Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants

Abstract

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B 00 and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with a-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.

Tissue-specific Expression and Dynamic Organization of SR Splicing Factors inArabidopsis

“…Whereas speckles exhibited limited overall movements within a constrained volume, their protein components were shown to rapidly exchange with the nucleoplasmic population. Similar to previous studies on SR45 (Ali et al, 2003) and atRSp31 (Docquier et al, 2004), a subset of speckles, at any given time, was observed to merge, bud, disassemble, or assemble at new sites. However, after blocking transcription, we found that almost all of the above-mentioned dynamic events ceased, indicating that these dynamic events are also related to transcriptional activities in the plant nuclei.…”

“…Nuclear compartmentalization is thought to facilitate the regulation of transcription and pre-mRNA splicing to generate mature transcripts (Lamond and Earnshaw, 1998). However, the presence of IGCs in plant nuclei has not been previously identified (Testillano et al, 1993;Acevedo et al, 2002;Cui and Moreno Díaz de la Espina, 2003;Docquier et al, 2004). Our observations demonstrated that two SF2/ASF and one SC35-like splicing factor localize to speckles that correspond to IGCs in plant nuclei upon TEM analysis.…”

“…In plants, spliceosomal components were found to localize in Cajal bodies (Beven et al, 1995;Boudonck et al, 1998Boudonck et al, , 1999Acevedo et al, 2002;Cui and Moreno Díaz de la Espina, 2003;Docquier et al, 2004) and in nuclear speckles (Lopato et al, 2002;Ali et al, 2003;Docquier et al, 2004). However, only the plant-specific SR proteins were studied in living cells from a limited number of cell types, and the expression of green fluorescent protein-tagged SR proteins were under the control of a highly active constitutive 35S promoter (Lopato et al, 2002;Ali et al, 2003;Docquier et al, 2004).…”

“…However, only the plant-specific SR proteins were studied in living cells from a limited number of cell types, and the expression of green fluorescent protein-tagged SR proteins were under the control of a highly active constitutive 35S promoter (Lopato et al, 2002;Ali et al, 2003;Docquier et al, 2004). To gain more insight into the expression and dynamic organization of SR splicing factors in a single organism, we visualized the expression and dynamic organization of SF2/ASF and SC35-like splicing factors under the control of their endogenous promoters in Arabidopsis by deconvolution and confocal microscopy.…”

“…Furthermore, members of a plant-specific family of SR proteins termed the atRSp31 family (atRSp31, atRSp40, and atRSp41) were isolated (Lopato et al, 1996). More recently, a plant-specific SR protein atRSZ33 was shown to interact with SR1/atSRp34, atRSZp21, atSRZp22 and SC35-like splicing factors (Lopato et al, 2002).In plants, spliceosomal components were found to localize in Cajal bodies (Beven et al, 1995;Boudonck et al, 1998Boudonck et al, , 1999Acevedo et al, 2002;Cui and Moreno Díaz de la Espina, 2003;Docquier et al, 2004) and in nuclear speckles (Lopato et al, 2002;Ali et al, 2003;Docquier et al, 2004). However, only the plant-specific SR proteins were studied in living cells from a limited number of cell types, and the expression of Article published online ahead of print.…”