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

Published: 13 November 2009

Additional files

Additional file 1:

Sam68 is efficiently immunoprecipitated by its cognate antisera. LNCaP cell nuclear extracts were subjected to immunoprecipitation (IP) using anti-Sam68 rabbit antisera. IP was carried out in the presence or absence (+/-) of antisera to Sam68 or normal rabbit IgG (negative control), with or without (+/-) Dynabeads Protein A, and either with or without (+/-) prior cross-linking of the antisera to Sam68 to the Dynabeads Protein A. Recovered material was subjected to Western analysis with the antisera to Sam68. Western analysis confirmed that Sam68 protein was efficiently immunoprecipitated by its cognate antisera, and that this was most efficient if the antisera were cross-linked to the Dynabeads Protein A prior to incubation with nuclear extracts (compare lanes 3 and 4). Sam68 protein was not pulled down by Dynabeads Protein A alone (lane 2).

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Additional file 2:

hnRNPs A1 or A2/B1 do not localise within SNBs. (A to D) Representative indirect immunofluorescence images of HeLa cells captured by confocal laser scanning microscopy using antibodies to hnRNPs A1, A2/B1, hnRNP L, hnRNP LL, and Sam68. hnRNPs A1 (A) and A2/B2 (B) exhibit a diffuse nucleoplasmic distribution but do not co-localise with Sam68 to SNBs (arrowed). (C) Sam68 co-localises with hnRNP L to SNBs (arrowed). (D) hnRNP LL exhibits a different subnuclear localisation to hnRNP L and does not co-localise to SNBs. (Bar = 10 μm).

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Additional file 3:

hnRNP G represses Sam68-mediated CD44 variable exon v5 inclusion. (A) Minigene pETv5 contains the CD44 variable exon v5 cloned downstream of a constitutively-active RSV promoter. Splicing events are shown by broken lines, and arrows show the location of primers for RT-PCR. (B) HEK293 cells were transfected with the pETv5 minigene (150 ng), and expression vectors for hnRNP G or GFP-Sam68 (500 ng). The gel images are representative of at least three independent experiments, from which densitometric assessment of RT-PCR product was performed to obtain means +/- standard error (shown in bar chart). The lane marked M shows the migration of the 1 Kb plus DNA ladder (Invitrogen). Ectopic expression of hnRNP G protein repressed CD44 variable exon v5 inclusion both in the presence and absence of ectopic Sam68 (compare lane 1 with lane 3, and lane 2 with lane 4).

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