Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, Université Paul Sabatier, IFR109, 118 route de Narbonne, 31062 Toulouse CEDEX 4, France
Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
(e-mail: tamas{at}ibcg.biotoul.fr)
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Introduction |
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Biogenesis of spliceosomal snRNPs |
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In the cytoplasm, the survival of motor neurons (SMN) protein complex facilitates the assembly of pre-snRNA with a heteroheptameric ring of seven Sm proteins - B/B', D1, D2, D3, E, F and G - to form the snRNP core complex (Paushkin et al., 2002). The Sm proteins bind to the conserved Sm-binding site (Sm, consensus RAUUU/GUUGR) of Pol-II-specific spliceosomal snRNAs. Binding of Sm proteins is a prerequisite for hypermethylation of the 7mG cap to 2,2,7-trimethylguanosine (TMG) by the Tgs1 methyltransferase, as well as for exonucleolytic removal of the 3' trailer sequences. The newly synthesized TMG cap and the associated Sm proteins provide the nuclear localization signal for the cytoplasmic core snRNP. Reimportation of Sm snRNPs into the nucleus is mediated by three factors: snurportin-1 (SPN1), which recognizes the TMG cap of the snRNA; importin ß, which associates with SPN1; and an unidentified import receptor that interacts with Sm proteins (Sm IR) (Palacios et al., 1997
).
After re-entering the nucleoplasm, the import proteins dissociate and the newly imported snRNPs transiently accumulate in Cajal bodies (Sleeman and Lamond, 1999). In the Cajal body, the U1, U2, U4 and U5 snRNAs undergo site-specific pseudouridylation (
) and 2'-O-methylation (m) directed by scaRNAs, which specifically accumulate in Cajal bodies (Jády et al., 2003
). In addition to the common Sm core proteins, the mature U1, U2, U4 and U5 snRNPs also contain species-specific RNP proteins. Since the snRNP-specific proteins appear to concentrate in Cajal bodies, they are believed to associate with core snRNPs in this organelle.
Finally, mature spliceosomal snRNPs accumulate in the interchromatin region in structures called splicing speckles. Usually, speckles are located close to actively transcribed genes. They probably provide storage sites for snRNPs or function in snRNP recycling (Lamond and Spector, 2003). It is important to note that several laboratories have reported low levels of nucleolar localization of Sm snRNPs under various conditions (Gerbi et al., 2003
; Sleeman and Lamond, 1999
). Unfortunately, the functional importance of transient accumulation of Sm snRNAs in the nucleolus remains conjectural. Biogenesis of the Pol-II-specific U11, U12 and U4atac minor spliceosomal snRNPs probably follows the maturation pathway of major Sm snRNPs.
In contrast to the Pol-II-specific Sm snRNAs, biogenesis of the Pol-III-transcribed U6 snRNA is confined to the nucleus. Before accumulating in the nucleoplasmic splicing speckles, the maturing U6 snRNA visits the nucleolus and Cajal body. Synthesis of U6 snRNA terminates within a short U stretch that serves as a transcription termination signal for Pol III. The 3'-terminal U stretch of the newly synthesized U6 RNA transiently associates with the La protein, which provides stability for the nascent RNA and facilitates snRNP assembly (Wolin and Cedervall, 2002). Later, the La protein is replaced by the doughnut-like heteromer of seven Sm-like proteins (Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7 and Lsm8) (Achsel et al., 1999
). The structure of the Lsm ring is not yet known.
During its maturation, the U6 snRNA also undergoes site-specific pseudouridylation and 2'-O-methylation. However, in contrast to Sm snRNAs, which are modified by scaRNPs in the Cajal body, 2'-O-methylation and probably also pseudouridylation of the U6 snRNA is directed by snoRNAs, which reside in the nucleolus. This indicates that U6 cycles through the nucleolus to undergo snoRNA-mediated modifications (Ganot et al., 1999; Lange and Gerbi, 2000
). Binding of the Lsm protein complex is essential for targeting of U6 snRNA to the nucleolus and, afterward, to the Cajal body (Gerbi and Lange, 2002
). The mature U6 and U4 snRNPs exist in a common U4-U6 disnRNP that also binds the U5 snRNP to form the U4-U6-U5 tri-snRNP. The recent finding that SART3/p110, a protein factor facilitating U4-U6 assembly, is concentrated in the Cajal body suggests that assembly of the U4-U6 snRNP may take place in this organelle (Stanek et al., 2003
). It is unclear whether U5 snRNP joins the U4-U6 di-snRNP in the Cajal body or later in the nucleoplasm. The mature U6 snRNA carries a
-monomethylphosphate (mpppG) cap structure (Singh and Reddy, 1989
), but it remains unknown at what stage of U6 biogenesis the mpppG cap is synthesized.
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Biogenesis of snoRNPs and scaRNPs |
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Except for the U3, U8 and U13 box C/D snoRNAs, which are transcribed from independent genes, all mammalian snoRNAs and scaRNAs are encoded within introns of split genes. The intronic sno/scaRNAs are processed from the spliced and debranched host introns by exonucleolytic trimming. It appears that binding of the sno/scaRNP proteins occurs on the host pre-mRNA. Moreover, the splicing machinery bound to the host pre-mRNA may facilitate sno/scaRNP assembly (Hirose et al., 2003). In the released host intron, exonucleolytic excision of the intronic snoRNA occurs close to the site of transcription and splicing of the host premRNA (Samarsky et al., 1998
). The bound RNP proteins probably protect the sno/scaRNA from processing exonucleases.
Before accumulating in the nucleolus, intronic box C/D snoRNAs transit through Cajal bodies. The recent finding that final 3' end trimming and synthesis of the TMG cap of the U3 box C/D snoRNA that is transcribed by Pol II from an independent gene occurs in the Cajal body suggests that the final steps of box C/D snoRNP maturation take place in this organelle (Verheggen et al., 2002). So far, no transient Cajal-body-specific accumulation has been reported for box H/ACA snoRNPs. Therefore, it remains uncertain whether this class of snoRNA is transported directly into the nucleolus or, like box C/D snoRNAs, they also transit through Cajal bodies.
The nucleolar accumulation of the two classes of snoRNAs is determined by the conserved C/D and H/ACA box elements themselves (Terns and Terns, 2002). However, the final destination of scaRNAs, although they carry C/D and/or H/ACA boxes, is the Cajal body. The box H/ACA scaRNAs contain a cis-acting localization signal, the CAB box, that is responsible for targeting these RNAs into the Cajal body (Richard et al., 2003
). Interestingly, the CAB box Cajal body localization signal is also present in human telomerase RNA, which has a box-H/ACA-RNA-like domain and accumulates in the Cajal body (Jády et al., 2004
; Zhu et al., 2004
). Presumably, box H/ACA scaRNPs and the telomerase RNP share a common CAB-box-associated trans-acting protein factor responsible for Cajal-body-specific accumulation of these RNPs. The molecular mechanism directing the Cajal-body-specific accumulation of box C/D scaRNAs is still unknown.
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References |
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