A noncoding RNA modulator potentiates phenylalanine metabolism in mice

Abstract

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair ^-/- and Pah ^R408W/R408W mice and improved the Phe tolerance of these mice.

Fig. 1.. Pair^−/− mouse mimics human PKU disease.

(A) lncRNA profiling of livers from E18.5 embryos and from 2-month-old adult mice (n = 3). 2210408F21Rik is highlighted in red. (B) Schematic of using CRISPR-Cas9 to generate a Pair^−/− mouse model. (C) Northern blot detection of the expression of Pair in the indicated mouse livers. β-actin was used as a loading control. (D) Representative images of 12-month-old Pair mice. (E and F) Comparison of body weights of the indicated female (E) and male (F) mice at the age of 3 to 12 weeks. Data are shown as mean ± SD, one-way ANOVA. (G) Blood Phe concentrations in the indicated mice was tested every 2 weeks starting from 4 weeks of age (n = 5 Pair^+/+, n = 5 Pair^−/−, n = 7 Pair^+/− animals). Data are shown as mean ± SD, one-way ANOVA. (H) Cumulative survival curve of cohorts of indicated littermates (log-rank test). (I) Cumulative seizure-free survival curve of cohorts of indicated littermates (log-rank test). (J) Representative images of Pair^−/− mice experiencing seizures. (K) Left: representative images of brains from indicated mice at the age of 12 months (n = 5). Right: scatter plots represent brain weight quantification for both female and male indicated littermates at the age of 12 months (n = 5). Mean ± SD, one-way ANOVA. (L) Coronal sections of adult mouse brains subjected to immunohistochemical staining and quantitative data for TH⁺ neurons in the substantia nigra compact/ventral tegmental area of the indicated mice at the age of 12 months (n = 5). Mean ± SD, one-way ANOVA. Top row, 10× magnification; bottom row, 200× magnification. n.s., not significant at P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.. Pair and HULC associate with PAH.

(A) Protein candidates interacting with Pair were revealed by LC-MS. The x-axis indicates different experimental groups. (B) PAH-binding sites along HULC and Pair identified by CLIP assay. The figure represents the read coverage along HULC and Pair transcripts obtained by mapping Sanger sequencing reads to the representative transcript. cDNA counts are shown. (C) Representative images of immunolabeling with fluorescent in situ hybridization to simultaneously detect colocalization of the indicated RNA and protein molecules in human primary hepatocytes. TUG1 and Hu were used as RNA and protein controls, respectively. Scale bars, 50 μm. (D) Resolution of SHAPE reactivity by capillary electrophoresis. Four-color electropherograms for the products of one multicolor run by 6-FAM-labeled NMIA modified RNA (+SHAPE), VIC-labeled control RNA (−SHAPE) of HULC nucleotides 178 to 202, NED-labeled ddA, and a PET-labeled ddT are shown. Bottom panel: normalized SHAPE reactivity of HULC nucleotides 178 to 202. x-axis indicates the sizes of capillary electrophoresis. Data are shown as mean ± SD of n = 15 independent experiments. (E and F) EMSA using recombinant His-tagged PAH and [γ-32P]–labeled Pair nucleotides 467 to 488 (E) or HULC nucleotides 180 to 202 (F) WT or mutant oligonucleotides. Unlabeled Pair or HULC WT or mutant RNA oligonucleotides were included as competitors. (G) MS2-TRAP assay using Pair^−/− hepatocytes expressing indicated plasmids performed by immunoblotting using the indicated antibodies. (H) Immunoblotting (bottom) or autoradiography (top) of CLIP assay using the indicated hepatocytes expressing the indicated plasmids.

Fig. 3.. HULC and Pair modulate the enzymatic activities of PAH.

(A) HULC binds to PAH to stabilize the allosteric Phe-induced open conformation of PAH. (B) Magnified view of HULC’s interaction with the regulatory domain of PAH. HULC A¹⁹¹ forms hydrogen bonds with His⁶⁴ and polar interaction with Thr⁶³. Phe forms stacking interaction with HULC A¹⁹¹. Color representation in both (A) and (B): green indicates PAH; orange and magenta indicate HULC; yellow indicates allosteric Phe; and cyan indicates Thr⁶³ and His⁶⁴. (C) HULC A¹⁹⁵ forms a hydrogen bond with Tyr¹⁶⁶; HULC A²¹⁴ forms a hydrogen bond with Arg¹⁵⁷; HULC G²⁰² forms a hydrogen bond with Tyr¹⁵⁴. Green indicates PAH; orange and magenta indicate HULC; and Cyan indicates Tyr¹⁵⁴, Arg¹⁵⁷, and Tyr¹⁶⁶. (D) Superimposition of the three-dimensional structures of HULC nucleotides 184 to 216 (cyan) and Pair nucleotides 467 to 484 (magenta) after docking to PAH (yellow). Pair A⁴⁷⁹, U⁴⁸⁰ and HULC U¹⁹⁰, A¹⁹¹ are indicated by arrows. (E) Streptavidin pull-down using Bio-Phe/Bio-BH₄ followed by immunoblotting detection using anti-PAH antibody in the indicated hepatocytes. (F) Fold change of peptides recovered from LiP-MS. Top panel: comparison of PAH WT only, PAH incubated with HULC, and PAH incubated with a HULC mutant sequence. Bottom panel: comparison of PAH incubated with HULC, PAH incubated with HULC and Phe, and PAH incubated with HULC and BH₄. The x-axis indicates the amino acid position of full-length PAH; the y-axis indicates the fold change of peptide recovery number. (G and H) ELISA measurement of the percentage of PAH-associated BH₄ (G) or PAH-associated Phe (H) in the indicated hepatocytes expressing the indicated mimics. Data are shown as mean ± SEM of n = 5 independent experiments, one-way ANOVA. n.s., not significant at P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.. HULC mimics facilitate PAH-Phe and PAH-BH₄ interactions.

(A and B) Log₂ of relative fold change of His-PAH WT/mutants and Biotin-Phe (A) or His-PAH WT/mutants and biotin-BH₄ (B) binding affinity in the presence of the indicated lncRNA mimics. The fold change was normalized using His-PAH WT in the presence of the LINK-A mimic. (C) Phe and Tyr concentrations in neonatal blood spots in the presence of His-tagged PAH WT or indicated mutants and the indicated lncRNA mimics. Data are shown as mean ± SD of n = 3 independent experiments, Student’s t test. The lncRNA mimic representing LINK-A nucleotides 1100 to 1117 was included as a negative control. (D) Determination of k_cat of recombinant PAH WT or indicated mutant proteins in the presence of the indicated mimics. Data are shown as the mean ± SD of n = 3 independent experiments, one-way ANOVA. n.s., not significant at P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.. GalNAc-HULC lncRNA mimics alleviate PKU symptoms.

(A) Graphic illustration of GalNAc-tagged HULC mimics (left) and the treatment schedules (right). (B) Blood Phe concentrations were monitored every day for short-term treatment in female and male Pair^−/− mice with the indicated mimics. Data were analyzed with Student’s t test. (C) Blood Phe concentrations were monitored every other day for medium-term treatment in female and male Pair^−/− mice treated with the indicated mimics. Data were analyzed with Student’s t test. (D) Blood Phe concentrations were monitored every day for short-term treatment in female or male Pah^R408W/R408W mice treated with the indicated mimics. Data were analyzed with Student’s t test. (E) Blood Phe concentrations were monitored every other day for medium-term treatment in female or male Pah^R408W/R408W mice treated with the indicated mimics. Data were analyzed with Student’s t test. (F) Measurement of blood tyrosine concentrations for medium-term treatment in female and male Pah^R408W/R408W mice treated with the indicated mimics, Data were analyzed with Student’s t test. (G and H) Phe clearance test (G) or area under the curve (AUC) (H) of female or male Pah^R408W/R408W mice subjected to a Phe-free diet and pretreatment with the indicated mimics. Data are shown as mean ± SD, Student’s t test. (I) Phe tolerance test of female or male Pah^R408W/R408W mice subjected to a Phe-free diet and pretreatment with indicated mimics for 3 days, followed by water containing 0, 0.75, 1.5, 3.0, or 6.0 mg/ml of Phe (with the dose increasing every 2 days). Data were analyzed with Student’s t test. (J) Blood Phe concentrations were monitored every day for short-term treatment in Pah^R408W/R408W mice subjected to sapropterin alone or in combination with the indicated mimics. Data are shown as mean ± SD, one-way ANOVA. n.s., not significant at P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.