Ribonuclease Activity of Rat Liver Perchloric Acid-soluble Protein, a Potent Inhibitor of Protein Synthesis

Abstract

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 M led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [␣-32 P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3 -monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5 -or 3 -32 P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the singlestranded regions of the molecule.Rat liver perchloric acid-soluble protein (L-PSP) 1 is a 136-amino acid protein that inhibits protein synthesis (1). Oka et al.(1) demonstrated that L-PSP, when added to a rabbit reticulocyte cell-free system, causes inhibition of a biphasic kinetic nature and also leads to the disaggregation of polysomes. This would be similar to the mode of inhibition of translation by the heme-regulated eukaryotic initiation factor 2␣ kinase (2) (however, see our results below). Based on these data, it was suggested that the protein inhibits the initiation step rather than the elongation step (1).A 14-kDa translational inhibitor protein remarkably similar to L-PSP has been characterized in human monocytes and mouse liver (3-5). A homology search revealed that these proteins belong to a new group of small proteins named the YER057c/YJGF family (3), which is of unknown physiological function. The protein sequences of these family members are highly conserved in prokaryotes (including cyanobacteria), fungi, and eukaryotes, suggesting that the proteins may be involved in a basic cellular process. Indeed, mRNA of the translational inhibitor p14.5, the human homologue of L-PSP, becomes significantly up-regulated with the induction of differentiation to macrophages (3), and the synthesis of PSP from rat kidney increases from the 17th fetal day to the fourth postnatal week and then enters a steady-state level (6). In contrast, the expression of PSP from rat kidney in renal tumor cells was down-regulated (6).Recently, Schmiedeknecht et al. (7) have identified the functional promoter of the human p14.5 translational inhibitor. They reported a head-to-head orientation of p14.5 with the gene for the protein subunit hPOP1 of RNase P and with RNase MRP ribonucleoproteins...

Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay

“…Among these, two factors, DCP1A and HRSP12, are known to be related to RNA-degrading ability: DCP1A promotes decapping activity of DCP2 (Coller and Parker 2004), and HRSP12 has an endoribonucleolytic ability (Morishita et al 1999). Several previous studies and the present data support the important roles of DCP1A-mediated decapping and HRSP12-mediated endoribonucleolytic cleavage in GMD.…”

“…and has endoribonuclease activity (Morishita et al 1999;Mistiniene et al 2003Mistiniene et al , 2005. Although it is unknown whether the trimeric structure of HRSP12 is critical for its endoribonucleolytic activity, it is possible that the trimerization of HRSP12 is involved in maintaining the structural integrity of a functionally active GMD complex.…”

Y-Box Binding Protein 1 and RNase UK114 Mediate Monocyte Chemoattractant Protein 1 mRNA Stability in Vascular Smooth Muscle Cells

“…Studies suggest that UK has multiple functions. For example, UK was reported to inhibit protein synthesis in vitro in rabbit reticulocyte lysate (35). Overexpression of UK suppressed cell proliferation of normal rat kidney epithelial cells without any influence on cell viability (20).…”

“…Overexpression of UK suppressed cell proliferation of normal rat kidney epithelial cells without any influence on cell viability (20). In addition, UK was reported to act as an endoribonuclease that inhibits translation by cleaving mRNA (35).…”

Redox Proteomics Uncovers Peroxynitrite-sensitive Proteins That Help Escherichia coli to Overcome Nitrosative Stress

“…RidA belongs to the YjgF/YER057c/UK114 superfamily, which is highly conserved across a wide range of organisms. This protein family has a variety of assigned functions, ranging from translational inhibition and activation of a proteinase, to nuclease and chaperone activity (33)(34)(35)(36). Recent studies identified an enamine/imine deaminase activity of RidA in Salmonella enterica (15).…”