Sm and U2B″ proteins redistribute to different nuclear domains in dormant and proliferating onion cells

Abstract

Monoclonal antibodies against the spliceosomal proteins Sm and U2B", and against p105, a protein component of interchromatin granules, were used to investigate the nuclear distribution of the splicing factors in Allium cepa L. meristematic cells. Confocal microscopy showed that in steady-state proliferating cells, the spliceosomal components were distributed into two nuclear domains: (i) a diffuse nucleoplasmic network similar to that formed by interchromatin granules and (ii) numerous Cajal bodies. These domains were the counterpart of the perichromatin fibrils and granules, interchromatin granules and Cajal bodies observed by electron microscopy after EDTA and bismuth oxynitrate stainings. Dormant cells showed a nuclear distribution of the proteins in small Cajal bodies and numerous micro-speckles, correlated with the distribution of ribonucleoproteins (RNPs) observed by electron microscopy. The spliceosomal proteins relocated to the diffuse nucleoplasmic network and Cajal bodies when the cells were released from dormancy by water soaking and they re-started their proliferative activity. Inhibition of RNA synthesis by 5,6-dichloro-1-beta- d-ribofuranosylbenzimidazole (DRB) treatment in proliferating cells demonstrated that the micro-speckles were not the morphological expression of a transcription block. Fractionation and confocal microscopy studies showed a differential association of the splicing factors with the nuclear matrix depending not only on the protein, but also on nuclear activity. Our results suggest a reversible relocation of the spliceosomal proteins between different sub-nuclear domains in physiological conditions. We report here an unusual nuclear domain in dormant nuclei, the micro-speckles, corresponding to storage sites for RNPs, which were rapidly mobilised after water imbibition.