Spliceosomal Small Nuclear Ribonucleoprotein Particles Repeatedly Cycle through Cajal Bodies

Abstract

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 Because snRNPs do not shuttle between the nucleus and cytoplasm (Änkö and Neugebauer, unpublished data), Sm ring assembly seems to occur early and only once in the lifetime of each snRNP.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...

Friend of Prmt1, FOP is a Novel Component of the Nuclear SMN Complex Isolated Using Biotin Affinity Purification

“…This does not conflict to the previous notion that FOP has a critical role in specific mRNA transcriptional regulation and mRNA export [27,28]. We do not exclude, however, other roles of FOP-SMN complex, such that it may prevent improperly processed mRNAs from transit through the nuclear speckle [20,21], or that FOP may keep the nuclear SMN complexes near the transcription sites on chromosome until it is used for recycling UsnRNPsafter splicing [22][23][24].…”

“…Although it has been well documented that improper process of mRNAs impedes transit of the mRNAs through the nuclear speckle [20,21], the mechanism how the mRNAs are retained in recycle splicing complex and impeded the transit remains undetermined. The splicing complexes including UsnRNPs that are recycled after each round of splicing into the cytoplasm, must pass again through the Cajal/Gems for re-assembly before they can take part in a further round of splicing [22][23][24].…”

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

“…For example, the U4/U6•U5 tri-snRNP is formed from three individual snRNPs (U4, U5 and U6) that are inactive for splicing before assembly. A transient intermediate, the U4/U6 di-snRNP, is highly concentrated in CBs and accumulates there when U4/U6•U5 tri-snRNP formation is blocked [14][15][16] . Partial rescue of the coilin morphant by the U4•U5/U6 snRNP implicates coilin in these late stages of assembly.…”

“…Spliceosomal snRNPs explore the nuclear volume by Brownian diffusion 3,15,43 . Given this, the rate at which immature snRNPs encounter partner proteins or other snRNPs will determine how quickly their assembly occurs.…”

Coilin: The first 25 years

“…20,21 Spliceosomal snRNPs are assembled and recycled in CBs. [22][23][24][25][26][27][28] Mathematical modeling and measurement of snRNP kinetics in CBs suggest that concentration of snRNPs in the CB increases snRNP assembly rate by a factor of 10. 29,30 Similarly, co-localization of snRNAs and scaRNAs, which guide snRNA modifications, in CBs might enhance snRNA modification efficiency.…”