Upf1/Upf2 regulation of 3' untranslated region splice variants of AUF1 links nonsense-mediated and A+U-rich element-mediated mRNA decay

Abstract

AUF1 is an RNA-binding protein that targets mRNAs containing A+U-rich elements (AREs) for rapid cytoplasmic turnover. Alternative pre-mRNA splicing produces five variants of AUF1 mRNA that differ in the composition of their 3'-untranslated regions (3'-UTRs). Previous work suggested that this heterogeneity in 3'-UTR sequence could regulate AUF1 expression by two potential mechanisms. First, AUF1 may regulate its own expression by binding to AREs in 3'-UTR splice variants that retain intron 9. The second potential mechanism, and the focus of this report, is regulation of a subset of 3'-UTR splice variants by the nonsense-mediated mRNA decay (NMD) pathway. Two of the five AUF1 mRNA 3'-UTR variants position the translational termination codon more than 50 nucleotides upstream of an exon-exon junction, creating a potential triggering signal for NMD in mammalian cells. Disruption of cellular NMD pathways by RNA interference-mediated knockdown of Upf1/Rent1 or Upf2/Rent2 or transfection of a dominant-negative Upf1 mutant specifically enhanced expression of these two candidate NMD substrate mRNAs in cells, involving stabilization of each transcript. Ribonucleoprotein immunoprecipitation experiments revealed that both Upf1 and Upf2 can associate with an NMD-sensitive AUF1 mRNA 3'-UTR variant in cells. Finally, quantitation of AUF1 mRNA 3'-UTR splice variants during murine embryonic development showed that the expression of NMD-sensitive AUF1 mRNAs is specifically enhanced as development proceeds, contributing to dynamic changes in AUF1 3'-UTR structures during embryogenesis. Together, these studies provide the first evidence of linkage between the nonsense- and ARE-mediated mRNA decay pathways, which may constitute a new mechanism regulating the expression of ARE-containing mRNAs.

FIG. 1.

Potential splicing variants of the AUF1 3′-UTR. Five possible AUF1 3′-UTR splice variants are shown based upon the exon-intron organization of the AUF1 gene. Arrows below the diagrams of variant mRNAs I to IV depict the locations of forward and reverse primers for qRT-PCR amplification of each specific splice variant.

FIG. 2.

Effects of Upf1 reduction on expression of endogenous AUF1 3′-UTR splice variants. (A) Western blot analysis of Upf1 levels. A two-hit strategy was used to transfect control or Upf1-specific siRNA into HeLa cells. A parallel culture was cotransfected with a plasmid encoding an siRNA-resistant Upf1 cDNA (Upf1^R) and Upf1 siRNA as described in Materials and Methods. A twofold dilution series of HeLa cytoplasmic lysate was probed with antibodies for Upf1 and α-tubulin to permit estimations of the Upf1 knockdown efficiency (left). Estimates of Upf1 protein levels in the cytoplasm of transfected cells are expressed as percentages of Upf1 in cells transfected with control siRNA (right). n.d., not detectable. (B) qRT-PCR analysis of changes in Upf1 mRNA levels resulting from two-hit transfection of Upf1 siRNA, with or without cotransfected Upf1^R, relative to control siRNA transfection, expressed as means ± SD (n = 3). **, P < 0.01 versus control siRNA. (C) Upf1-dependent changes in expression of endogenous AUF1 3′-UTR splice variants. Total RNA was isolated from the transfected cells described in panel A or from cells transfected with a single hit of Upf1 siRNA and analyzed by qRT-PCR, using primer pairs specific for individual AUF1 3′-UTR splice variants (see Fig. 1). The bars indicate the means ± SD (n = 4), where the level of each AUF1 variant mRNA is relative to that measured in cells transfected with the control siRNA (dotted line) **, P < 0.01 versus control siRNA. The same RNA samples were utilized to perform the qRT-PCRs shown in panels B and C.

FIG. 3.

Identification of AUF1 3′-UTR elements required for regulation by Upf1. Schematics of firefly luciferase-AUF1 3′-UTR chimeric mRNAs are shown on the left and are numbered for text reference. AUF1 exon and intron sequences are labeled, and the ABS within intron 9 are indicated by a black box. In constructs where intron 9 cannot be excised by splicing, the intron sequence is shaded. HeLa cells were first transfected with control or Upf1-specific siRNA, followed 48 h later by cotransfection of luciferase-AUF1 3′-UTR constructs and a Renilla luciferase control vector. Two days following luciferase transfections, firefly and Renilla luciferase mRNA and activity levels were measured as described in Materials and Methods. For each chimeric mRNA, bars represent the means ± SD (n = 3) of firefly luciferase mRNA levels (middle panel) or activities (right panel) relative to those for the control siRNA transfection following normalization to Renilla luciferase mRNA and activity, respectively. *, P < 0.05; **, P < 0.01 versus control siRNA.

FIG. 4.

Control of luciferase-AUF1 3′-UTR splice variant expression by a dominant-negative Upf1 mutant. Schematics of firefly luciferase-AUF1 3′-UTR chimeric mRNAs are shown as described in the legend to Fig. 3 (left). HeLa cells were cotransfected with luciferase-AUF1 3′-UTR constructs and a Renilla luciferase control vector in the absence (white bars) or presence of an expression vector encoding wild-type Upf1 (gray bars) or the dominant-negative Upf1 R844C mutant (black bars). At 2 days posttransfection, firefly and Renilla luciferase mRNA and activity levels were analyzed as described in Materials and Methods. For each reporter construct, bars represent the means ± SD (n = 3) of firefly luciferase mRNA levels (middle panel) or activities (right panel) relative to those for cotransfections lacking ectopic Upf1 (or the R844C mutant) following normalization to Renilla luciferase mRNA and activity, respectively. *, P < 0.05; **, P < 0.01 versus control.

FIG. 5.

Effects of Upf2 reduction on expression of endogenous AUF1 3′-UTR splice variants. (A) Western blot analysis of Upf2 levels. A two-hit strategy was used to transfect control or Upf2-specific siRNA into HeLa cells or to cotransfect a combination of Upf2 siRNA and siRNA-resistant Upf2 cDNA (Upf2^R) as described in Materials and Methods. Estimation of the Upf2 knockdown efficiency was performed by probing immunoblots of cytoplasm from each transfected cell population with antibodies for Upf2 and α-tubulin (right panel) and by comparison to a twofold dilution series of cytoplasmic lysate from nontransfected HeLa cells (left panel). Estimates of Upf2 protein levels in each cell population are expressed as percentages of the Upf2 in cells transfected with control siRNA. (B) Changes in Upf2 mRNA levels resulting from two-hit transfection of Upf2 siRNA, with or without cotransfected Upf2^R, relative to those with control siRNA, measured by qRT-PCR and expressed as means ± SD (n = 3). **, P < 0.01 versus control siRNA. (C) Upf2-dependent changes in expression of endogenous AUF1 3′-UTR splice variants. Total RNA was isolated from the transfected cells described in panel A or from cells transfected with a single hit of Upf2 siRNA and analyzed for individual AUF1 3′-UTR variant mRNAs by qRT-PCR. The bars indicate the means ± SD (n = 4), where the level of each mRNA variant is shown relative to that measured in cells transfected with the control siRNA (dotted line). **, P < 0.01 versus control siRNA. The same RNA samples were utilized to perform the qRT-PCRs shown in panels B and C.

FIG. 6.

Association of Upf1 and Upf2 with AUF1 3′-UTR sequences. (A) Analyses of Upf1 association. HeLa cells were transiently transfected with plasmid pFLAG-Upf1 or a control vector lacking Upf1 sequences (pFLAG), together with luciferase reporter vectors lacking (pGL3-Promoter) or containing (pGL3-Ex9:In9:Ex10) the AUF1 exon 9-intron 9-exon 10 sequence downstream of the luciferase coding region. At 2 days posttransfection, whole-cell lysates were prepared and fractionated by ribonucleoprotein immunoprecipitation (IP), using anti-FLAG antibodies or control mouse IgG (mIgG) as described in Materials and Methods. Immunoprecipitated material was analyzed by Western blotting to validate anti-FLAG-dependent recovery of FLAG-Upf1 (top), while RT-PCR was used to identify copurifying firefly luciferase mRNA (bottom). Lane 1 is a 100-bp ladder, and the position of the 500-bp marker is noted to the left of the panel. (B) Analyses of Upf2 association. HeLa cells were cotransfected with plasmid pT7-Upf2 and either pGL3-Promoter or pGL3-Ex9:In9:Ex10. Cell lysates were prepared and immunoprecipitated, using control IgG or anti-T7-tag antibody. Immunoprecipitated material was analyzed by Western blotting for Upf2 and by RT-PCR for luciferase mRNA, essentially as described for panel A.

FIG. 7.

Decay kinetics of endogenous AUF1 3′-UTR splice variants following reduction of Upf1 or Upf2. The turnover rates of endogenous AUF1 3′-UTR variant I, II, III, and IV mRNAs were measured using DRB time course assays following two-hit transfections of control (solid circles, solid lines), Upf1 (open circles, dotted lines), or Upf2 (triangles, dashed lines) siRNA into HeLa cells. Levels of each AUF1 3′-UTR mRNA variant were normalized to that of GAPDH mRNA and plotted as the percent AUF1 variant mRNA remaining as a function of time following DRB treatment. Nonlinear regression analysis yielded first-order decay constants (k) and associated cellular mRNA half-lives, which are listed in Table 1.

FIG. 8.

Dynamic expression of AUF1 3′-UTR splice variants during murine embryogenesis. Total RNA was purified from C57BL/6 embryos at developmental stages E5.5, E9.5, E13.5, and E16.5 as described in Materials and Methods. qRT-PCR was performed using RNAs from each developmental stage to quantify mRNA levels of AUF1 splice variant II (A), variant III (B), and variant I (C). Bars represent the means ± SD for levels of each AUF1 mRNA variant measured from four separate embryos at each stage, normalized to GAPDH mRNA and shown relative to the expression of each AUF1 variant mRNA at stage E5.5. *, P < 0.05; **, P < 0.01.

FIG. 9.

Model for regulation of AUF1 expression and possible downstream pathways by NMD. NMD was first described as a mechanism to prevent the synthesis of truncated proteins by degrading mRNAs containing PTCs. Emerging models indicate that NMD may also regulate a variety of naturally occurring mRNAs, including AUF1 mRNA, as demonstrated in the current work. By controlling the production of cellular AUF1, NMD may thus indirectly influence a broad variety of biological pathways regulated through the various AUF1 isoforms, including telomere maintenance, cell growth, extracellular and intracellular signaling, and possibly many others. This model is discussed further in the text.