Progranulin upstream open reading frames of different 5’ leaders affect downstream protein expression

Abstract

Heterozygous loss-of-function (LOF) mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD) by a mechanism of haploinsufficiency. Progranulin expression is regulated at multiple levels, including by its 5' untranslated region (UTR). Two different GRN 5' leaders contain upstream open reading frames (uORFs). These non-coding elements could contribute to the regulation of GRN expression by acting as cis repressors of translation. Selected from the Flanders-Belgian FTD patient cohort, 302 unrelated patients (mean age at onset 64.9 ± 9.8 years, range 29 to 84 years, 46.3% female) without a GRN LOF mutation. The GRN cohort of LOF mutation carriers comprised members of the GRN founder family (n=83, mean onset age 63.1 ± 7.7 years) and carriers of other LOF mutations (n=53, mean onset age 58.9 ± 7.1 years). Targeted resequencing of uORF regions of GRN transcripts by amplicon target amplification assays (https://www.agilent.com). Luciferase reporter assays and semi-quantitative Real-Time PCR for characterization of GRN uORFs and identified variants. Western blotting for investigation of uORF-mediated translation. Quantification of alternative splicing by analyzing two brain RNA-seq datasets from healthy controls (AMP-AD Knowledge Portal, https://adknowledgeportal.synapse.org/). We identified the presence of an alternative 5' UTR in human brain. Quantification of the alternative splicing in the exon1-intron1 splicing-site suggests low expression for the alternative spliced 5' UTR with notable variability between samples in both datasets. Abolishment of each uORF (n=3) of the alternative 5' UTR increases protein expression, with a mild impact on mRNA levels. The two ATG-initiated uORFs were capable of initiating translation. Targeted resequencing of the uORF regions identified two genetic variants, one in each 5' UTR. Both variants increase downstream protein levels, with the main 5' UTR variant rs76783532 causing a significant 1.5-fold increase in protein expression (p=0.03), with no impact on mRNA levels. The alternatively spliced GRN 5'UTR contains 3 uORFs that can regulate downstream protein expression by acting on the translational level. The variant identified in the main 5'UTR, significantly increases protein expression by altering uORF codon identity. These results promote further experimental work to elucidate the potential mechanistic involvement of GRN uORFs in GRN associated FTLD.