Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms

Abstract

The A+U-rich element (ARE) in the 3' non-coding region (3' NCR) of short-lived cytokine mRNAs binds several regulatory proteins, including hnRNP D/AUF1, which comprises four isoforms of 37, 40, 42 and 45 kDa. ARE-mRNA degradation involves ubiquitin-proteasome activity, and one or more AUF1 proteins are thought to be ubiquitinated. Here we have characterized the mechanism for differential ubiquitination and degradation of the different AUF1 protein isoforms. We demonstrate in an in vitro ubiquitination system that the p37, followed by the p40 protein, are strongly ubiquitinated, whereas the p42 and p45 forms are not. Over expression in cells of enzymes that control the ubiquitin cycle were found to control p37 and p40 AUF1 protein levels through ubiquitination and proteasome activity, but not p42 and p45 forms. The p42 and p45 AUF1 proteins share a C-terminal exon 7 that is not found in the p37/p40 isoforms. Our studies show that exon 7 blocks ubiquitination and rapid degradation of AUF1 proteins, whereas its deletion permits ubiquitination to occur and promotes rapid turnover of AUF1 proteins. Thus, the stabilities of AUF1 isoforms are differentially controlled by insertion of an alternate exon that regulates ubiquitin targeting activity.

Figure 1

Structure of the four AUF1 protein isoforms. Shown is a diagram of the four isoforms of AUF1 representing the common core sequence containing RNA-binding domains 1 and 2 (RBD1 and RBD2) and a glutamine-rich element (Q), exons 2 and 7. Amino acid positions are provided relative to the core p37 AUF1 sequence. The amino acid sequence of exon 2 and exon 7 is provided below the p45 AUF1 isoform.

Figure 2

AUF1 is an in vivo substrate for ubiquitination. HeLa cells were transiently transfected with vector HA-UB₈, which synthesizes a polypeptide containing eight fused ubiquitins with an N-terminal HA tag, or vector alone. Cells were lysed in NP-40 lysis buffer containing 20 mM NEM, AUF1 protein or translation initiation factor eIF4E were immunoprecipitated from equal amounts of protein lysate using anti-AUF1 antibodies that recognize all four isoforms (p37, p40, p42 and p45), or anti-eIF4E antibodies, and samples were subjected to immunoblot analysis using anti-AUF1, anti-eIF4E or anti-HA antibodies. Polyubiquitin (HA-UB₈) conjugates are indicated.

Figure 3

In vitro ubiquitination of AUF1 isoforms. Individual AUF1 protein isoform mRNAs corresponding to p37, p40, p42 or p45 were transcribed and translated in vitro in a rabbit reticulocyte lysate with [³⁵S]methionine, equal labeled amounts of each AUF1 isoform were mixed with HeLa cell extract containing ubiquitin conjugating enzymes E1, E2 and E3 in the presence or absence of exogenous ubiquitin and an ATP regenerating system (40,41). Ubiquitin conjugation reactions were analyzed by SDS–PAGE and fluorography. Ubiquitination of AUF1 was detected by slower electrophoretic mobility, and results were quantified by digital densitometry. Typical results are shown.

Figure 4

Control of AUF1 protein levels by the ubiquitination reaction. Overexpression of deubiquitinating enzyme UBPY facilitates removal of polyubiquitin from degraded peptides and accelerates protein degradation by enhancing new rounds of ubiquitination. Overexpression of deubiquitinating enzyme UNP inhibits ubiquitin-dependent protein degradation by prematurely removing polyubiquitin from undegraded proteins. HeLa cells were transiently transfected with a UBPY or UNP expression vector or vector alone. Equal amounts of protein extracts were resolved by gel electrophoresis and immunoblotted with antisera to AUF1, eIF4E, p53 and UBPY or UNP. Lanes marked UBPY or UNP represent duplicate samples.

Figure 5

Half-life analysis of AUF1 protein isoforms. HeLa cells were metabolically labeled with [³⁵S]Met-Cys for 1 h followed by a chase with unlabeled medium for the times shown. When added, proteasome inhibitor MG132 was present throughout labeling and chase. AUF1 or eIF4E were immunoprecipitated from equal amounts of cell lysate, resolved by SDS–PAGE, fluorographed, and quantified by densitometry. Decay profiles were calculated by densitometry of autoradiograms and data plotted as log10 relative protein changes from time 0.

Figure 6

In vitro ubiquitination of AUF1 controlled by exon 7. p45 and p37 AUF1 protein isoform mRNAs were transcribed and translated in vitro in a rabbit reticulocyte lysate with [³⁵S]methionine, equal labeled amounts of each AUF1 isoform were immunoprecipitated with antibodies to AUF1 and mixed with the HeLa cell ubiquitin conjugating system. The p45ΔCTD mutant was deleted of the entire C-terminus, including exon 7. The p45Δ1/2CTD mutant was deleted of the C-terminal half of the CTD but retains exon 7. Ubiquitin conjugation reactions were analyzed by SDS–PAGE and fluorography. Ubiquitination of AUF1 was detected by slower electrophoretic mobility, and results were quantified by digital densitometry. Typical results are shown.