Control of Cajal body number is mediated by the coilin C-terminus

Abstract

Cajal bodies (CBs) are nuclear suborganelles implicated in the post-transcriptional maturation of small nuclear and small nucleolar RNAs. The number of CBs displayed by various cell lines and tissues varies, and factors that control CB numbers within a given cell have yet to be described. In this report, we show that specific regions within the C-terminus of coilin, the CB marker protein, are responsible for regulating the number of nuclear foci. Despite the fact that the coilin N-terminal domain is responsible for its self-oligomerization activity, truncation or mutation of predicted sites of phosphorylation in the conserved C-terminal region leads to a striking alteration in the number of nuclear bodies. Similarly, coilin constructs from various species display differential propensities to form nuclear foci when expressed in heterologous backgrounds. We mapped the domain responsible for this variability to the coilin C-terminus utilizing chimeric proteins. Furthermore, the activities responsible for regulating coilin self-association must reside in the nucleus, as constructs lacking critical nuclear localization sequences fail to form foci in the cytoplasm. Factors controlling the putative signal transduction cascade that phosphorylates coilin are also discussed. The results point to a model whereby phosphorylation of the coilin C-terminus regulates the availability of the N-terminal self-interaction domain.

Gemin3Is an Essential Gene Required for Larval Motor Function and Pupation inDrosophila

“…Furthermore, immunofluorescence of S2 cells cotransfected with dSMN (GFP-dSMN) and Dhh1 (myc-dGem3) constructs revealed complete colocalization throughout the cytoplasm and in nuclear Cajal bodies ( Figure 2C). Interestingly, dSMN overexpression formed cytoplasmic aggregates, similar to its human ortholog (Shpargel et al, 2003). On the basis of these and other experiments (see below), we conclude that Dhh1 is the Drosophila ortholog of Gemin3.…”

Targeting SMN to Cajal bodies and nuclear gems during neuritogenesis

“…6d-f) or high levels (Fig. 6g-i) of GFP-SMN showed no additional SMN-positive nuclear bodies, although cytoplasmic accumulations were readily detectable (Shpargel et al 2003;Sleeman et al 2003). Moreover,GFP-SMN expression did not affect the timecourse of differentiation toward a neuron-like phenotype.…”

“…Surprisingly, the total number of nuclear bodies was similarly refractive to GFP-SMN overexpression in dexamethasone-treated UR61 cells. These findings are curious because high levels of overexpression of myc-tagged and GFP-tagged SMN in HeLa cells resulted in a large increase in the numbers of both nuclear and cytoplasmic SMN foci (Hebert et al 2002;Shpargel et al 2003;Sleeman et al 2003). While it is possible that similar high levels of SMN overexpression cannot be achieved in UR61 cells, it appears that differentiation-induced formation of gems in UR61 is not simply the result of increased SMN expression levels.…”

“…To induce neuron-like differentiation, cultures were exposed to 0.2 μM dexamethasone for 12, 24, 36 and 48 h (Guerrero et al 1988). Transfection was performed with the plasmid construct pGFP-SMN, as previously described (Shpargel et al 2003;Sleeman et al 2003). Untreated and dexamethasone-treated UR61 cells were transfected for 18 h using FuGene 6 transfection reagent (Roche) according to the manufacturer's instructions.…”

“…Thus overexpression of SMN in undifferentiated UR61 cells might plausibly saturate the number of CB-binding sites and lead to formation of gems. In fact, previous investigations using HeLa cells had shown that overexpression of wild-type SMN can lead to production of additional nuclear and cytoplasmic SMN foci (Shpargel et al 2003;Sleeman et al 2003). We therefore transiently expressed GFP-SMN in uninduced UR61 cells and monitored the number of CBs using anti-coilin antibodies.…”