes were fixed in 2 % PFA (Serva), permeabilized in 0.2-0.5 % Triton X-100 (ICN Biomedicals), blocked with 1 % blocking reagent (Roche) and incubated with primary and secondary Abs as described previously (Khodyuchenko et al 2012). Oocytes were mounted in antifade DAВCO mounting medium (Мerck), containing 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ ml; Sigma).…”

“…Immunofluorescent staining of fixed nuclei was performed as described previously (Khodyuchenko et al 2012). Nuclei were incubated for 1 h with primary Abs diluted in PBS, washed with PBS for 10 min and incubated with the appropriate secondary Abs diluted in PBS for 1 h. Commercial blocking solutions were not applicable for GV incubation due to the substantial nuclear shrinkage and loss of 3D morphology.…”

“…Likewise, an avian oocyte nucleus could also be employed as a model to explore the three-dimensional (3D) organisation of chromosomes and other nuclear structures (Maslova and Krasikova 2011;Khodyuchenko et al 2012;Maslova and Krasikova 2012). The oocyte nucleus was first described by Jan Evangelista Purkyně who examined transparent structure inside chicken oocytes in the early 1830s and named this organelle 'vesicula germinativa', or the germinal vesicle (GV) (De La Fuente 2006).…”

The Cajal body and the nucleolus: “In a relationship” or “It's complicated”?

“…A study using prolonged nocodazole treatment to induce micronucleation of rat-kangaroo kidney cells reported a number of micronuclei containing CB-like structures but not nucleoli. 125 In pigeon oocytes, which lack functional nucleoli, CB-like structures, containing coilin and snRNPs but lacking fibrillarin, have been detected, 126 suggesting that CBs may, on occasion, be independent from nucleoli.…”