On-line Abstract Book

A Proteomic Approach for the Identification of Nuclear Proteins Involved in the Regulation of HIV-1 RNA Splicing

Alain Van Dorsselaer¹, Sarah Sanglier¹, Laetitia Fouillen¹, Virginie Marchand², Christelle Aigueperse², Iouri Motorine², Christiane Branlant²

Correspondence address: Alain Van Dorsselaer, CNRS - Louis Pasteur University, LSMBO, 25 rue Becquerel, Strasbourg, 67087 France.

Keywords: Complex; MS/MS, Liquid Chromatography; Proteomic; RNA.

Novel aspect: Identification of proteins complexes that interact with RNA.

 

Production of HIV-1 proteins depends upon a complex alternative splicing of the viral primary transcript. HIV-1 multiplication is strictly dependent on the use of some of the splicing sites (A2, A3, A4, A5, A7). It is thus highly important to study the molecular mechanisms involved in the regulation of HIV-1 RNA splicing.

The aim of this work is to use a systematic proteomic approach for the search of nuclear proteins involved in the regulation of HIV-1 RNA splicing. The proteomic approach developed is based on the fractionation of the complexes formed between a fragment of the viral RNA (SLS2) containing the splicing site A7 and a HeLa cell nuclear extract.
Protein complexes were isolated using the MS² purification method. After maltose elution of the complexes, the associated proteins were further separated by 1D gel electrophoresis. Protein identification by mass spectrometry was performed by combining peptide mass fingerprinting by MALDI-TOF and nanoLC-MS/MS analyses.
In agreement with previous studies, hnRNP A1 protein known to downregulate site A7 use was identified in the protein complexes. Unexpectedly, one of the identified tightly bound proteins was the nucleolin, an RNA binding protein highly abundant in nucleolus. Its direct binding on viral SLS2 RNA was demonstrated by gel-shift assays. Other hnRNP (K, Q, R and U) proteins were also identified in the proteins complexes. The possible implication of all these proteins in HIV-1 splicing regulation are under investigation.