The polypyrimidine tract binding protein regulates desaturase alternative splicing and PUFA composition

Abstract

The Δ6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling.

Fig. 1.

FADS2 codes for a desaturase that adds cis double bonds to the growing fatty acid chains in omega-3 (n-3) and omega-6 (n-6) long chain polyunsaturated fatty acid synthesis. Nomenclature refers to the number of carbons and double bonds (e.g., 20:5n-3 has 20 carbons and 5 double bonds, with n-3 describing distance of the double bonds from the terminal methyl carbon of the fatty acid). The end products of the biosynthetic pathway give rise to eicosanoids and docosanoids with opposing effects.

Fig. 2.

Western blot showing eluted proteins from an RNA pull-down assay (first two lanes) run in parallel with whole nuclear extract (NE) as a positive control. PTB was pulled down by an RNA oligo containing the FADS2 putative binding site (wt) but not by a mutated version (mut), with no nonspecific binding of β-actin.

Fig. 3.

(A) RT-PCR results for a time course of PTB siRNA knockdown in SK-N-SH neuroblastoma cells, showing increasing FADS2 AT1 (■) with decreasing PTB (□). Integrated densities of gel bands were normalized to actin and expressed as the ratio of knockdown/control. (B) Quantitative real-time PCR showing fold change in FADS gene transcript expression in HepG2 cells for PTB knockdown compared with nontargeting siRNA control, n = 3 biological and technical replicates. * P < 0.01

Fig. 4.

The combination of splicing and transcriptional regulation determines FADS2 AT1 levels in vivo. (A) Splicing regulation is suggested by inverse correlation of PTB and FADS2 AT1 in the majority of neonatal baboon tissues examined (■), with R² = 0.87 for trend excluding tissues with low PTB (△). Axes represent RT-PCR-integrated densities normalized to β-actin. (B) Tissues with low PTB levels (△) closely follow the trendline relating FADS2 AT1 with FADS2 CS (R² = 0.74 for trend including all points), suggesting transcription-level regulation (pre-mRNA abundance) is the primary determinant of FADS2 AT1 levels in the three tissues with low PTB.

Fig. 5.

Lower correlation between PTB and FADS3 transcripts. (A) Slight inverse correlation of FADS3 AT7 and PTB in the majority of neonatal baboon tissues examined (■), with R² = 0.44 for trend excluding tissues with low PTB (△). Axes represent RT-PCR-integrated densities normalized to β-actin. (B) Low correlation of FADS3 AT1 and PTB, with R² = 0.26 for trend excluding tissues with low PTB (△).

Fig. 6.

Fatty acid composition of liver-derived HepG2 cells, showing fold change in concentration of each fatty acid with PTB siRNA compared with nontargeting control siRNA. Gray bars represent treatments in regular media, and black bars are media supplemented with 25 μM 22:5n-3. All changes shown were significant at P < 0.05, except for 18:3n-6 in regular media. (A) Omega-6 (n-6) fatty acids. (B) Omega-3 (n-3) fatty acids. (C) Summary of total fatty acids and ratios of fatty acids with opposing effects.