A CHOP-regulated microRNA controls rhodopsin expression

Abstract

Using genome-wide microribonucleic acid (microRNA [miRNA]) expression profiling, bioinformatics, and biochemical analyses, we identified miR-708, an endoplasmic reticulum (ER) stress-inducible miRNA whose expression is regulated by the transcription factor CCAAT enhancer-binding protein homologous protein (CHOP) in vertebrates. miR-708 is encoded within an intron of the CHOP-regulated gene Odz4, a member of the highly conserved teneurin family of developmental regulators. Odz4 and mir-708 expression is coregulated by CHOP, and the two transcripts are coexpressed in the brain and eyes of mice, suggesting common physiological functions in these tissues. We validated rhodopsin as a target of miR-708 through loss- and gain-of-function experiments. Together, our data implicate miR-708 in the homeostatic regulation of ER function in mammalian rod photoreceptors, whereby miR-708 may help prevent an excessive rhodopsin load from entering the ER. Hence, miR-708 may function analogously to other unfolded protein response controls that throttle protein influx into the ER to avoid ER stress through mechanisms, such as general translational attenuation by protein kinase RNA-like ER kinase or membrane-bound messenger RNA decay by inositol-requiring enzyme 1.

Figure 1.

CHOP regulates miR-708 expression during ER stress. (A) Heat maps and Venn diagrams of miRNAs differentially regulated during ER stress in Chop^+/+ and Chop^−/− MEFs. The applied criterium for differential expression was a more than twofold change in treated versus untreated conditions, represented as logarithmic values. Red, increase in differential expression during ER stress; Green, decrease in expression. miR-708 is indicated in bold. Cells were treated with 5 µg/ml tunicamycin (Tm) or 500 nM thapsigargin (Tg) for 24 h. UT, untreated. (B) Scatter plots illustrating the changes in expression of the miRNAs in A. (C) RNase protection assay in Chop^+/+ MEFs treated with 5 µg/ml Tm or 500 nM Tg for 24 h. The loading control used was miR-16. (bottom) Quantification of the data (miR-708/miR-16). Error bars are SDs of two independent experiments. (D) TaqMan miRNA assay of miR-708 (normalized to snoRNA 202) in Chop^+/+ and Chop^−/− MEFs treated with 5 µg/ml Tm. Error bars are SDs of three independent experiments.

Figure 2.

miR-708 is a conserved intronic miRNA highly expressed in neuroectodermal tissues. (A) Schematic of the locus encoding Odz4 indicating that mir-708 resides within its first intron. University of California, Santa Cruz Genome Browser conservation in mammals is shown. (B, top) Sequence alignment of the miR-708 stem loop in mammals (Mmu, Mus musculus; Rno, Rattus norvegicus; Hsa, Homo sapiens; Ppy, Pongo pygmaeus; Cfa, Canis familiaris; and Eca, Equus caballus). The guide (miR-708) and passenger strands (miR-708*) are outlined in black boxes. (bottom left) Stem loop structure and mature duplex of murine miR-708. (C) Phylogenetic tree of bilateral animals in which Odz/Teneurin homologues are found. Chop homologues are found only in amphibians and mammals, and miR-708 homologues are found only in mammals (blue box). Bioinformatics analyses were performed using the HomoloGene database (National Center for Biotechnology Information). (D) RT-PCR analyses in Chop^+/+ and Chop^−/− MEFs treated with 5 µg/ml Tm for 24 h. Grp78 mRNA indicates activation of the UPR. The loading control used was β-actin (Actb). (E) Gene expression analyses of miR-708 (TaqMan miRNA assay) and Odz4 (qRT-PCR) in adult mouse tissues normalized to snoRNA 202 and Rps26, respectively. Variations in their relative expressions (rel. expression) can be attributed to (a) undetected Odz4 isoforms, (b) differential regulation of the miRNA and host gene, and/or (c) experimental variation between TaqMan and SYBR green–based assays. Error bars are SDs of two independent experiments. sk., skeletal; sm., small.

Figure 3.

Mature miR-708 is loaded on the RISC. (A) Immunoprecipitation (IP) of FLAG-tagged Ago2 (FLAG-Ago2) from 3T3 fibroblasts stably expressing it. (right) 3T3 cells transduced with an empty vector. FT, flow through. WB, Western blot. (B) TaqMan miRNA assay of miR-708 from FLAG-immunoprecipitated fractions obtained from lysates of the cells in A. Error bars are SDs of two independent experiments. *, P < 0.0005; **, P < 0.008. P-values were derived from a t test for independent samples.

Figure 4.

miR-708 targets rhodopsin for posttranscriptional inhibition. (A, top) Schematic of full-length mouse rhodopsin mRNA. (bottom) Sequence alignment of the region containing the predicted conserved miR-708 site in mammals. The gray box represents the putative site complementary to the seed sequence; the black dotted line encircles the entire putative site. Mmu, M. musculus; Hsa, H. sapiens; Ptr, Pan troglodytes; Mml, Macaca mulatta; Rno, R. norvegicus; Ocu, Oryctolagus cuniculus; Cfa, C. familiaris; Fca, Felis catus; Bta, Bos taurus; and Dno, Dasypus novemcinctus. (B) Immunoblots of lysates from 293T cells transfected with plasmids encoding full-length mouse Rho or GFP along with an miR-708 antagomir (anta.) or mimic. Overexpression of rhodopsin results in the expected aggregates observed when resolved by SDS-PAGE. GRP78 was used to show activation of the UPR. GFP was used as a control for transfection efficiency and off-target effects of the antagomir/mimic. The loading control used was GAPDH. Numbers indicate the fold change in expression normalized to GAPDH. (C) Autoradiograms of 239T cells transfected with a plasmid encoding full-length mouse Rho along with an antagomir or scrambled control and pulse labeled with [³⁵S]methionine (³⁵S-Met) for 1 h. (left) Lysates immunoprecipitated (IP) with an anti-RHO antibody. (right) Total lysates. Numbers indicate relative amounts of radiolabeled rhodopsin normalized to total lysate. (D) Same experiment as in C except the 239T cells were transfected with a plasmid encoding full-length mouse Rho in which the miR-708 seed in the 3′UTR was replaced with a scrambled sequence (miR-708 seed mutant).