The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation

Abstract

The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5 untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat Cationic amino acid transporter 1 (Cat-1) is a high-affinity Na ϩ -independent transporter of L-arginine and L-lysine belonging to system yϩ (12, 62). Growth factors, hormones, and nutrients can modulate its expression level (20,53). Expression of the Cat-1 gene increases during stress in a manner that involves phosphorylation of translation initiation factor 2␣ (eIF2␣) (24). During such conditions, expression of the Cat-1 gene is regulated at the level of (i) mRNA synthesis via the transcription factor ATF4, which binds an amino acid response element in the first exon of the gene (54); (ii) mRNA stability via the binding of the nucleocytoplasmic protein HuR to an AU-rich element present within its 3Ј untranslated region (UTR) (94); and (iii) translation via a cap-independent mechanism of initiation via an internal ribosome entry site (IRES) (23).IRES-dependent translation involves the recruitment of ribosomes independently of the m 7 G cap at the 5Ј end of the mRNA followed by initiation downstream of the ribosome binding site (46). We showed that increased translation of the Cat-1 mRNA during amino acid starvation requires the translation of a 48-amino-acid open reading frame (ORF) in the 5Ј UTR of the mRNA, introducing the concept of a "dynamic IRES" (23,93). In the absence of upstream ORF (uORF) translation, the Cat-1 IRES remains in an inactive conformation. During starvation, translation of the uORF unwinds this secondary structure, leading to formation of a conformation that has IRES activity. Previous studies have also proposed that the active conformation is stabilized by proteins (IRES transactivating factors [ITAFs]) that are either synthesized or modified by processes that require eIF2␣ phosphorylation. We also showed that decreased translation elongation rates of the uORF in fed cells result in a prolonged half-life of this active remodeled structure, mimicking the role of ITAFs in amino acid-starved cells (21). Ribosome stalling within the uORF can occur physiologically during stresses that cause increased phosphorylation of elongation factor 2 and therefore decreased translation elongation rates (63). These two mechanisms o...

eIF2α Phosphorylation Tips the Balance to Apoptosis during Osmotic Stress

“…Other Methods-Nuclear and cytosolic fractions were prepared from cultured cells as described previously (21). Immunoblot analysis was performed using standard procedures.…”

“…Cytoplasmic extracts (500 g of protein) were immunoprecipitated with 4 g of anti-FLAG or mouse control antibody (Santa Cruz Biotechnology) as described previously (21), except that Dynabeads protein G (Invitrogen) were used. mRNAs were analyzed by RT-qPCR as described below.…”

Expression of cationic amino acid transporters, carcass traits, and performance of growing pigs fed low-protein amino acid-supplemented versus high protein diets

“…Lys and Arg are transported by the Na-independent systems b 0,+ and CAT-1 (Majumder et al, 2009); these are the major cationic AA transporters in mammals (Palacin et al, 2001;Broer, 2008). The transporter b 0,+ is mainly expressed in epithelial cells and functions as antiporter exchanging Leu for Lys; the absorption of Lys is coupled with the efflux of Leu.…”

“…Both Arg and Lys are absorbed by the enterocyte, at high affinity, by the Na-independent transporters b 0,+ (Majumder et al, 2009) and CAT-1 (Hatzoglou et al, 2004). The b 0,+ system is the most important transporter for cationic AA expressed in epithelial cells, whereas CAT-1 is expressed in non-epithelial cells (Palacin et al, 2001).…”

Insights into the roles of hnRNP A2/B1 and AXL in non-small cell lung cancer

“…The superfamily is composed of >20 members, the majority of which bind to the splicing sequences located within introns and exons to perform regulatory roles (6). Among them, the principle members are hnRNP A1, A2, A3 and B1.…”