Thyroid hormone (TH) is required for limb development in Xenopus laevis. Specific cell types in the growing limb were targeted for expression of a dominant negative form of the TH receptor by sperm-mediated transgenesis. Limb muscle development, the innervation of muscle from the spinal cord, and cartilage growth can be inhibited without affecting patterning of the limb or differentiation of other cell types. Remodeling of the skin occurs late in metamorphosis after the limb has formed. The coordination of these independent programs is affected in part by the control that TH exerts over DNA replication in all cell types of the limb.

Cajal bodies (CBs) are complex organelles found in the nuclei of a wide variety of organisms, including vertebrates, invertebrates, plants, and yeast. In most cell types CBs are <1 mum in diameter, severely limiting the range of experimental observations that can be made on them. By contrast, CBs in the amphibian oocyte nucleus (also called the germinal vesicle) are 2-10 mum in diameter. We have taken advantage of this large size to carry out kinetic studies on coilin, a protein that is specifically enriched in CBs. We labeled coilin with photoactivatable green fluorescent protein and analyzed the movement of the protein by confocal microscopy in unfixed germinal vesicles isolated in oil. We showed that coilin leaves the CB relatively slowly (minutes rather than seconds) with kinetics similar to earlier measurements on its entrance. We also showed that coilin diffuses very slowly within the CB, consistent with its being in a large macromolecular complex. Finally, we found that the movement of coilin is not directly affected by the transcriptional state of the nucleus or ongoing nucleocytoplasmic exchange. These data on the kinetics of coilin reinforce the conclusion that CB components are in a constant state of flux, consistent with models that postulate an active role for CBs in nuclear physiology.

Cajal bodies (CBs) are nuclear organelles that occur in a variety of organisms, including vertebrates, insects, and plants. They are most often identified with antibodies against the marker protein coilin. Because the amino acid sequence of coilin is not strongly conserved evolutionarily, coilin orthologues have been difficult to recognize by homology search. Here, we report the identification of Drosophila melanogaster coilin and describe its distribution in tissues of the fly. Surprisingly, we found coilin not only in CBs but also in histone locus bodies (HLBs), calling into question the use of coilin as an exclusive marker for CBs. We analyzed two null mutants in the coilin gene and a piggyBac insertion mutant, which leads to specific loss of coilin from the germline. All three mutants are homozygous viable and fertile. Cells that lack coilin also lack distinct foci of other CB markers, including fibrillarin, the survival motor neuron (SMN) protein, U2 small nuclear RNA (snRNA), U5 snRNA, and the small CB-specific (sca) RNA U85. However, HLBs are not obviously affected in coilin-null flies. Thus, coilin is required for normal CB organization in Drosophila but is not essential for viability or production of functional gametes.

During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.

The organization of the cell nucleus into specialized compartments is important for nuclear function. We address the significance of compartmentalization by studying the Cajal body, an evolutionarily conserved nuclear organelle proposed to be involved in such diverse functions as assembly of the spliceosome, assembly of the transcription machinery, and modification of spliceosomal small nuclear RNAs. The Cajal body is typically identified by the presence of coilin, a protein of poorly defined function. Here, we demonstrate that coilin is not a unique Cajal body marker but also occurs in a related yet distinct nuclear organelle known as the histone locus body in both Drosophila and Xenopus. We stress the importance of multiple markers not only for identification of nuclear bodies but also for assessing their functional significance.

The Cajal body (CB) is a nuclear organelle present in all eukaryotes that have been carefully studied. It is identified by the signature protein coilin and by CB-specific RNAs (scaRNAs). CBs contain high concentrations of splicing small nuclear ribonucleoproteins (snRNPs) and other RNA processing factors, suggesting that they are sites for assembly and/or posttranscriptional modification of the splicing machinery of the nucleus. The histone locus body (HLB) contains factors required for processing histone pre-mRNAs. As its name implies, the HLB is associated with the genes that code for histones, suggesting that it may function to concentrate processing factors at their site of action. CBs and HLBs are present throughout the interphase of the cell cycle, but disappear during mitosis. The biogenesis of CBs shows the features of a self-organizing structure.

The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 2'-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 2'-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.

The spliceosomal small nuclear RNAs (snRNAs) are modified post-transcriptionally by introduction of pseudouridines and 2'-O-methyl modifications, which are mediated by box H/ACA and box C/D guide RNAs, respectively. Because of their concentration in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific (sca) RNAs. In the cell, scaRNAs are associated with the WD-repeat protein WDR79. We used coimmunoprecipitation with WDR79 to recover seven new scaRNAs from Drosophila cell lysates. We demonstrated concentration of these new scaRNAs in the CB by in situ hybridization, and we verified experimentally that they can modify their putative target RNAs. Surprisingly, one of the new scaRNAs targets U6 snRNA, whose modification is generally assumed to occur in the nucleolus, not in the CB. Two other scaRNAs have dual guide functions, one for an snRNA and one for 28S rRNA. Again, the modification of 28S rRNA is assumed to take place in the nucleolus. These findings suggest that canonical scaRNAs may have functions in addition to their established role in modifying U1, U2, U4, and U5 snRNAs. We discuss the likelihood that processing by scaRNAs is not limited to the CB.

Despite long-term exploration into ribosomal RNA gene functioning during the oogenesis of various organisms, many intriguing problems remain unsolved. In this review, we describe nucleolus organizer region (NOR) activity in avian oocytes. Whereas oocytes from an adult avian ovary never reveal the formation of the nucleolus in the germinal vesicle (GV), an ovary from juvenile birds possesses both nucleolus-containing and non-nucleolus-containing oocytes. The evolutionary diversity of oocyte NOR functioning and the potential non-rRNA-related functions of the nucleolus in oocytes are also discussed.

The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved post -transcriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast Saccharomyces cerevisiae and mouse. Furthermore, in S. cerevisiae, Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system, we tested enzymatic activity of Pus1p from the fission yeast Schizosaccharomyces pombe, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the frog Xenopus tropicalis. We demonstrated that Pus1 Delta from C. elegans has no enzymatic activity on U2 snRNA when expressed in yeast cells, whereas in similar experiments, position 44 in yeast U2 snRNA (equivalent to position 43 in vertebrates) is a genuine substrate for Pus1p from S. cerevisiae, S. pombe, Drosophila, Xenopus, and mouse. However, when we analyzed U2 snRNAs from Pusl knockout mice and the pus14 S. pombe strain, we could not detect any changes in their modification patterns when compared to wild -type U2 snRNAs. In S. pombe, we found a novel box H/ACA RNA encoded downstream from the RPC10 gene and experimentally verified its guide RNA activity for positioning psi 43 and psi 44 in U2 snRNA. In vertebrates, we showed that SCARNA8 (also known as U92 scaRNA) is a guide for U2 -psi 43 in addition to its previously established targets U2-psi 34/psi 44.