Regulation of Unr Expression by 5'- and 3'-Untranslated Regions of its mRNA through Modulation of Stability and IRES Mediated Translation

Abstract

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein that can act as a regulator of mRNA stability and IRES-mediated translation. Unr, a member of the cold-shock domain (CSD) protein super-family, is ubiquitously expressed, with variable abundance, in different tissues or during embryonic development. Prokaryotic and eukaryotic coldshock protein expression is highly regulated at both the transcriptional and post-transcriptional levels. Here we analyzed the role of the 5'-and 3'-untranslated regions (UTR) of unr mRNA in post-transcriptional regulation of Unr expression. We show that, in vitro, unr 3'-UTR specifically destabilizes unr transcripts. Accordingly, in vivo, the half-life of unr messages deleted of noncoding regions is increased by ~3.6 fold, resulting in an enhanced steady-state level of Unr protein. We also show that the 5'-UTR exhibits IRES activity both when translated in vitro and in transiently transfected cells. This IRES activity displays cell type specificity with a higher efficiency in HeLa and HuH7 than in ES cells. Moreover, Unr IRES activity was higher in unr-/than in unr +/+ ES cells, indicating that Unr negatively regulates its own IRES activity. Our studies further reveal that Unr specifically interacts with its own mRNAs in vivo. These results suggest that a feedback control of mRNA translation is involved in regulating Unr expression.

Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells

“…Unr contains an IRES element located in the 5 0 untranslated region (UTR) of unr mRNAs (Dormoy-Raclet et al, 2005), which could maintain Unr synthesis under stress conditions (Carter and Sarnow, 2000;Henis-Korenblit et al, 2000). Finally, Unr activity, as determined by its capacity to stimulate the poliovirus IRES activity (Boussadia et al, 2003), was unaffected following irradiation of ES cells.…”

“…For each gene, real-time PCR reactions were performed in triplicate using the iCycler IQ real-time PCR detection system (Bio-Rad, Marnes la coquette, France), as described previously (Dormoy-Raclet et al, 2005). Sequences of primers used in Q PCR were mCaspase-3 (L: 5 0 -GAG ATG GCT TGC CAG AAG AT-3 0 , R: 5 0 -TAA CGC GAG TGA GAA TGT GC-3 0 ), mp53 (L: 5 0 -AAGTCCTTTGCCCTGAACTG-3 0 , R: 5 0 -CTGTAGCATG GGCATCCTTT-3 0 ), mMrpl27 (L: 5 0 -TGG CAC TCA GCG TCA GTT TA-3 0 , R: 5 0 -GGG CAC GTA GAC ATC TTT CG-3 0 ), and mGadd45g (L: 5 0 -GTT GAT TCA GGC GTT CTG CT-3 0 , R: 5 0 -GCT CTC CTC GCA GAA CAA AC-3 0 ).…”

Functional domains of Drosophila UNR in translational control

“…In both cases, UNR interacts with PABP within complexes that bind to distinct regions in the target transcripts. Mammalian UNR also regulates translation driven by the internal ribosome entry sites (IRESs) of a number of viral and cellular transcripts, including rhinovirus, poliovirus, c-myc, PITSLRE protein kinase, the pro-apoptotic factor Apaf-1, and UNR itself (Hunt et al 1999;Boussadia et al 2003;Evans et al 2003;Mitchell et al 2003;Brown and Jackson 2004;Dormoy-Raclet et al 2005;Tinton et al 2005;Schepens et al 2007). At least in the case of Apaf-1, hUNR acts as an RNA chaperone, changing the conformation of the IRES to make it accessible to the activator PTB and, ultimately, the ribosome .…”

Global assessment of GU-rich regulatory content and function in the human transcriptome

“…This well-differentiated hepatoma cell line responds to a variety of pro-inflammatory cytokines and has been used for hepatitis, cytokine and post-transcriptional studies. [16][17][18][19] First, GFP reporter constructs fused to 3'UTRs containing natural GREs of the genes TGIF2, SMC5, CDK6, JUN and TNFRS1B were tested and showed enhanced mRNA decay (Fig. 4A).…”

“…It is hepatoma cell line that has been previously used for hepatitis, cytokine and post-transcriptional studies. [16][17][18][19] Transfection was performed with GFP reporter constructs with the indicated 3'UTRs using Lipofectamine 2000 reagent (Invitrogen). For RNA analysis experiments, transfections were ©2 0 1 1 L a n d e s B i o s c i e n c e .…”