Molecular cloning and characterization of a translational inhibitory protein that binds to coding sequences of human acid beta-glucosidase and other mRNAs

Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, a prototypical inherited metabolic error for enzyme and gene therapy. An 80- kDa cytoplasmic protein, termed TCP80, was found to inhibit GCase mRNA tran slation in mammalian cells by binding to RNA-coding regions. The TCP80 cDNA was cloned by screening an expression library with the GCase-coding region RNA. The cDNA sequence was nearly identical to those for M-phase phosphopr otein (MPP4; 99%) and for the IL-2 enhancer binding protein (NF90; 96%), Ex pression of the carboxy-terminal third, TCP30, showed it to be an RNA-bindi ng protein that bound to a 184-nt fragment of GCase-coding sequence near th e 5' end of the mature mRNA. When added to reactions, a large molar excess of TCP30 diminished the translation inhibition of GCase RNA by cytoplasmic TCP80, TCP50, expressed from the NH2-terminal two-thirds of TCP80, did not bind to nor inhibit the translation of GCase RNA. Reconstitution of in vitr o translation inhibition of GCase RNA required intact human TCP80 heterolog ously expressed in insect cells. Time course analyses show that TCP80 funct ions at the initiation phase of GCase mRNA translation, probably by inhibit ing its binding to polysomes. Seven additional RNAs were isolated by specif ic binding to TCP30 including those for aldolase B, complement protein 8 ga mma-subunit, fibronectin receptor beta 1, ABL, lactate dehydrogenase A, fib rinogen gamma-chain, and peroxisomal proliferator-activated receptor or. In vitro translation of their RNAs was inhibited by TCP80. These studies show that TCP80 has RNA-binding (TCP30) and inhibitory (TCP50) domains that fun ction to modulate translation of several mRNAs, TCP80 is Likely identical t o MPP4 and NF90, but has previously undescribed roles in cellular function. (C) 1999 Academic Press.