The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

Abstract

Coiled bodies are nuclear organelles that contain components of at least three RNAprocessing pathways: pre-mRNA splicing, histone mRNA 3Ј-maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3Ј-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2BЉ-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2BЉ gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body. INTRODUCTIONThe coiled body was first described by Ramon y Cajal (1903), who called it the "nucleolar accessory body" because of its association with the nucleolus. This nuclear organelle was later reidentified by electron microscopy and renamed the "coiled body" because of its appearance as loosely packed coiled fibrils (Monneron and Bernhard, 1969). Subsequent studies detected coiled bodies in animal and plant nuclei, showing that it is a conserved structure (Moreno Diaz de la Espina et al., 1980;Seite et al., 1982;Schultz, 1990).Coiled bodies have been shown to contain splicing small nuclear ribonucleoproteins (snRNPs) and small nuclear RNAs (snRNAs), a subset of nucleolar components -including fibrillarin, Nopp140, NAP57, and U3 small nucleolar ribonucleoprotein (U3 snoRNP) -and the protein p80 coilin, which has been widely used as a marker for coiled bodies (Lamond and Earnshaw, 1998;Matera, 1998). The function of coiled bodies is still under debate, but several hypotheses have been proposed that are not necessarily mutually exclusive. Because coiled bodies do not contain DNA, nascent pre-mRNA, heterogeneous nuclear RNPs (hnRNPs), or the SC-35 splicing factor, which is required for splicing in vitro, it has been argued that they are not directly involved in transcription and pre-mRNA splicing . However, it has been speculated that coiled bodies may be sites of splicing factor assembly or recycling, may play a role in histone mRNA 3Ј processing (Gall et al., 1995;Lamond and Earnshaw, 1998), or may be involved in all these activities. As coiled bodies are frequently observed at the nucleolar periphery and also in the nucleoplasm and within nucleoli (Malatesta et al. 1994;Ochs et al., 1994), they may also act as nuclear transport or sorting structures. It has been shown recently that coiled bodies are also involved in processing or transport of small nucleolar RNA (snoRNA) pr...

Cell Biology of the Arabidopsis Nuclear siRNA Pathway for RNA-directed Chromatin Modification

“…These findings indicate that the nucleolar subdomain where AGO4 colocalizes with RDR2, DCL3, NRPD1b, and various siRNAs are Cajal bodies, or related entities. The fact that these AGO4 bodies can be found inside the nucleolus, or on the outer edge of the nucleolus, is consistent with other observations of the dynamic nature of Cajal bodies (Boudonck et al 1999;Cioce and Lamond 2005). Previously described associations of Cajal bodies with chromatin, such as histone genes and telomeres, to deliver histone mRNA processing or telomerase RNPs, respectively, suggest that the trafficking of RISCs to heterochromatic loci via Cajal body intermediates is plausible.…”

The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation

“…Differences in the HYL1 protein level affect miRNA metabolism and mRNA stability but not dsRNA-mediated PTGS. HYL1 is predominantly a nuclear protein and is detected in nuclear bodies and ring-like structures reminiscent of Cajal or coiled bodies, known to be involved in nuclear RNA processing in association with the nucleolus (45,46). One-third to one-half of the HYL1 protein in crude extracts is in the form of an Ϸ300-kDa complex.…”

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

“…Recent studies have shown that CB components reside for relatively short times in CBs, suggesting that molecular events occur in CBs in the context of a dynamic exchange of factors with the surrounding nucleoplasm (Handwerger et al, 2003;Dundr et al, 2004). In addition, CBs themselves are highly dynamic, in that they move, split, and fuse during the lifetime of the cell (Andrade et al, 1993;Boudonck et al, 1999;Platani et al, 2000); moreover, their structure is dependent on active transcription and metabolic activity, suggesting that a constant supply of substrates is required to maintain CB integrity (Carmo-Fonseca et al, 1992;Boudonck et al, 1998;Shpargel and Matera, 2005;Lemm et al, 2006). Although it is formally possible that the occurrence of CBs in nuclei is an indirect outcome of physiological state, speculation has focused in recent years on the possibility that concentration of components facilitates key steps in gene expression.…”