Similar Regulation of Human Inducible Nitric-oxide Synthase Expression by Different Isoforms of the RNA-binding Protein AUF1

Abstract

The ARE/poly-(U) binding factor 1 (AUF1), a protein family consisting of four isoforms, is believed to mediate mRNA degradation by binding to AU-rich elements (ARE). However, evidence exists that individual AUF1 isoforms may stabilize AREcontaining mRNAs. The 3-untranslated region of the human inducible nitric-oxide synthase (iNOS) contains five AREs, which promote RNA degradation. We have recently shown that the RNA-binding protein KSRP is critically involved in the decay of the iNOS mRNA. In this study we examined the effects of the individual AUF1 isoforms on iNOS expression. Overexpression of each AUF1 isoform reduces iNOS expression on mRNA and protein levels to the same extent by modulation of mRNA stability. Accordingly, knockdown of all or individual AUF1 isoforms by an RNA interference approach enhances iNOS expression. The AUF1 effect on iNOS expression is dependent on the iNOS 3-untranslated region sequence, as demonstrated in transfection experiments with a reporter mRNA. Binding studies showed that all AUF1 isoforms interact with the same AU-rich region in the iNOS-3-untranslated region. Cytokine stimulation altered intracellular AUF1 binding activities. These data demonstrate that AUF1 is an important factor that promotes iNOS mRNA degradation. Furthermore, all individual AUF1 isoforms act in a similar manner.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells

“…The attenuation of the NFκB signalling resulted in reduced induction of iNOS and diminished production of the chemokines, CXCL2/MIP2 and CCL2/MCP1. In other cell types, AUF1 destabilises iNos mRNA [17]. Although our data did not reach statistical significance, in MIN6 cells treated with siRNAs directed against AUF1, iNos mRNA tended to be more stable (ESM Fig.…”

“…AUF1 overproduction and downregulation To experimentally increase AUF1 levels, MIN6 cells were transiently transfected with plasmids expressing enhanced green fluorescent protein (EGFP)-labelled constructs of each individual AUF1 isoform [17]. Reduction of the level of the selected isoforms was achieved using siRNA duplexes directed against: exon 2, targeting p45 and p40 (siAUF1 p45p40 ); exon 7, targeting p45 and p42 (siAUF1 p45p42 ); and a sequence astride exon 3 and 4, targeting all isoforms (siAUF1 all ).…”

The regulation of mRNA stability in mammalian cells: 2.0

“…By contrast, for p16 INK4 mRNA, HuR and AUF1 act as cofactors to promote mRNA degradation (Chang et al, 2010). Regulation of inducible nitric oxide synthase ( iNOS ) expression involves five RBPs: AUF1, HuR, KSRP, polypyrimidine tract-binding protein (PTB), and TTP (Fechir et al, 2005; Linker et al, 2005; Pautz et al, 2006; Pautz et al, 2009). TTP does not bind the ARE directly, but rather, it binds KSRP, which does bind the ARE and competes with HuR for binding the same site within the ARE.…”

Alternatively Expressed Domains of AU-rich Element RNA-binding Protein 1 (AUF1) Regulate RNA-binding Affinity, RNA-induced Protein Oligomerization, and the Local Conformation of Bound RNA Ligands

“…Suppression of AUF1 using siRNA-based approaches is most frequently associated with stabilization of mRNA substrates, consistent with a general mRNA-destabilizing role for at least some AUF1 proteins (17,64,65). However, ectopic overexpression of AUF1 proteins has prompted much more variable conclusions, associated with both mRNA-destabilizing (18) and -stabilizing (66) roles.…”