Progranulin Transcripts with Short and Long 5′ Untranslated Regions (UTRs) Are Differentially Expressed via Posttranscriptional and Translational Repression*

Background: Haploinsufficiency of progranulin is a major cause of familial frontotemporal lobar degeneration. Results: Expression of progranulin is differentially repressed by a unique 5′ untranslated region. Conclusion: Expression of progranulin is tightly controlled at the translational and transcriptional level. Significance: Understanding the physiological regulation of progranulin expression may allow the development of therapeutic approaches to restore reduced levels of progranulin in patients. 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.

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.
Neurodegenerative diseases such as Alzheimer disease, Parkinson disease, and frontotemporal lobar degeneration (FTLD) 3 are a major threat to our aging society. Among them, FTLD is the second most common cause of dementia in patients under the age of 65 (1,2). FTLD is a disease spectrum known to show a major clinical and pathological overlap with amyotrophic lateral sclerosis (ALS) (3). Pathologically different subtypes of FTLD are defined by the deposition of disease-characterizing proteins (3). About 40% of FTLD patients develop tau-positive inclusions (FTLD-tau) (3). The remaining patients are characterized by tau-negative, ubiquitin-positive nuclear or cytoplasmic aggregates. TDP-43 is the most frequently deposited protein in tau-negative FTLD cases (FTLD-TDP) (4). TDP-43 is a nucleic acid binding protein that normally localizes to the nucleus. During the disease, TDP-43 is frequently deposited within the cytosol, accompanied by its nuclear clearance (4). The risk for FTLD-TDP is increased dramatically in patients with a haploinsufficiency for progranulin (GRN) (5,6). Genetic linkage analysis and exome sequencing identified a large number of non-sense mutations that all lead to GRN haploinsufficiency (7). In addition, missense mutations were also found (8 -10), which can lead to cytoplasmic missorting of GRN because of a dysfunctional signal peptide or to misfolding and, consequently, to a reduction of GRN secretion (7,11,12). Therefore, all FTLD-TDP-associated GRN mutations result in reduced GRN levels. Homozygous mutations in the GRN gene that cause a complete absence of GRN cause neuronal ceroid lipofuscinosis, suggesting that lysosomal dysfunction may also underlie, to some extent, FTLD pathology in patients with GRN haploinsufficiency (13). Interestingly, compounds that inter-fere with lysosomal acidification and autophagy, such as bafilomycin A1 or chloroquine, lead to a strong increase in GRN levels and may rescue GRN haploinsufficiency (14). GRN expression is increased not only during lysosomal stress but also in several other pathological conditions such as inflammation, wound healing, tumor genesis, and type 2 diabetes (7). The cellular mechanisms that allow the rapid and robust increase of GRN levels are poorly understood. However, upon interference with lysosomal function, posttranscriptional mechanisms appear to be involved (see, for example, Ref. 14).
Here we identified GRN transcripts with short (38 -93 nucleotides) and long (219 nucleotides) 5Ј UTRs. We have shown previously that translation of the ␤-amyloid precursor proteinprocessing enzymes ␤-secretase (␤-site amyloid precursor protein-cleaving enzyme 1 (BACE1)) and a disintegrin and metalloprotease 10 (ADAM10)) mRNAs are both repressed by their long 5Ј UTRs (15,16). Encouraged by these findings, we investigated whether GRN 5Ј UTRs of different lengths may differentially affect GRN translation and levels of GRN mRNA. We provide evidence that the 5Ј UTR of GRN contributes to translational repression and mRNA stability.

EXPERIMENTAL PROCEDURES
cDNA Constructs-The human GRN ORF and the 5Ј UTR and 3Ј UTR were amplified by PCR from HEK 293T cell cDNA and subcloned into the BamHI and EcoRV restriction sites of the inducible pcDNA5/FRT/TO (Hygro) expression vector for the Flp-In TM System (Life Technologies, Invitrogen, Darmstadt, Germany). GRN cDNA constructs were generated either by triple PCR or by site-directed mutagenesis (Stratagene, La Jolla, CA). All GRN cDNA constructs contained the endogenous Kozak consensus sequence (CAGACC) (17) in front of the start codon and a myc tag at the 3Ј end of the ORF containing a stop codon. The GRN 5Ј UTR with or without a GRN signal peptide was cloned in front of EGFP by triple PCR, and the final PCR product was subcloned into the BamHI and EcoRV restriction sites of the pcDNA5/FRT/TO (Hygro) expression vector. Note that, in addition to the indicated 5Ј UTR, the transcripts of all cDNA constructs harbor at their 5Ј end 100 vector-derived nucleotides from the region between the putative transcriptional start and the BamHI cloning site. All cDNA constructs were verified by DNA sequencing.
Quantifying mRNA with Real-time Quantitative PCR-For quantitative RT-PCR, total RNA preparation and reverse transcription were performed as described before (14). Quantitative RT-PCRs were carried out on a 7500 Fast real-time PCR system (Applied Biosystems, Carlsbad, CA) with TaqMan technology using human GRN (Hs00963703, exon boundary 3-4), and human GAPDH (4326317E) primer sets (Applied Biosystems). For each cDNA construct, at least three independent samples were analyzed. Reverse-transcribed GRN cDNA was normalized to endogenous GAPDH and expressed as a ratio to the 5Ј3Ј UTR cDNA construct (see Fig. 2 A) levels using the 2 Ϫ⌬⌬Ct method.
In Vitro Transcription and Translation-The indicated cDNA constructs in pcDNA4 MycHis with a stop codon before the His tag were linearized, purified by agarose gel electrophoresis, eluted with diethylpyrocarbonate-treated H 2 O, and cDNA concentration was adjusted to 0.5 g/l. Capped mRNAs were generated using the mMessage mMachine kit (Life Technologies, Ambion, Darmstadt, Germany). After template digestion with DNaseI, the mRNAs were purified with the RNeasy kit (Qiagen, Hilden, Germany), and RNA concentration was adjusted to 0.5 g/l. The size and integrity of the mRNAs were assessed by gel electrophoresis. In vitro translation reactions were performed in nuclease-treated rabbit reticulocyte lysate as described by the manufacturer (Promega, Madison, WI).
Cell Culture, Stable Cell Lines, and Transient Transfection-The Flp-In TM T-REx TM 293 cell line is a HEK 293 cell line containing a single integrated Flp recombination target (FRT) site. For stable, tetracycline-inducible, isogenic expression, each construct cloned in pcDNA5/FRT/TO (Hygro) was cotransfected with 9/10 cDNA of a Flp recombinase expression plasmid (pOG44, all from Invitrogen). Pooled stable cell lines were selected and cultured with 150 g/ml hygromycin in DMEM with Glutamax I (Invitrogen), supplemented with 10% (v/v) fetal calf serum (Invitrogen) and penicillin/streptomycin (PAA Laboratories, Pasching, Austria). For plateaued expression levels, cell lines were treated with 0.2 g/ml tetracycline (Sigma-Aldrich, Munich, Germany) for 24 h before the start of the experiment. Transient transfection of HEK293T cells was carried out using Lipofectamine TM 2000 (Invitrogen).
Preparation of Conditioned Media, Cell Lysates, and Immunoblotting-Conditioned media and cell lysates were prepared and analyzed as described previously (14). Briefly, conditioned media were centrifuged at 15,000 ϫ g for 15 min at 4°C and either subjected directly to standard 10% SDS-PAGE or to GRN ELISA. After washing with PBS, cells were lysed in ice-cold STEN lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.6), 2 mM EDTA, and 2% Nonidet P-40, supplemented with protease inhibitor mixture (Sigma-Aldrich)) and clarified by centrifugation. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. For detection, the indicated antibodies were used. Bound antibodies were visualized by horseradish peroxidase-conjugated secondary antibody using an enhanced chemiluminescence technique (GE Healthcare). Signals for quantification were detected by a luminescent image analyzer (LAS-4000, Fujifilm Life Science, Tokyo, Japan) and evaluated with Multi GaugeV3.0 software.
ELISA for Human GRN-Secreted GRN in conditioned medium was quantified by sandwich ELISA using streptavidincoated 96-well multiarray plates and the Meso Scale Discovery Sector Imager 2400 for the readout, as described previously (14).
Statistics-Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA). For comparison of two groups, unpaired Student's t test was used with a significance level of 0.05. For multiple comparisons, one-way analysis of variance (ANOVA) was used to determine significance, followed by Tukey's post hoc test.

RESULTS
Identification of GRN 5Ј UTRs-We first searched for potential 5Ј UTRs by rapid amplification of cDNA ends in HEK 293 cells and could demonstrate the presence of a 219-nucleotidelong 5Ј UTR (data not shown) as annotated (NM 002087) (Fig.  1, A and B). Furthermore, the 219-nucleotide-long 5Ј UTR could be amplified by PCR from an adult human brain cDNA (OriGene Technologies, Rockville, MD) (data not shown). In addition, we identified several shorter 5Ј UTRs by 5Ј rapid amplification of cDNA ends that were in line with previous computational predictions (XM005257254/XM005257255) (Fig. 1A). We also found a so far undescribed alternative splice variant of GRN mRNA. This variant skips the exon1/intron1 splice site and includes 271 nucleotides of the intron 1 (XM005257253) (Fig. 1, A and C).
Repression of GRN Expression by Its 5Ј UTR-In contrast to the short transcripts, the 219-nucleotide GRN 5Ј UTR (i.e. long 5Ј UTR) contains two AUG start codons (Fig. 1, A and B), which are within a single short uORF. Such short uORFs are frequently found in transcripts that are regulated via translational mechanisms (18 -22). Therefore, we investigated whether the FIGURE 1. Annotated variants of the GRN 5Ј UTR. A, sequence of the 219 nucleotides of the GRN 5Ј 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. 5Ј 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 5Ј 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 5Ј 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).
219-nucleotide GRN 5Ј UTR may affect expression of the GRN protein. To do so, we generated GRN variants with and without the 219-nucleotide 5Ј UTR ( Fig. 2A). We also added the GRN 3Ј UTR to the 5Ј UTR variants to investigate any possibility of additional effects ( Fig. 2A). To achieve isogenic expression, we used stably transfected Flp-In TM T-REx TM HEK 293 cells in this and all other experimental setups. Strikingly, GRN lacking its 5Ј UTR showed a strongly increased expression in cell lysates as well as in conditioned media compared with the GRN variants containing the 5Ј UTR or the 5Ј and 3Ј UTR (Fig. 2B). Therefore, these results demonstrate that GRN protein expression is repressed by the 219-nucleotides long 5Ј UTR. Although GRN containing only the 3Ј UTR showed increased expression compared with GRN with the 5Ј UTR or the 5Ј and 3Ј UTR, the expression was still significantly lower than GRN lacking both UTRs (Fig. 2B). This suggests that the 3Ј UTR may also contribute to repression of GRN expression. However, the addition of the 3Ј UTR to 5Ј UTR GRN (5Ј3Ј UTR GRN) did not further lower GRN expression levels (Fig. 2B).
To provide further evidence that the 5Ј UTR of GRN is directly involved in reducing GRN expression, we fused the GRN 5Ј UTR to an EGFP reporter (Fig. 3A). Because GRN is a secreted protein, we added the GRN signal peptide to EGFP, allowing its translocation into the endoplasmic reticulum and, consequently, its secretion (Fig. 3A). In line with the results shown above, the 5Ј UTR significantly repressed expression of EGFP in cell lysates (Fig. 3B). Furthermore, production of secreted EGFP was also strongly repressed by the GRN 5Ј UTR sequence (Fig. 3B), whereas the GRN 3Ј UTR had no effect on EGFP expression (Fig. 3B). Therefore, the 5Ј UTR exhibits an intrinsic repressor function for GRN expression that is independent of the downstream coding sequence.
The GRN 5Ј UTR Interferes with GRN Translation and mRNA Stability-With the experiments described above, we could not distinguish the effects of the UTRs on mRNA levels or protein translation. To prove that the 5Ј and 3Ј UTR of GRN interferes with protein translation, we performed an in vitro translation assay using equal amounts of in vitro transcribed GRN mRNA (Fig. 4A). Upon in vitro translation of GRN mRNA lacking the 5Ј UTR, robust amounts of protein were obtained, whereas mRNAs containing the 5Ј UTR were much less effi- Secreted GRN was analyzed using an ELISA assay described previously (14). For all quantifications, data are mean Ϯ S.D. (n Ն 3 independent experiments). ****, p Ͻ 0.0001, n.s., not significant. ANOVA followed by Tukey's multiple comparison test was used to determine significance. ciently translated (Fig. 4B). In contrast, the presence of the 3Ј UTR did not repress GRN translation (Fig. 4B), supporting the conclusion that only the 5Ј UTR, but not the 3Ј UTR, selectively represses GRN translation. Because the presence of the GRN 3Ј UTR resulted in reduced GRN expression levels (see Fig. 2B), we investigated whether reduced mRNA levels of the stably transfected isogenic HEK cell line were responsible for the low GRN expression of the 3Ј UTR construct. Analysis of mRNA revealed significantly reduced levels not only for the variant, which contains the 3Ј UTR, but also for the variant 5Ј UTR (Fig. 4C). Therefore, both UTRs reduce GRN mRNA, most likely via mRNA destabilization. Differences in transcription were not expected because we used an isogenic system. Therefore, the 5Ј UTR represses GRN production via translational inhibition and mRNA reduction.
A 50-Nucleotide Stretch of the UTR Is Required for Repression of GRN Expression-To directly determine which sequence stretch within the 5Ј UTR is responsible for the inhibition of GRN expression, we generated a variety of serial deletion constructs (Fig. 5A). The deletions included one cDNA construct that lacked nucleotides 1-125 of the 5Ј UTR (⌬125 UTR). This construct, which is similar to the shorter transcript described in Fig. 1A (XM005257255), lacks both AUG codons present in the long variant. Expression analysis revealed that the first 75 nucleotides of the 5Ј UTR of GRN could be removed without significant consequences for translational inhibition (Fig. 5B). Interestingly, removing an additional 50 nucleotides (Fig. 5A,  ⌬125 UTR) resulted in significantly increased expression and completely abolished repression of GRN expression similar to the constructs missing the entire 5Ј UTR (Fig. 5B, no UTR) or containing only a very short 5Ј UTR (Fig. 5B, ⌬182). Therefore, all identified GRN transcripts with shorter 5Ј UTRs that lacked the first 125 nucleotides (⌬125 UTR), including the annotated variants XM005257255 and XM005257254, failed to repress GRN expression in contrast to the GRN transcript with the 219-nucleotide 5Ј UTR. This also suggests that a short sequence stretch of 50 nucleotides might be required for inhibition of GRN expression. To confirm that this sequence is indeed required for repression of GRN expression, we deleted nucleotides 75-125 (Fig. 6A, ⌬75-125). In line with the data shown above, this short sequence stretch is indispensable for repression of GRN expression by the 5Ј UTR of GRN (Fig. 6B). Because the stretch between nucleotides 75-125 contains the two AUG start codons described above, we mutated both separately (data not shown) and in combination (Fig. 6A, 5Ј AUA -UTR). Although the mutation of a single start codon had no effect on expression (data not shown), the mutation of both AUGs resulted in a slight but significant increase of GRN expression compared with the long 5Ј UTR of GRN (Fig. 6B).
The incomplete rescue of GRN expression upon mutagenesis of the two start codons implicates overlaying effects on translation and mRNA levels. To distinguish between these two options, we first analyzed mRNA levels. Indeed, the mRNA level of the 5Ј AUA UTR variant was similar to that of the 5Ј UTR variant and 3-fold lower than for GRN containing no UTR or the deletion variant ⌬ 75-125 -UTR (Fig. 6C). Therefore, reduced mRNA levels might be responsible for the impaired rescue of expression of the 5Ј AUA -UTR variant. To prove that the mutagenesis of the two AUGs is capable of increasing protein translation, we performed an in vitro translation assay using equal amounts of in vitro transcribed GRN mRNA. Although the 5Ј UTR severely abolished in vitro translation of GRN, mutagenesis of the two AUGs was sufficient to obtain GRN levels similar to those translated from GRN mRNA containing no 5Ј UTR (Fig. 6D). Therefore, the two AUGs in the 5Ј UTR of GRN repress protein translation. However, because mRNA levels of the 5Ј AUA -UTR variant are still as low as with the long 5Ј UTR, protein expression failed to reach the level of the GRN variant lacking the entire 5Ј UTR.
Both AUGs are in the same frame, followed by an in-frame stop codon at position 159 -161 (Fig. 1B). To further investigate the role of the uORF on repression of GRN, we investigated whether nucleotides 76 -161 containing the entire uORF (Fig.  7A) are sufficient to suppress GRN expression. Indeed, the stretch of 50 nucleotides is sufficient to fully repress GRN expression (Fig. 7B). Moreover, similar findings were obtained when the same sequence was fused to the EGFP reporter (data not shown). uORFs in conjunction with appropriate secondary structures are characteristic for UTRs with translational repression properties (18 -20). Therefore, these findings suggest that either ribosomes may be stalled at the upstream start codons and, consequently, be unable to efficiently initiate translation of GRN similar to the translational inhibition of BACE1 (23) or that leaky scanning or impaired reinitiation contribute to translational inhibition. Therefore, we mutagenized the two AUG codons within the uORF of the 5Ј UTR to AUA (76 -161 AUA ) (Fig. 7A) and investigated the consequences on expression of GRN. Mutation of the two AUGs significantly increased expression but not to the levels of the variant lacking a 5Ј UTR (Fig. 7B). Therefore, we investigated the potential effects on mRNA levels. Indeed, the mRNA level of the AUA (76 -161 AUA ) variant was reduced strongly compared with mRNA without the 5Ј UTR, suggesting that reduction of mRNA prevented the full rescue of protein expression of the AUA (76 -161 AUA ) variant (Fig. 7C).
To prove that the two AUGs are involved in repression of protein translation, we performed an in vitro translation assay using equal amounts of in vitro transcribed GRN mRNA (Fig. 7D). Indeed, in vitro translation of mRNA containing nucleotides 76 -161 was almost completely repressed (Fig. 7E). Strikingly, mutagenesis of the two AUGs was sufficient to abolish the translational inhibition (Fig. 7E). Therefore, the two AUGs contribute significantly to repression of GRN expression by reducing its translation.
Initiation of Translation Occurs at the Two AUGs within the uORF of GRN-To provide further evidence that the AUGs of the uORF are used for translation initiation, we generated cDNA constructs of the uORF in-frame with EGFP lacking the uORF stop codon and the EGFP start codon (76 -158). This should allow monitoring of the protein production if, indeed, one of the two AUGs in the 5Ј UTR is used for initiation of translation. In addition, we also individually mutated each AUG (76 -158 AUA1 and 76 -158 AUA2 ) of the uORF or both in combination (76 -158 AUA1,2 ) (Fig. 8A). Translation can indeed be initiated at both upstream AUGs because expression of EGFP 76 -158 allowed translation of two peptides with a slightly higher molecular weight than EGFP (Fig. 8B). When both AUGs of the uORF are present, the first AUG is used preferen-tially as a start codon, as indicated by the stronger expression of the slightly higher molecular weight variant (Fig. 8B). Mutation of the first AUG (76 -158 AUA1 ) abolishes the expression of the higher molecular weight variant and results in increased expression of the lower molecular weight variant, whereas mutation of the second start codon (76 -158 AUA2 ) allows only the expression of the larger variant (Fig. 8B). For initiation of translation, at least one AUG of the uORF is required because the mutation of both AUGs (76 -158 AUA1,2 ) results in a complete loss of EGFP expression. Taken together, these findings demonstrate that ribosomes can indeed use the two AUGs for initiation of translation.
An Alternatively Spliced Novel GRN Transcript That Contains a Short uORF Represses GRN Expression-Because the alternatively spliced transcript described in Fig. 1, A and C, lacks the nucleotide stretch shown above to be required for translational repression but contains an alternative uORF, we expected that this 5Ј UTR variant should also be capable of repressing GRN translation. To investigate potential effects on GRN expression by this alternatively spliced 5Ј UTR, we gener- ated a stable Flp-In TM T-REx TM HEK 293 cell line (5Ј alt UTR). Expression analysis revealed that the alternative 5Ј UTR suppressed GRN protein expression in a similar way as the long 5Ј UTR (Fig. 9B). Moreover, the 5Ј alt UTR also caused a reduction of mRNA levels similar to the long 5Ј UTR (Fig. 9C). To prove that the alternative 5Ј UTR also affects translational repression, we performed in vitro translation experiments using equal amounts of in vitro transcribed mRNAs (Fig. 9D). This revealed that the alternative 5Ј UTR reduced translational efficiency as strongly as the long 5Ј UTR (Fig. 9E). Therefore, not only the long 5Ј UTR described originally but also the alternative 5Ј UTR described here for the first time reduce mRNA levels and repress protein translation in parallel.

DISCUSSION
Deregulation of GRN is frequently associated with pathological conditions. Enhanced expression of GRN is observed upon lysosomal stress, inflammation, wound healing, and tumorigenesis, whereas reduced levels of GRN are associated with a significantly enhanced risk for FTLD-TDP (7). Expression of GRN is controlled on several levels. Proteolysis of GRN to granulin peptides and its inhibition by endogenous protease  . ***, p Ͻ 0.001; n.s., not significant. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. Note that the 5Ј alt UTR efficiently represses GRN expression.
inhibitors appears to be involved in inflammation (24). Levels of secreted GRN are regulated via receptor-mediated internalization (25). Finally, acute up-regulation of GRN levels via transcriptional (26) and posttranscriptional mechanisms (14) has been observed upon treatment with certain compounds thought to be useful in rescuing GRN haploinsufficiency in FTLD patients. However, very little is known about how GRN levels are maintained under physiological conditions. We identified a set of GRN mRNAs containing different 5Ј UTRs including a so far not described alternatively spliced variant (Fig. 1). Interestingly, the 219-nucleotide 5Ј UTR as well as the alternatively spliced variant harbor a potential uORF. Such 5Ј UTRs with uORFs have been shown to affect the translation of several mRNAs (18 -20). In line with these findings, we observed that translation of GRN is repressed selectively by the two 5Ј UTR variants containing uORFs. In contrast, the shorter transcripts without an uORF occurring in vivo, including transcripts XM005257255 and XM005257253, are not translationally repressed (Fig. 5). In addition to a translational repression, the presence of the 5Ј UTR also contributes to a reduction of mRNA levels, most likely by reducing their stability. Therefore, the 5Ј UTR affects GRN expression at two levels: mRNA stability as well as translational efficacy.
We propose the following models for translational repression of GRN via its long and alternative 5Ј UTR (Fig. 10). First, 40 S ribosomal subunits associated with the eIF2-GTP-Met-tRNA ternary complex scan the GRN mRNA for initiation codons, and translation will be initiated at the first AUG. Initiation at the second and third AUG (the latter being the authentic start codon for translation of GRN) may be hindered by a secondary structure located between the first two initiation codons and result in stalling of ribosomal scanning (Fig. 10,  stalled ribosomes). Bioinformatics analysis revealed a stable loop structure between the two AUGs (Fig. 11A). Similarly, a very stable secondary structure is also formed by the 5Ј UTR of the alternative transcript (Fig. 11B). Reinitiation at the third AUG codon is, therefore, a relatively unlikely event, and, consequently, translation of GRN is not very efficient, i.e. repressed by its 5Ј UTR. In contrast, scanning of alternative transcripts lacking uORFs will result in efficient translation from the first and authentic start codon. This model is consistent with mechanisms involved in translational control of the transcription factors ATF4 or ATF5 mRNA (27). However, in line with the data in Fig. 8, our findings are also consistent with a second mechanism involving leaky scanning (Fig. 10, leaky scanning), whereby the 40 S ribosomal subunits associated with the eIF2-GTP-Met-tRNA ternary complex scans the 5Ј UTR, and translation is initiated, to some extent, at the first and second AUG, therefore reducing translational efficacy at the third AUG. Finally, reinitiation after aborted translation of the uORF may also be considered (Fig. 10, reinitiation).
On the basis of our findings, one may speculate that GRN transcripts with a short 5Ј UTR could cause a long term increase of GRN expression and that, under certain pathogenic conditions, initiation sites of GRN transcription or splicing may be changed and, consequently, result in dramatically different protein levels of GRN. Interestingly, Puoti et al. (28) just published the first FTLD-associated GRN mutation within the 5Ј UTR. Strikingly, this mutation is located at the exon 1/intron 1 splice 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. site and results in reduction of GRN mRNA and protein levels (28).
Repression of GRN expression is likely to be supported by RNA binding proteins. Such proteins may be degraded under stress conditions and, therefore, indirectly facilitate translation as a fast response, for example, to injury. In addition, in that regard, it is also interesting to note that many proteins that are genetically associated with ALS or FTLD, such as TDP-43 (29), Fused in sarcoma (30,31), heterogeneous ribonucleoprotein (hnRNP) A1, and hnRNP A2B1 (32), are well known RNAbinding proteins involved in translation, mRNA transport, and RNA splicing. If any of these proteins are involved in translational repression of GRN remains to be shown. However, it is tempting to speculate that pharmaceutical interference with such binding proteins could be used to therapeutically modulate GRN expression under various pathological conditions.