Gene expression and cell growth are modified by silencing SUMO2 and SUMO3 expression

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Abstract

Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. Here we report that blocking SUMO2 and SUMO3 conjugation by silencing their expression markedly modifies gene expression. A microRNA-based RNAi system was used to specifically silence SUMO2 and SUMO3 expression simultaneously and stably transfected neuroblastoma B35 cells expressing dual SUMO2/3 microRNA were created. In cells stably expressing SUMO2/3 microRNA, mRNA levels of 105 and 58 known genes were significantly up- and down-regulated, respectively. About 20% of differentially regulated genes were associated with pathways involved in cell growth and differentiation. Cell division was significantly suppressed in SUMO2/3 miRNA expressing cells. Elucidating what effect the silencing of SUMO2/3 expression has on gene expression will help to identify the impact of SUMO2/3 conjugation on the various cellular pathways.

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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