RNAse L and genome expression during early period of the rat liver regeneration

Partial hepatectomy (PHE) stimulates an intact part of the liver to exchange the existing «quiescence» program for the proliferative one. Several changes between 0.5 and 3 h after PHE are considered as manifestations of the abolishment of «quiescence» program' a temporal decrease of either RNA synthesis or accumulation of the newly formed RNA; a restricted variability of RNA transcripts; a partial retention of newly synthesized RNA within the nuclei thereby providing less RNA for the cytoplasm; previously obtained data about transient dissociation of ribosomes from endoplasmic reticulum. An involvement of the 2' ,5' -oligo( A Synthetase—RNAse L system in the process is suggested by upand down-regulation of 2',5'-oligo( A)synthetase activity in the cytoplasm and in the nucleus, respectively, and by up-regulation of RNAse L content in regenerating liver. The production of IFN alp, an inducer of the 2',5'-oligo( A Synthetase—RNAse L system, is also increased during transition period. A specific role of sinusoidal cells, the main producers of IFN alp in the liver, in the abolishment of the old program is strongly suggested.

Introduction.Extensive damage of liver parenchyma stimulates an intact part of the liver to exchange the existing «quiescence» program to the proliferative one with eventual restoration of liver mass and function.What kind of mechanisms is responsible for the abolishment of the old program is an intriguing question concerning not only regenerating liver but the transition periods of eukaryotic cell activity in general.
There are several evidences of partial restriction of genome expression during the transition period of liver regeneration e. g. temporal loss of ribosomes from endoplasmic reticulum [1 J, reciprocal alterations of 2',5'-oligo(A)synthetase (2 , ,5'-OAS) activity in nuclei and cytoplasm [2] etc.The 2,5-OAS is a limiting enzyme of 2,5-OAS-RNAse L system [3] that is present in cells of higher vertebrates.The system is responsible for RNA degradation during viral infection and is induced by interferons a/p (IFN alp) [3 ].Its role in the absence of infection remains still obscure.This paper addresses general charac teristics of genome expression in regenerating liver at the RNA level and the production of IFN a/p and RNAse L, the inducer and the target of 2,5-OAS activity, respectively.Materials and Methods.Pretreatment of the ani mals.Male Wistar rats (200-250 g) were used throughout.Partial (2/3) hepatectomy (PHE) and sham operation (ShO) were performed by standard procedures [4].Livers were extirpated at the times indicated and processed as described below.
Investigation of intracellular distribution of newly synthesized RNA. 3 H-orotic acid (13.5 Ci/mmole) was injected intraperitoneally (3 /*Ci/g of body weight) at the times indicated.For each time point liver samples from five animals were fixed, processed for electron autography [5 J and analyzed at the microscope JEM-100 B. Silver grains were counted over extranucleolar part of the nucleus, nucleolus, mitochondria and endoplasmic reticulum of fifty hepatocytes from each sample.
Detection of kinetic complexity of nuclear RNA.Kinetic complexity of nuclear RNA was evaluated in hybridization reaction of the trace amount of unique DNA sequences with the excess of RNA according to [6,7].
Evaluation of RNAse L and IFN al$ production.RNAse L was quantified by specific radiocovalent 2',5'-A affinity labelling.The reaction was performed with the liver extract with the subsequent PAGE/SDS [8].Activity of interferon a/p was detected in S10 fraction of liver homogenate (1/10 w/v) by its ability to inhibit cytopathogenic action of vesicular stomatitis virus in the cultivated test cells [9 ].
Results.Intracellular distribution of newly synthe sized RNA.The synthesis and accumulation of newly formed RNA were evaluated from the number of silver grains over corresponding cellular compartments after pulse (15 min) and more prolonged CH-orotic acid injected immediately after PHE) labeling, res pectively.The RNA synthesis and transit into cyto plasm occur in two stages.Immediately after PHE the indices increase (Fig. 1, A).The subsequent changes are specific for each compartment.The gradual inc rease of silver grains over extranucleolar part of the nucleus coincides with the normalization of their amount over the nucleolus and mitochondria and transient decrease over ER (Fig. 1, A). Prolonged labeling reveals partial retention of newly synthesized RNA within the nuclei thereby temporally providing less RNA for the cytoplasm (Fig. 1,2?).
Kinetic complexity of nuclear RNA.The nuclear RNA from the liver in 3 h after PHE hybridizes with lower percent of unique DNA sequences than nuclear RNA from intact animals (7.9 % versus 11.4 %) (Fig. 2).As kinetic complexity of unique sequences of rat DNA is equal to 1.9-10 9 base pairs [8] the kinetic complexity of nuclear RNA from intact and partially hepatectomized rats comprises 4.3 10 s and 3.0-10 8 nucleotides correspondingly.
RNAse L and IFN a/p production.PHE induces an increase of 2',5'-oligo(A) affinity labelling (Fig. 3) and therefore content of enzyme RNAse L. Antiviral activity of liver cytosol from partially hepatectomized rats also increases with the maximal value in 0.5 h after operation.An active S10 fraction dilution from intact rats and rats in 0.5 h after PHE is equal to 1/80 and 1/320 correspondingly.
Discussion.Several findings of this work are interpreted to result from partial restriction of genome expression and loss of the previous phenotype: a temporal decrease of either RNA synthesis or accu-  The potential role of 2' ,5-OAS-RNAse L sys tem in this process is supported by the first indica tions that production of RNAse L and IFN alp is up-regulated.We hypothesize the following sequence of events after PHE providing the abolishment of «quiescence» program at the RNA level.The bio logical effect of the 2',5'-OAS-RNAse L system is stimulated by interferon a/p that induces trans cription of genes encoding 2',5'-OAS and RNAse L [3].The activated cytoplasmic synthetase [2] con verts more ATP to PP, and to oligoadenylates.The latter bind to RNAse L and activate its catalytic functions.The preferential ability of RNAse L to destroy the polysomes by the degradation of mes senger RNA [10] is compatible with the reorga nization of ribosomes above-mentioned [1] and with the general idea about the mechanisms of «quiesсепсе» program exchange for proliferative one.In the nuclei, on the contrary, the decreased catalytic activi ty of 2',5'-A-OAS may protect the «new» messages from degradation.
The participation of sinusoidal cells in the pro- cess, the main producers of IFN alp in the liver [11], is in line with their regulatory role during hepatocytes transition from quiescence to proliferation [12].

Fig. 1 .
Fig. 1.The average number of silver grains over compartments of singular hepatocyte: J -extranucleolar part of nucleus; 2 -nuc leolus; 3 --mitochondria; 4 -endoplasmic reticulum; t t and t 0 are indicated and zero time, respectively; Л and В -3 H-orotic acid was injected 0.25 h before slaughter and immediately after PHE, respectively