Article
- The EMBO Journal (2004) 23, 3000 - 3009
- doi:10.1038/sj.emboj.7600296
Published online: 15 July 2004
Subject Categories:
RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies
Nina Schaffert1, Markus Hossbach1, Rainer Heintzmann2, Tilmann Achsel3 and Reinhard Lührmann1
- Department of Cellular Biochemistry, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany
- Department of Molecular Biology, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany
- IRCCS Fondazione Santa Lucia, Neurobiologia, Via Ardeatina 306, 00179 Rome, Italy
Correspondence to:
Reinhard Lührmann,
Department of Cellular Biochemistry, Max-Planck-Institute of Biophysical Chemistry, Am Fa
berg 11, 37077 Göttingen, Germany. Tel.: +49 551 201 1407; Fax: +49 551 201 1197; E-mail: reinhard.luehrmann@mpi-bpc.mpg.de
Received 2 April 2004; Accepted 8 June 2004
Abstract
Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.
Keywords:
- Cajal bodies,
- hPrp31,
- pre-mRNA splicing,
- retinitis pigmentosa,
- snRNP biogenesis
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