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. 2004 Sep 27;166(7):1015-25.
doi: 10.1083/jcb.200405160.

Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer

David Stanĕk  1 , Karla M Neugebauer
Affiliations

Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer

David Stanĕk et al. J Cell Biol. .

Abstract

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are required for pre-mRNA splicing throughout the nucleoplasm, yet snRNPs also concentrate in Cajal bodies (CBs). To address a proposed role of CBs in snRNP assembly, we have used fluorescence resonance energy transfer (FRET) microscopy to investigate the subnuclear distribution of specific snRNP intermediates. Two distinct complexes containing the protein SART3 (p110), required for U4/U6 snRNP assembly, were localized: SART3.U6 snRNP and SART3.U4/U6 snRNP. These complexes segregated to different nuclear compartments, with SART3.U6 snRNPs exclusively in the nucleoplasm and SART3.U4/U6 snRNPs preferentially in CBs. Mutant cells lacking the CB-specific protein coilin and consequently lacking CBs exhibited increased nucleoplasmic levels of SART3.U4/U6 snRNP complexes. Reconstitution of CBs in these cells by expression of exogenous coilin restored accumulation of SART3.U4/U6 snRNP in CBs. Thus, while some U4/U6 snRNP assembly can occur in the nucleoplasm, these data provide evidence that SART3.U6 snRNPs form in the nucleoplasm and translocate to CBs where U4/U6 snRNP assembly occurs.

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Figures

Figure 1.
Figure 1.
Model of U4/U6 snRNP assembly in Cajal bodies. SART3 interacts with newly synthesized U6 snRNP or with the U6 snRNP released after splicing in the nucleoplasm. The complex is then transported to Cajal bodies where formation of the U4/U6 snRNP occurs. SART3 disassociates from the U4/U6 snRNP as the U4/U6•U5 tri-snRNP complex is formed (also potentially in CBs). During formation of the active spliceosome, the tri-snRNP disassembles; U5 and U6 snRNPs play an essential role during splicing while the U4 snRNP leaves the spliceosome.
Figure 2.
Figure 2.
FRET detection in CBs using coilin. HeLa cells were cotransfected with (A) coilin–CFP and coilin–YFP or (B) CFP–coilin and YFP–coilin. Cells were fixed after 24 h and FRET efficiencies were measured by acceptor photobleaching in CBs (A) and in the nucleoplasm and CB (B). Unbleached areas in the nucleoplasm and unbleached CBs were used as a negative control. (C) Plot of FRET efficiencies (average of means of two independent experiments ± SEM) between coilin–coilin pairs. FRET efficiencies were measured in the bleached area within the nucleoplasm (the white box). In the case of CBs, FRET efficiencies were measured in the area of the CB only. FRET efficiency was calculated from CFP fluorescence before and after bleaching: FRETefficiency [%] = (CFPafter − CFPbefore) × 100/CFPafter. Very low levels of coilin–CFP/coilin–YFP pair in the nucleoplasm did not allow a FRET analysis in this compartment. The decrease of CFP fluorescence in unbleached CBs is due to bleaching during CFP detection. Bars, 5 μm.
Figure 3.
Figure 3.
Fluorescently tagged proteins assemble into snRNPs. (A) Model of the SART3•U4/U6 snRNP complex. (B) HeLa cells were transfected with SART3, hPrp3, hPrp4, 61K, or LSm7 that have been tagged fluorescently. Total RNA was metabolically label by [32P]orthophosphate and snRNAs were immunoprecipitated by the anti-Sm antibody (anti-Sm) or anti-GFP antibodies (anti-GFP), which cross-reacts with CFP. The anti-Sm antibody precipitated all major snRNAs, anti-GFP antibodies precipitated preferentially U4 and U6 snRNAs. Positions of snRNAs are indicated on the left and 5.8S and 5S rRNAs on the right. The 5.8S rRNAs (migrating as a doublet slightly above U1 snRNA) and 5S rRNA (migrating as a single band above U5 snRNAs) were common contaminants in our immunoprecipitations but were for unknown reasons underrepresented in GFP only controls. NT, nontransfected control.
Figure 4.
Figure 4.
SART3 preferentially associates with the U4/U6 snRNP proteins in CBs. (A and B) HeLa cells were cotransfected with YFP–SART3 and either (A) CFP–hPrp4 or (B) CFP–hPrp3. FRET was subsequently measured in the nucleoplasm and in CBs within the same cell by acceptor photobleaching. (C) Plot of averages of means from three independent experiments between SART3 and the U4/U6 snRNP proteins 61K, hPrp3, or hPrp4. FRET efficiencies between YFP–SART3 and the U4/U6 snRNP proteins were, in all cases, three to five times higher in CBs than in the nucleoplasm. hPrp3 revealed 2.5 times lower FRET signal in CBs when coexpressed with SART3 tagged at the COOH terminus (SART3–YFP). As controls, FRET efficiencies were measured in the nucleoplasm and CBs of cells coexpressing either CFP–hPrp3/YFP–hPrp4, which form a stable complex or fibrillarin–CFP/YFP–SART3, which both localize to CBs but no interaction has been identified between them (average of two independent experiments). Bars, 5 μm.
Figure 5.
Figure 5.
SART3•U4/U6 snRNP complexes in the absence of functional coilin. (A) MEF 42coilin−/− were cotransfected with YFP–SART3 and CFP–61K and FRET was measured in the defined area in the nucleoplasm. MEF 42coilin−/− were cotransfected with PA–GFP–mcoilin, YFP–SART3, and CFP–61K and FRET efficiencies were measured in the nucleoplasm and CBs. Mcoilin was detected by indirect immunofluorescence. (B) Plot of average of means of two independent experiments between YFP–SART3 and CFP–61K in MEF 26coilin+/+ and MEF 42coilin−/−. MEF 26coilin +/+ revealed a high FRET signal in both the nucleoplasm and CBs with FRET efficiency two times higher in CBs than the nucleoplasm. MEF 42coilin−/− exhibited a high FRET signal in the nucleoplasm, which was comparable to the signal in CBs of MEF 26coilin +/+. After restoration of CBs by expression of exogenous mcoilin, FRET signal in the nucleoplasm decreased to ∼60% of the level detected in newly formed CBs. Bar, 5 μm.
Figure 6.
Figure 6.
SART3 interacts with U6 snRNP proteins in the nucleoplasm. (A) HeLa cells were cotransfected with SART3–CFP and YFP–LSm7. FRET was measured by acceptor photobleaching in the nucleoplasm and in a CB within the same cell. (B) Plot of averages of means from two independent experiments between SART3 and LSm proteins. The efficiency of FRET was measured as shown in panel A and the average of means and standard error of the mean were calculated. All YFP–LSm proteins revealed a positive FRET signal with SART–CFP in the nucleoplasm but not in CBs. FRET signal in the nucleoplasm decreased four times when YFP–LSm7 was coexpressed with CFP–SART3 and nine times when coexpressed with the ΔCT10ΔRRM–CFP mutant. Bar, 5 μm.
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