Biochemical and Biophysical Research Communications
Gene expression and cell growth are modified by silencing SUMO2 and SUMO3 expression
Introduction
Small ubiquitin-like modifier (SUMO) conjugation is a post-translational protein modification that was identified 12 years ago [1]. Four SUMO paralogues have been identified, denoted SUMO1–4. Of these, SUMO2 and SUMO3 are highly homologous and are, therefore, referred to as SUMO2/3. SUMO is conjugated to the ε amino group of lysine residues of target proteins thereby modifying their activity, stability and cellular localization. The SUMO conjugation pathway is massively activated in cells exposed to various stress conditions of high clinical relevance, including oxidative stress, thermal stress (hypo- and hyper-thermia), and transient cerebral ischemia [2], [3], [4], [5], [6], [7], [8]. It is, therefore, of key interest to us to understand the possible significance of the activation of the SUMO conjugation pathway in determining the fate of cells exposed to stressful conditions.
Proteomic analyses have revealed that many of the SUMO conjugation target proteins are transcription factors or other nuclear proteins modulating gene expression [9]. Any marked change in levels of SUMO conjugated proteins can, therefore, be expected to have a major impact on gene expression. To date no information has been published on the effect of SUMO conjugation on global gene expression. One strategy towards elucidating how gene expression is modified by SUMO conjugation is to silence SUMO expression and perform genomic analyses. We decided to focus on SUMO2/3 conjugation, because this pathway is dramatically activated both in-vitro and in-vivo in cells exposed to various forms of cellular stress conditions. We have created stably transfected cell lines expressing designed microRNA (miRNA) that effectively silence SUMO2/3 expression, and have analyzed the effects of silencing SUMO2/3 expression on the cellular transcription pattern and cell proliferation.
Section snippets
Materials and methods
Experiments were performed on B35 cells, a neuroblastoma cell line [10]; by courtesy of Dr. P.F. Maness, University of North Carolina, Chapel Hill, USA. To produce miRNA-based RNAi vectors, the pcDNA6.2-GW/EmGFP-miR (Invitrogen) containing enhanced green fluorescent protein (EGFP) and a blasticidin resistance cassette was used as vector backbone. We screened for effective target sequences in transient transfection experiments. The miRNA expression vectors miR-SUMO2 and miR-SUMO3 containing the
Results
First, we screened various SUMO2 and SUMO3 pre-miRNA sequences to identify the most effective one in silencing SUMO2 and SUMO3 expression, respectively. B35 cells were transiently co-transfected with miR-SUMO2 or miR-SUMO3 constructs and plasmids expressing HA-tagged SUMO2 or SUMO3, respectively. An example of this miRNA sequence screening is illustrated in Fig. 1A. Cells were transfected with HA-SUMO3 and constructs expressing miR-Neg, miRNA related to SUMO2 (miR-SUMO2) and two miRNA sequences
Discussion
One strategy towards identifying genes where the expression is modified by SUMO conjugation is to elucidate the effects of silencing of SUMO expression on global gene expression. Whereas conjugated forms of mouse SUMO2 and SUMO3 share 96.7% amino acid identity, they have only 69.3% nucleotide sequence identity. The miRNA approach used in the study enabled us to create a dual miRNA silencing vector miR-SUMO2/3 expressing both designed miR-SUMO2 and miR-SUMO3. Using this construct, we have
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