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. 2014 Sep 12;289(37):25879-89.
doi: 10.1074/jbc.M114.560128. Epub 2014 Jul 23.

Progranulin transcripts with short and long 5' untranslated regions (UTRs) are differentially expressed via posttranscriptional and translational repression

Anja Capell  1 , Katrin Fellerer  2 , Christian Haass  3
Affiliations

Progranulin transcripts with short and long 5' untranslated regions (UTRs) are differentially expressed via posttranscriptional and translational repression

Anja Capell et al. J Biol Chem. .

Abstract

Frontotemporal lobar degeneration is associated with cytoplasmic or nuclear deposition of the TAR DNA-binding protein 43 (TDP-43). Haploinsufficiency of progranulin (GRN) is a major genetic risk factor for frontotemporal lobar degeneration associated with TDP-43 deposition. Therefore, understanding the mechanisms that control cellular expression of GRN is required not only to understand disease etiology but also for the development of potential therapeutic strategies. We identified different GRN transcripts with short (38-93 nucleotides) or long (219 nucleotides) 5' UTRs and demonstrate a cellular mechanism that represses translation of GRN mRNAs with long 5' UTRs. The long 5' UTR of GRN mRNA contains an upstream open reading frame (uORF) that is absent in all shorter transcripts. Because such UTRs can be involved in translational control as well as in mRNA stability, we compared the expression of GRN in cells expressing cDNAs with and without 5' UTRs. This revealed a selective repression of GRN translation and a reduction of mRNA levels by the 219-nucleotide-long 5' UTR. The specific ability of this GRN 5' UTR to repress protein expression was further confirmed by its transfer to an independent reporter. Deletion analysis identified a short stretch between nucleotides 76 and 125 containing two start codons within one uORF that is required and sufficient for repression of protein expression. Mutagenesis of the two AUG codons within the uORF is sufficient to reduce translational repression. Therefore initiating ribosomes at the AUGs of the uORF fail to efficiently initiate translation at the start codon of GRN. In parallel the 5' UTR also affects mRNA stability; thus two independent mechanisms determine GRN expression via mRNA stability and translational efficiency.

Keywords: Amyotrophic Lateral Sclerosis (ALS) (Lou Gehrig Disease); Frontotemporal Lobar Degeneration; Neurodegeneration; Neurodegenerative Disease; Neuroinflammation; Progranulin; Protein Expression; TAR DNA Binding Protein 43; Translational Control.

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Figures

FIGURE 1.
FIGURE 1.
Annotated variants of the GRN 5′ UTR. A, sequence of the 219 nucleotides of the GRN 5′ UTR derived from human brain cDNA. This annotated variant (NM002087) and start sites of predicted shorter variants (XM005257255 and XM005257254) are indicated, as well as a predicted splice variant (XM005257253) containing a short sequence stretch of exon 1, followed by an insertion of 271 nucleotides of intron 1 (green box) at the exon 1/2 boundary. 5′ ends identified by rapid amplification of cDNA ends are marked by arrows. Green arrows indicate mRNAs containing the intron 1-derived sequence stretch. ORFs are highlighted in red, and AUG start codons are circled. B, schematic of the 219-nucleotide-long GRN 5′ UTR. Two AUG initiation codons (positions 90 and 120), the corresponding uORF, and the stop codon (nucleotides 159–161) are indicated. C, schematic of the alternative spliced GRN 5′ UTR (XM005257253). This splice variant contains a short stretch of exon 1 (nucleotides 190–212), followed by 271 nucleotides derived from intron 1 containing two AUGs (circled) and one short uORF (156–176).
FIGURE 2.
FIGURE 2.
GRN expression is inhibited by its 5′-UTR. A, cDNA constructs used to evaluate the effects of the 5′ and the 3′ UTR on GRN expression. B, isogenic expression of the cDNA constructs shown in A in HEK 293 cells. GRN expression was investigated in cell lysates by Western blotting using an anti-myc or anti-GRN antibody. Actin was used as loading control. Quantitation of the expression levels of GRN in cell lysates and of secreted GRN is shown in the bottom panel. Levels are normalized to the expression of the 5′-3′ UTR. Secreted GRN was analyzed using an ELISA assay described previously (14). For all quantifications, data are mean ± S.D. (n ≥ 3 independent experiments). ****, p < 0.0001, n.s., not significant. ANOVA followed by Tukey's multiple comparison test was used to determine significance.
FIGURE 3.
FIGURE 3.
The GRN 5′ UTR inhibits expression of an EGFP reporter. A, schematic showing cDNA constructs used to evaluate the effects of the 5′ UTR, 3′ UTR, and both 5′3′ UTR of GRN on EGFP expression. To allow secretion of EGFP, the coding sequence of EGFP was fused to the signal peptide (SP) derived from GRN (SPEGFP). B, isogenic expression of SPEGFP variants with and without the GRN 5′ UTR, 3′ UTR, and 5′3′ UTR. Actin was used as a loading control. Bottom panel, expression levels of SPEGFP in cell lysates and corresponding media were quantified. All quantifications were normalized to 5′3′ UTR constructs. Data are mean ± S.D. (n ≥ 3 independent experiments). **, p < 0.01; ****, p < 0.0001; n.s., not significant. ANOVA followed by Tukey's multiple comparison test was used to determine significance.
FIGURE 4.
FIGURE 4.
Inhibition of GRN expression occurs at the translational and mRNA levels. A, mRNAs encoding the indicated GRN variants were transcribed in vitro. B, equal amounts of in vitro transcribed mRNAs (∼1 μg, adjusted to the number of nucleotides per individual mRNA) were used for in vitro translation. Note that the 3′ UTR does not affect GRN translation, whereas the 5′ UTR strongly represses GRN translation. Bottom panel, quantitation of the expression levels of GRN upon in vitro translation of equal amounts of mRNA normalized to expression of the 5′3′ UTR cDNA construct. C, quantitation of GRN mRNA in stable cell lines expressing the indicated cDNA constructs. Levels are normalized to expression of the 5′3′ UTR cDNA construct. Data are mean ± S.D. (n ≥ 3 independent experiments). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant. Note that the 5′ and 3′ UTR contributes to a destabilization of GRN mRNA in cells. ANOVA followed by Tukey's multiple comparison test was used to determine significance.
FIGURE 5.
FIGURE 5.
Identification of a 50-nucleotide sequence that is sufficient to repress GRN expression. A, schematic of the sequential deletions constructed to identify the sequence responsible for translational inhibition within the 5′ UTR. B, isogenic expression of the deletion constructs shown in A reveals a sequence between nucleotides 75 and 125 to be sufficient for translational repression of GRN. Bottom panel, quantitation of the expression levels of various deletion constructs normalized to the 5′ UTR construct. Data are mean ± S.D. (n ≥ 3 independent experiments). ***, p < 0.001; ****, p < 0.0001; n.s., not significant. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test.
FIGURE 6.
FIGURE 6.
A 50-nucleotide stretch within the 5′ UTR of GRN is required for translational inhibition and lowering of mRNA levels. A, schematic of investigated cDNA constructs. B, deletion of nucleotides 75–125 (Δ75–125) is sufficient to abolish repression of GRN expression, whereas mutagenesis of both in-frame AUGs within the 5′ UTR increases GRN expression to a significantly lower extent. Bottom panel, quantitation of the expression levels of the GRN variants shown in the top panel. Expression is normalized to the 5′ UTR cDNA construct. Data are mean ± S.D. (n ≥ 3 independent experiments). *, p < 0.05; ****, p < 0.0001; n.s., not significant. C, quantitation of GRN mRNA in cell lines expressing the indicated cDNA constructs. Levels are normalized to expression of the 5′ UTR cDNA construct. Data are mean ± S.D. (n ≥ 3 independent experiments). ****, p < 0.0001; n.s., not significant. Note that deletion of nucleotides 75–125 (Δ75–125) is sufficient to block the reduction of mRNA levels, whereas mutation of the two AUGs within the 5′ UTR fails to stabilize mRNA levels. D, equal amounts of mRNAs transcribed in vitro encoding the indicated GRN variants were translated in vitro. For quantitation, levels of in vitro translated GRN protein were normalized to the 5′ UTR variant. Deletion of nucleotides 75–125 (Δ75–125) as well as mutagenesis of the two AUGs within the 5′ UTR leads to a complete loss of translational inhibition. Data are mean ± S.D. (n ≥ 3 independent experiments). ***, p < 0.001; n.s., not significant. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test.
FIGURE 7.
FIGURE 7.
AUGs in the uORF are responsible for translational repression of GRN but not for reducing mRNA levels. A, schematic of the investigated cDNA constructs. B, isogenic expression of the cDNA constructs shown in A. Actin was used to verify equal loading. Bottom panel, quantitation of the expression levels of the GRN variants normalized to the 5′ UTR. Levels are normalized to expression of the 5′ UTR cDNA construct. Data are mean ± S.D. (n ≥ 3 independent experiments). ***, p < 0.001; ****, p < 0.0001; n.s., not significant. C, quantitation of GRN mRNA in cell lines expressing the indicated cDNA constructs normalized to the 5′ UTR construct. Data are mean ± S.D. (n ≥ 6 independent experiments). *, p < 0.05; ****, p < 0.0001. D, mRNAs encoding the indicated GRN variants were transcribed in vitro, and equal amounts of in vitro transcribed mRNAs (∼1 μg, adjusted to the number of nucleotides per individual mRNA) were used in E for in vitro translation. Bottom panel, quantitation of the expression levels of GRN upon in vitro translation normalized to expression of the 5′ UTR cDNA construct. Data are mean ± S.D. (n ≥ 3 independent experiments). ****, p < 0.0001; n.s., not significant. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test.
FIGURE 8.
FIGURE 8.
Both AUGs in the uORF can initiate translation. A, schematic of the uORF in-frame with EGFP containing a mutated start codon. The two mutated uORF start codons are indicated (AUA1 and AUA2). B, transient expression of EGFP cDNA or cDNA constructs containing nucleotides 76–158 of the 5′ UTR including both AUGs (76–158), either one (76–158AUA1 and 76–158AUA2), or both (76–158AUA1,2) mutated AUGs. Actin was used as a loading control. Note that both start codons of the uORF are functional because they are capable to initiate protein translation. The top and bottom bands represent proteins translated from the first and second AUG of the uORF. When both AUGs are mutated, no translation product can be detected.
FIGURE 9.
FIGURE 9.
A novel alternatively spliced variant containing two AUGs within its 5′ UTR also represses GRN expression. A, schematic of the GRN 5′ UTR splice variant. B, isogenic expression of GRN variants in the presence and absence of the 5′ UTR or the alternative 5′alt UTR. Actin was used as a loading control. Bottom panel, quantitation of the expression levels of GRN. Data are mean ± S.D. (n ≥ 3 independent experiments). ***, p < 0.001; ****p < 0.0001; n.s., not significant. C, quantitation of GRN mRNA in cell lines expressing the indicated cDNA constructs normalized to the 5′ UTR construct. Data are mean ± S.D. (n ≥ 3 independent experiments). *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. D, mRNAs encoding the indicated GRN variants were transcribed in vitro, and equal amounts of in vitro transcribed mRNAs (∼1 μg, adjusted to the number of nucleotides per individual mRNA) were used for in vitro translation. E, bottom panel, quantitation of expression levels of GRN upon in vitro translation of equal amounts of mRNA. For all quantifications, data are mean ± S.D. (n = 3 independent experiments). ***, p < 0.001; n.s., not significant. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. Note that the 5′alt UTR efficiently represses GRN expression.
FIGURE 10.
FIGURE 10.
Model explaining the dual mechanism of GRN expression control via translational inhibition and lowering of mRNA levels. Translational inhibition may be due to stalled ribosomes, leaky scanning, and/or inefficient reinitiation at the authentic start codon of GRN.
FIGURE 11.
FIGURE 11.
Predicted secondary structure of the GRN 5′1–219 UTR (A) and the 5′alt UTR (B). Shown is the centroid structure encoding base pair probabilities. The bases are colored violet (0) for low and red (1) for high base-pairing probabilities. For the structure prediction, RNAfold 2.0, provided by ViennaRNA Web Services, was used. The start codons within the 5′ UTR are indicated by arrows.
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