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Open Access Research article

Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies

Prabhakar Rajan12, Caroline Dalgliesh1, Cyril F Bourgeois3456, Monika Heiner7, Kaveh Emami8, Emma L Clark9, Albrecht Bindereif7, James Stevenin3456, Craig N Robson9, Hing Y Leung2* and David J Elliott1*

Author affiliations

1 Institute of Human Genetics, Newcastle University, Central Parkway, Newcastle-upon-Tyne, NE1 3BZ, UK

2 Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK

3 IGBMC Department of Functional Genomics, Illkirch, F-67400, France

4 INSERM U596, Illkirch, F-67400, France

5 CNRS UMR 7104, Illkirch F-67400, France

6 University of Strasbourg, Strasbourg, F-67000, France

7 Institute of Biochemistry, Department of Biology and Chemistry, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

8 North East Proteome Analysis Facility, Devonshire Building, Newcastle University, Devonshire Terrace, Newcastle-upon-Tyne, NE1 7RU, UK

9 Northern Institute for Cancer Research, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK

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Citation and License

BMC Cell Biology 2009, 10:82  doi:10.1186/1471-2121-10-82

Published: 13 November 2009

Abstract

Background

Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids.

Results

We used proteomics to search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. The interaction between Sam68 and hnRNP L proteins was observed in a cell line which exhibits low frequency of SNBs suggesting that this association also takes place outside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons.

Conclusion

Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs.