UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes

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Abstract

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.

Introduction

Cardiac hypertrophy occurs in response to various factors including mechanical, hemodynamic and pathological stimuli [1]. While this response is initially adaptive, it can result in pathologic hypertrophy and subsequent heart failure. Regression or inhibition of cardiac hypertrophy may lead to improved outcomes as was the case in the HOPE trial in which the angiotensin converting enzyme (ACE) inhibitor ramipril was shown to decrease the development of and cause regression of electrocardiographic markers of left ventricular hypertrophy (LVH) [2]. These changes were independent of blood pressure reduction and were associated with reduced risk of death, myocardial infarction, stroke and congestive heart failure. Enhanced protein synthesis is a critical component of cardiac hypertrophy. Cardiac hypertrophy in vivo, and cardiac myocyte hypertrophy in vitro are characterized by increases in total protein content, increased cell size and up-regulation of fetal genes including atrial natriuretic peptide and B-type natriuretic peptide [3]. Initiation of translation, peptide chain elongation and translation termination are the main stages of protein synthesis. Increased rates of protein synthesis in cardiomyocytes correlate with an increase of activity of translation initiation factors [3].

UAP56 is an ATP dependent RNA helicase that also has ATPase activity [4], [5], utilizing the energy derived from ATP hydrolysis to unwind double stranded RNA. UAP56 is a DExD/H box protein and is phylogenetically grouped with eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases [5], [6], [7]. UAP56 was initially identified in an analysis of genes centromeric to HLA-B in the human major histocompatibility complex and was named BAT1 (HLA-B associated transcript 1) [8]. BAT1 was rediscovered as an essential RNA splicing factor and renamed UAP56 [9]. UAP56 is required for spliceosome assembly and mRNA export from the nucleus to the cytoplasm [10], [11], [12]. Binding of ATP to lysine 95 (K95) of UAP56 is required for mRNA export [11]. Depletion of HEL, the Drosophila equivalent of UAP56 has been shown to inhibit protein synthesis [10]. Given the role of UAP56 in RNA splicing and mRNA export, we hypothesized that UAP56 plays an important role in protein synthesis and subsequent hypertrophy. In the present study we found that UAP56 regulates protein synthesis and cardiomyocyte growth an effect that is blocked by helicase dead mutant UAP56.

Section snippets

Methods

Cell culture. Neonatal rat cardiomyocytes were isolated from the cardiac ventricles of 2- to 3-day-old Sprague–Dawley rat neonates as described previously [13]. Briefly, the ventricular tissue parts were excised and rinsed in Hank’s balanced salt solution (HBSS) prior to digestion with multiple rounds of collagenase type II (Worthington). Cells were collected by centrifugation, resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin

UAP56 increases protein synthesis in HeLa cells

To assess the effect of UAP56 on protein synthesis, WT UAP56 was overexpressed in HeLa cells and [3H] leucine incorporation was measured. In cells stimulated with 20% FBS, WT UAP56 increased protein synthesis 2.0 ± 0.27-fold compared with control vector (Fig. 1A). We next examined the effect of UAP56 lysine 95 mutants on protein synthesis. Lysine 95 of UAP56 is part of motif I, a conserved DExD/H box protein motif that is important for the helicase and ATPase activity of UAP56 (Fig. 1B) [16].

Discussion

This is the first report to show that UAP56 is an important regulator of protein synthesis and cellular hypertrophy. The results presented here demonstrate that overexpression of UAP56 increases protein synthesis while the knockdown of UAP56 inhibits it. The results with UAP56 mutants also suggest that ATP binding and the ATPase and helicase activity of UAP56 are important in the regulation of protein synthesis. These findings are consistent with other reports showing that UAP56 plays an

Sources of funding

This study was supported by NIH Grant HL80938 to J.D.A.

Disclosures

None.

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