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Published 27 September 2004. doi:10.1083/jcb.200405160
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 166, Number 7, 1015-1025
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Article

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

David Stanek and Karla M. Neugebauer

Max Planck Institute of Molecular Cell Biology and Genetics

Address correspondence to K.M. Neugebauer, Pfotenhauerstrasse 108, 01307 Dresden, Germany. Tel.: (49) 351-210 2589. Fax: (49) 351-210 1209. email: neugebau{at}mpi-cbg.de

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.

Key Words: Cajal body; snRNP; pre-mRNA splicing; coilin; FRET

Abbreviations used in this paper: CB, Cajal body; CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; MEF, mouse embryonic fibroblast; PA-GFP, photoactivatable green fluorescent protein; snoRNP, small nucleolar ribonucleoprotein particle; snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein particle.


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