mRNA accumulation in the Cajal bodies of the diplotene larch microsporocyte

Abstract

In microsporocytes of the European larch, we demonstrated the presence of several mRNAs in spherical nuclear bodies. In the nuclei of microsporocytes, we observed up to 12 bodies, ranging from 0.5 to 6 μm in diameter, during the prophase of the first meiotic division. Our previous studies revealed the presence of polyadenylated RNA (poly(A) RNA) in these bodies, but did not confirm the presence of nascent transcripts or splicing factors of the SR family. The lack of these molecules precludes the bodies from being the sites of synthesis and early maturation of primary transcripts (Kołowerzo et al., Protoplasma 236:13-19, 2009). However, the bodies serve as sites for the accumulation of splicing machinery, including the Sm proteins and small nuclear RNAs. Characteristic ultrastructures and the molecular composition of the nuclear bodies, which contain poly(A) RNA, are indicative of Cajal bodies (CBs). Here, we demonstrated the presence of several housekeeping gene transcripts--α-tubulin, pectin methylesterase, peroxidase and catalase, ATPase, and inositol-3-phosphate synthase--in CBs. Additionally, we observed transcripts of the RNA polymerase II subunits RPB2 and RPB10 RNA pol II and the core spliceosome proteins mRNA SmD1, SmD2, and SmE. The co-localization of nascent transcripts and mRNAs indicates that mRNA accumulation/storage, particularly in CBs, occurs in the nucleus of microsporocytes.

Fig. 1

a–k FISH reaction using probes complementary to 11 larch mRNAs reveal high concentrations of the studied mRNAs in the oval, regular nuclear bodies. In the nucleus outside the nuclear bodies and in the cytoplasm, the mRNA is dispersed. l Multiplex FISH reaction using 15 probes complementary to 11 larch mRNAs (see MM, Table 2). Bars, 10 μm

Fig. 2

a–l Double labeling of mRNA of distinct genes: pectin methylesterase (PME) mRNA (a), mRNA of the second subunit of RNA polymerase II (RPB2) (e), mRNA of the core spliceosomal Sm protein D2 (i), and U2 snRNA (b, f, j, respectively). Merge—mRNAs present in CBs (c, g, k). Nuclei were stained with DAPI. All corresponding DAPI images were collected using widefield fluorescence and deconvolution software. Bars, 10 μm. m–o Ultrastructural localization of mRNA. For better visualization of mRNA localization at the ultrastructural level, results of in situ hybridization, in which several probes for mRNA to three genes coding Sm protein (m, o) or a multiplex reaction (n) were shown. m A high level of labeling in the microporocytes was observed primarily in a Cajal body (cb) (arrows, outline). n A lack of mRNA in dense nuclear bodies (db). The gold particles are present in the nucleoplasm and in CBs in single form (arrows). o Studied mRNA was not present in CBs in the somatic cells of tapetum, which surround the microsporocytes; the signal was only observed in the nucleoplasm (arrows, outline), outside dense chromatin (chr), and the nucleolus (Nu). Bars, 0.5 μm

Fig. 3

a–n Double labeling of a newly formed transcript (green, 90-min BrU incubation time) and poly(A) RNA (red) in microsporocytes during the middle diplotene (stages I–VII). Nuclei were stained with DAPI (blue). The DAPI images were collected using widefield fluorescence and deconvolution software. Cajal bodies (cb, arrowheads), nucleus (N), cytoplasm (C). Detection of ribosomal RNA 26S (o) and 5S (p) conducted in the last period highly accumulated these RNAs both in the nucleolus (Nu) and the cytoplasm. Bars, 10 μm