No abstract

Cajal bodies (CBs) are evolutionarily conserved nuclear structures involved in the metabolism of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). CBs are not present in all cell types, and the trigger for their formation is not yet known. Here, we depleted cells of factors required for the final steps of snRNP assembly and assayed for the presence of stalled intermediates in CBs. We show that depletion induces formation of CBs in cells that normally lack these nuclear compartments, suggesting that CB nucleation is triggered by an imbalance in snRNP assembly. Accumulation of stalled intermediates in CBs depends on the di-snRNP assembly factor SART3. SART3 is required for both the induction of CB formation as well as the tethering of incomplete snRNPs to coilin, the CB scaffolding protein. We propose a model wherein SART3 monitors tri-snRNP assembly and sequesters incomplete particles in CBs, thereby allowing cells to maintain a homeostatic balance of mature snRNPs in the nucleoplasm.

A combination of mathematical modeling and live-cell measurements was applied to determine the dynamics of small nuclear ribonucleoprotein (snRNP) formation in Cajal bodies of living cells. Our results indicate that a substantial fraction of tri-snRNPs is formed in Cajal bodies in cells with many Cajal bodies per nucleus.

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6⅐U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs. INTRODUCTIONPre-mRNA splicing is a two-step transesterification reaction catalyzed by the spliceosome, a large complex assembled from preformed subcomplexes, called spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and hundreds of additional proteins (Jurica and Moore, 2003). In turn, the five major spliceosomal snRNPs, U1, U2, U4, U5, and U6, each consist of a single small nuclear RNA (snRNA) and specific set of proteins. Among the shared protein components of snRNPs are the seven Sm proteins, which are assembled as a stable, heteroheptameric ring on the RNA polymerase II-transcribed snRNAs: U1, U2, U4, and U5. After transcription, these snRNAs are transported to the cytoplasm, where the Sm ring is assembled on snRNAs by the SMN complex. Subsequently, the 5Ј ends of the snRNAs are hypermethylated to generate the trimethyl-guanosine cap, which together with SMN, promotes snRNP nuclear import (reviewed in Will and Luhrmann, 2001;Matera and Shpargel, 2006;Tycowski et al., 2006). Because snRNPs do not shuttle between the nucleus and cytoplasm (Änkö and Neugebauer, unpublished data), Sm ring assembly seems to occur early and only once in the lifetime of each snRNP. A related heteroheptameric ring, consisting of seven "like-Sm" (LSm) proteins, is assembled on U6, an RNA polymerase III transcript, which is thought to remain in the nucleus for all assembly steps (Achsel et al., 1999;Mayes et al., 1999;Kiss, 2004;Listerman et al., 2007).Once back in the cell nucleus, snRNPs first accumulate in CBs before distributing throughout the nucleoplasm, where splicing occurs (Sleeman and Lamond, 1999;Sleeman et al., 2001;Neugebauer, 2002). This suggests a role for CBs in nuclear steps of snRNP maturation, a prediction borne out by the following set of observations. First, po...