There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co-and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated sF3b155 (P-sF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin-and nucleoplasm-associated P-sF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRnA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-sF3b155 antibodies, coupled with transcription inhibition and a block in splicing after sF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of posttranscriptionally spliced mRnA from speckles is coupled to the nuclear mRnA export pathway. our data provide new insights into when and where splicing occurs in cells.

To better understand intranuclear-targeting mechanisms, we have studied the transport of U3 snoRNA in human cells. Surprisingly, we found that PHAX, the snRNA export adaptor, is highly enriched in complexes containing m7G-capped U3 precursors. In contrast, the export receptor CRM1 is predominantly bound to TMG-capped U3 species. In agreement, PHAX does not export m7G-capped U3 precursors because their caps become hypermethylated in the nucleus. Inactivation of PHAX and CRM1 shows that U3 first requires PHAX to reach Cajal bodies, and then CRM1 to be routed from there to nucleoli. Furthermore, PHAX also binds the precursors of U8 and U13 box C/D snoRNAs and telomerase RNA. PHAX was previously shown to discriminate between small versus large RNAs during export. Our data indicate that the role of PHAX in determining the identity of small RNAs extends to nonexported species, and this appears critical to promote their transport within the nucleus.

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs. INTRODUCTIONThe interphase nucleus contains many morphologically distinct substructures, called nuclear bodies, which are nonmembrane-bound, stable cellular compartments involved in the fidelity and efficiency of gene expression. One of the most extensively studied nuclear bodies, the Cajal body (CB), was originally identified more than 100 years ago (Cajal, 1903). CBs are present in the nucleus of vertebrates, invertebrates and plants in varying number and size depending on the cell type (Gall, 2000;Cioce and Lamond, 2005). They are molecularly defined by the presence of the protein coilin, which was discovered through the analysis of patient autoimmune sera Raska et al., 1991). The composition of CBs is very diverse, but a number of factors involved in RNA processing are found enriched within them: 1) spliceosomal U small nuclear ribonucleoproteins (snRNPs) and associated factors (Spector et al., 1992); 2) small Cajal-body-specific (sca)RNAs which guide posttranscriptional modifications of spliceosomal U snRNAs (Darzacq et al., 2002;Kiss et al., 2002;Jády et al., 2003); 3) small nucleolar (sno)RNPs involved in processing of pre-rRNA (Narayanan et al., 1999;Verheggen et al., 2002;Boulon et al., 2004); 4) U7 snRNP, which is required for the 3Ј trimming of histone mRNAs (Frey and Matera, 1995); and 5) basal transcription factors (Schul et al., 1998;Gall et al., 1999).The CB marker coilin is a multi-interacting protein that binds, among other things, directly to some of the spliceosomal Sm proteins (Hebert et al., 2001;Xu et al., 2005), and the snoRNP biogenesis factor Nopp140 (Isaac et al., 1998). In addition, coilin contains an N-terminal self-oligomerization domain (Hebert et al., 2000). It has been proposed t...