Conserved function of the long noncoding RNA Blnc1 in brown adipocyte differentiation

Abstract

Objective: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological processes. Recent work has demonstrated that the inducible lncRNA Blnc1 stimulates thermogenic gene expression during brown and beige adipocyte differentiation. However, whether Blnc1 is functionally conserved in humans has not been explored. In addition, the molecular basis of the Blnc1 ribonucleoprotein complex in thermogenic gene induction remains incompletely understood. The aims of the current study were to: i) investigate functional conservation of Blnc1 in mice and humans and ii) elucidate the molecular mechanisms by which Blnc1 controls the thermogenic gene program in brown adipocytes.

Methods: Full-length human Blnc1 was cloned and examined for its ability to stimulate brown adipocyte differentiation. Different truncation mutants of Blnc1 were generated to identify functional RNA domains responsible for thermogenic gene induction. RNA-protein interaction studies were performed to delineate the molecular features of the Blnc1 ribonucleoprotein complex.

Results: Blnc1 is highly conserved in mice and humans at the sequence and function levels, both capable of stimulating brown adipocyte gene expression. A conserved RNA domain was identified to be required and sufficient for the biological activity of Blnc1. We identified hnRNPU as an RNA-binding protein that facilitates the assembly and augments the transcriptional function of the Blnc1/EBF2 ribonucleoprotein complex.

Conclusions: Blnc1 is a conserved lncRNA that promotes thermogenic gene expression in brown adipocytes through formation of the Blnc1/hnRNPU/EBF2 ribonucleoprotein complex.

Keywords: ATP5A, ATP synthase, H+ transporting, mitochondrial F1 complex, alpha 1; BAT, brown adipose tissue; Blnc1; Brown adipocyte differentiation; Brown fat; Cox7a1, cytochrome c oxidase subunit 7A1; Dio2, deiodinase, iodothyronine type II; EBF2; EBF2, early B cell factor 2; Elovl3, elongation of very long chain fatty acids like 3; FABP4, fatty acid binding protein 4; PPARγ, peroxisome proliferator-activated receptor gamma; Ppargc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Pparα, peroxisome proliferator-activated receptor alpha; Prdm16, PR domain zinc finger protein 16; RACE, rapid amplification of cDNA ends; SDHB, succinate dehydrogenase complex iron sulfur subunit B; Thermogenesis; UQCRC2, ubiquinol-cytochrome c reductase core protein II; Ucp1, uncoupling protein 1; lncRNA.

Figure 1

Role of human Blnc1 in brown adipogenesis. (A) Genomic structure of the mouse and human Paqr9/Blnc1 locus (not drawn to scale). (B) Sequence conservation between mBlnc1 and hBlnc1. (C) Coding potential analysis using CPC. Note that negative values reflect a low probability of protein-coding. (D) qPCR analyses of gene expression during differentiation of brown preadipocytes transduced with vector (open) or hBlnc1 (brown) retrovirus. (E) Gene expression of differentiated brown adipocytes treated without (−) or with (+) isoproterenol for 4 h. Data represent mean ± sd. *p < 0.05, hBlnc1 vs. vector.

Figure 2

Human Blnc1 augments mitochondrial respiration in brown adipocytes. (A) Immunoblots of total lysates from brown adipocytes treated with vehicle (Veh) or isoproterenol (Iso) for 6 h. (B) Oil Red O and MitoTracker staining of differentiated brown adipocytes. Scale bar = 100 μm. (C) Mitochondrial DNA content measured by qPCR in brown adipocytes overexpressing vector (open) and hBlnc1 (brown). (D) Oxygen consumption rate (OCR) in the absence or presence of FCCP. Data represent mean ± sd. *p < 0.05, hBlnc1 vs. vector.

Figure 3

Human Blnc1 rescues RNAi knockdown of mouse Blnc1. (A) Differentiated brown adipocytes expressing control (Vec) or shRNA targeting mBlnc1 (#1 and #2) were treated with vehicle (Veh) or isoproterenol (Iso) for 4 h. Gene expression was analyzed by qPCR. (B) Immunoblots of brown adipocyte lysates. Data represent mean ± sd. *p < 0.05, hBlnc1 vs. Vec; #p < 0.05, hBlnc1/siBlnc1 vs. Vec/siBlnc1.

Figure 4

Functional analysis of mouse Blnc1 domains. (A) Predicted secondary structure of mBlnc1 using RNAfold web server. Three segments predicted to form stem-loop structures were indicated by red cycles. (B) Diagram of mBlnc1 truncation mutants. (C) Gene expression analysis of differentiated brown adipocytes expressing vector (Vec), full-length mBlnc1 (FL), or individual truncation mutants treated with Veh or Iso. (D) Immunoblots of brown adipocyte lysates. Data represent mean ± sd. *p < 0.05, vector vs. Vec.

Figure 5

A conserved RNA domain in mouse and human Blnc1 promotes brown adipogenesis. (A) Diagram showing the FL and RD1 Blnc1 constructs. (B) qPCR analysis of differentiated brown adipocytes expressing vector (Vec), full-length mBlnc1 (mFL) or hBlnc1 (hFL), or respective RD1 fragments (mRD1 and hRD1). (C) Immunoblots of brown adipocyte lysates. Data represent mean ± sd. *p < 0.05, vs. Vec.

Figure 6

Physical interaction between Blnc1 and hnRNPU. (A–B) IP/qPCR analyses of mBlnc1 (A) or hBlnc1 (B) in RNA isolated from anti-Flag immunocomplexes or input from transiently transfected HEK293 cells. (C–D) Immunoblots of total lysates and precipitated proteins on streptavidin beads (SA) from transiently transfected HEK293 cells. (E) Immunoblots of total lysates and anti-HA immunocomplex. (F) Immunoblots of Myc-EBF2 in lysates and streptavidin beads (SA) from transfected HEK293 cells. (G) IP/qPCR analyses of mBlnc1 in RNA isolated from anti-Myc immunocomplex or input from transiently transfected HEK293 cells.

Figure 7

HnRNPU facilitates the assembly of the Blnc1/EBF2 transcriptional complex. (A) qPCR analysis of differentiated brown adipocytes transduced with Vector (Vec) or shRNAs targeting hnRNPU (#1 and #2) in combination of vector (open) or mBlnc1 (brown) overexpression. (B) Immunoblots of brown adipocyte lysates. (C) qPCR analysis of differentiated brown adipocytes transduced with Vector (Vec) or shRNAs targeting hnRNPU (#1 and #2) in combination of vector (open) or EBF2 (brown) overexpression. Data represent mean ± sd. *p < 0.05 si#1 and #2 vs. Vec. (D) Immunoblots of brown adipocyte lysates. (E) Model depicting the induction of thermogenic gene program by the Blnc1 ribonucleoprotein transcriptional complex.