Isolation and Characterization of a Folate Receptor mRNA-binding trans-Factor from Human Placenta

Abstract

The interaction of an 18-base cis-element in the 5-untranslated region of human folate receptor (FR)-␣ mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539 -25547). This trans-factor was isolated to apparent homogeneity as a 43-and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA ciselement in the 3-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as ␣CP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.

Incrimination of Heterogeneous Nuclear Ribonucleoprotein E1 (hnRNP-E1) as a Candidate Sensor of Physiological Folate Deficiency

“…Indeed, Western blots of cytosolic S-100 fractions of HeLa-IU 1 cells using this antiserum (see Fig. 5B) confirmed a major band corresponding to hnRNP-E1 and a minor band corresponding to hnRNP-E2 (and no other contaminant proteins); this pattern was not dissimilar to the doublet found on SDS-PAGE of hnRNP-E1 isolated from human placenta (12) or Western blots of rat brain probed with anti-hnRNP-E1 and -E2 antiserum (31). Because there was no change in the expression of hnRNP-E1 per mg of cytosolic (S-100 fraction) proteins from HeLa-IU 1 -HF and HeLa-IU 1 -LF cells on Western blots (Fig.…”

“…Similar methods were also employed to evaluate the biosynthesis of hnRNP-E1 using antihnRNP-E1 antiserum (12) substituted for anti-FR antiserum (24) (24). Our anti-hnRNP-E1 peptide antiserum (12) developed against the hnRNP-E1 peptide sequence SLAQYLINARLSSEKGMGC contained shared sequences (underlined) with hnRNP-E2 (a closely related protein with 83% homology in its total amino acid sequence), predicting that this antiserum would recognize shared epitopes in both proteins, which are expressed in these HeLa cells (30). Indeed, Western blots of cytosolic S-100 fractions of HeLa-IU 1 cells using this antiserum (see Fig.…”

“…Initially, we identified an interaction between an 18-base cis-element in the 5Ј-UTR of FR-␣ mRNA and a trans-factor protein that was critical for the biosynthesis of FR (11). This trans-factor was later identified as hnRNP-E1 (12), which is also known as poly(rC)-binding protein 1 (PCBP1) and ␣CP1 (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Subsequent studies led to a model that established a linkage between perturbed folate metabolism and coordinated up-regulation of FR at the translational level (24); in this scheme, the accumu-lated intracellular homocysteine resulting from folate deficiency triggered the interaction of FR-␣ mRNA cis-element with hnRNP-E1, which then stimulated the biosynthesis and up-regulation of FR (24).…”

Influence of Physiologic Folate Deficiency on Human Papillomavirus Type 16 (HPV16)-harboring Human Keratinocytes in Vitro and in Vivo

“…(ii) This domain binds other members of the hnRNP family, including hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract-binding protein (13). (iii) Earlier, we purified hnRNP-E1 using poly(U)-Sepharose affinity chromatography (9), suggesting that hnRNP-E1 would bind the HPV16 57-nucleotide poly(U)-rich cis-element. Subsequent studies on the binding of purified recombinant GSThnRNP-E1 to HPV16 57-nucleotide poly(U)-rich cis-element in the presence of physiological concentrations of L-homocysteine, as well as mutation studies involving this cis-element, indicated the specificity and importance of this interaction (Fig.…”

“…Earlier we had determined that up-regulation of folate receptors in cervical cancer cells during folate deficiency involves the binding of hnRNP-E1 to an 18-base cis-element in the 5Ј-untranslated region of folate receptor-␣ mRNA, which triggers an increase in folate receptor biosynthesis at the translational level (8,9). Because this RNA-protein interaction was stimulated by homocysteine, which accumulates intracellularly during folate deficiency, this work established a link between perturbed folate metabolism and coordinated translational regulation of folate receptors (10).…”