Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells

Abstract

Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded. We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency. A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo. l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide element within the 5'-untranslated region of mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this element or by small interfering RNA to mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams. This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1.

PolyC-binding proteins enhance expression of the CDK2 cell cycle regulatory protein via alternative splicing

“…Such alterations in post-translational modifications and partitioning of PCBP1, could serve to impact on its availability/ability to alter splicing events in response to external environmental cues. This connection is further highlighted by the relationship of PCBP1 RNA-binding activities to levels of cellular nutrients, such as iron and folate ( 24 , 25 ). The level of iron loading may toggle PCBP1 RNA binding activity in a fashion similar to that well documented for the IREBP proteins ( 62 ) and can modulate cell cycle regulation through CDK2 ( 63 ).…”

A PCBP1–BolA2 chaperone complex delivers iron for cytosolic [2Fe–2S] cluster assembly

“…We then conducted a structure-function analysis of the BolA2–KH3 complex using site-directed mutagenesis of the KH3 domain. After examining the crystal structure of KH3, we engineered substitution mutations in (i) a conserved cysteine (Cys293) reported to undergo iron-dependent homocysteinylation 34 , (ii) residues identified by modeling GSH binding on KH3, (iii) residues in the vicinity of Cys293 that could serve as N-, O-, and S-based Fe(II) ligands 35 , and (iv) residues coordinating RNA in the structure of a KH3–RNA complex 36 . Wild-type and mutant versions of KH3 were expressed in cells and examined for their capacity to co-precipitate BolA2 ( Fig.…”

“…The Cys293 residue that is critical for iron binding on KH3 was previously identified as a site that modulates the RNA binding activity of PCBP1. In folate-deficient placental cells, PCBP1 (also called hnRNP E1) is reversibly homocysteinylated on Cys293, which enhances its affinity for folate receptor mRNA and increases the expression of folate receptor 34 , 44 , 45 . In these studies, iron and glutathione reduces the affinity of PCBP1 for mRNA, which is likely due to the coordination of Fe–GSH.…”

Protein interaction and functional data indicate MTHFD2 involvement in RNA processing and translation

“…While the consequences of interaction between MTHFD2 and hnRNPs are not yet clear, one possibility is that this interaction may allow nuclear MTHFD2 to exert some control over gene expression; indeed, there are many examples of hnRNP-interacting proteins influencing gene regulation via hnRNPs [ 45 – 47 ]. Moreover, hnRNPs may mediate signals on metabolic status: in particular, hnRNP-E1 has been reported to regulate gene transcription in response to folate starvation [ 48 ]. We also observed interactions with components of the small ribosomal subunit, and with several heat-shock proteins (HSPs) that assist with the folding and transportation of target proteins.…”