Degradation Of Ribosomal RNA Research Articles

Degradation of ribosomal RNA during starvation: Comparison to quality control during steady-state growth and a role for RNase PH

Ribosomal RNAs are generally stable in growing Escherichia coli cells. However, their degradation increases dramatically under conditions that lead to slow cell growth. In addition, incomplete RNA molecules and molecules with defects in processing, folding, or assembly are also eliminated in growing cells in a process termed quality control. Here, we show that there are significant differences between the pathways of ribosomal RNA degradation during glucose starvation and quality control during steady-state growth. In both processes, endonucleolytic cleavage of rRNA in ribosome subunits is an early step, resulting in accumulation of large rRNA fragments when the processive exoribonucleases, RNase II, RNase R, and PNPase are absent. For 23S rRNA, cleavage is in the region of helix 71, but the exact position can differ in the two degradative processes. For 16S rRNA, degradation during starvation begins with shortening of its 3' end in a reaction catalyzed by RNase PH. In the absence of this RNase, there is no 3' end trimming of 16S rRNA and no accumulation of rRNA fragments, and total RNA degradation is greatly reduced. In contrast, the degradation pattern in quality control remains unchanged when RNase PH is absent. During starvation, the exoribonucleases RNase II and RNase R are important for fragment removal, whereas for quality control, RNase R and PNPase are more important. These data highlight the similarities and differences between rRNA degradation during starvation and quality control during steady-state growth and describe a role for RNase PH in the starvation degradative pathway.

A role for a bacterial ortholog of the Ro autoantigen in starvation-induced rRNA degradation

Cellular adaptations to stress often involve changes in RNA metabolism. One RNA-binding protein that has been implicated in RNA handling during environmental stress in both animal cells and prokaryotes is the Ro autoantigen. However, the function of Ro in stress conditions has been unknown. We report that a Ro protein in the radiation-resistant eubacterium Deinococcus radiodurans participates in ribosomal RNA (rRNA) degradation during growth in stationary phase, a form of starvation. Levels of the Ro ortholog Rsr increase dramatically during growth in stationary phase and the presence of Rsr confers a growth advantage. Examination of rRNA profiles reveals that Rsr, the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNP) and additional nucleases are all involved in the extensive rRNA decay that occurs during starvation of this bacterium. We show that Rsr, PNP, and an Rsr-PNP complex exhibit increased sedimentation with ribosomal subunits during stationary phase. As the fractionation of PNP with ribosomal subunits is strongly enhanced in the presence of Rsr, we propose that Ro proteins function as cofactors to increase the association of exonucleases with certain substrates during stress.

Me, myself and I

Flowering plants have diversified and conquered the land. To do so, they have evolved strategies to avoid incest and promote compatible outcrossing. Philip Hunter explores some of the latest research in this area and its application to agriculture and horticulture.

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

Apoptotic signals induce specific degradation of ribosomal RNA in yeast

Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast.

Molecular Characterization of Three New Mutant Enzymes of Pyrimidine 5′-Nucleotidase Causing Hereditary Hemolytic Anemia.

Pyrimidine 5′-nucleotidase (P5′N-1) is a dephosphorylating enzyme that catalyzes the hydrolysis of various pyrimidine nucleoside 5′-monophosphates, particularly UMP and CMP, to produce the corresponding nucleosides. In RBC the reaction is essential for the removal of the nucleotides mainly arising from ribosomal RNA degradation during final erythroid maturation. Hereditary P5′N-1 deficiency is the third most common enzymopathy causing hereditary non-spherocytic hemolytic anemia. The disorder is transmitted as an autosomal recessive trait and is usually characterized by mild-to-moderate hemolytic anemia and accumulation of pyrimidine nucleotides within the erythrocyte. The enzyme is strongly inactivated by heavy metals; thus P5′N-1 deficiency can be acquired as a result of lead poisoning. The P5′N-1 gene is localized on 7p15-p14 and the cDNA has been cloned and sequenced. 24 different mutations have been identified so far, most of them at the homozygous level. Recently, five pathological variants of P5′N-1 have been in-depth characterized, and the molecular bases of the P5′N-1 deficiency has been elucidated. To unravel the cause of the P5′N-1 deficiency found in patients with hemolytic anemia and homozygous for 3 newly identified missense mutations (c.187T>C, c.469G>C, c.740T>C; Balta et al, Blood ASH 2006, 108:3743; Manco et al, Haematologica 2006, 91:266–267), we have undertaken a functional analysis of the 3 mutant enzymatic forms. The C63R, G157R and I247T proteins were produced as recombinant forms, purified and biochemically characterized. All enzymes were altered, although to a different extent, either in their catalytic efficiency or in thermal stability, the G157R being the most impaired enzyme. Catalytic efficiency of all mutants turned expecially towards UMP (about 50 to 200 times), owing to the increased Km values (about 10–25 times higher). The kinetic behaviour vs CMP was partly affected, the catalytic activity being moderately reduced (Kcat lowered to 5–20%). The G157R protein was highly heat unstable, halving the activity in about 23 min at 37°C, whereas C63R and I247T mutants at the same temperature maintained fully activity for more than 2 hours. However, at higher temperature also C63R and I247T mutants resulted less stable than the wild-type enzyme losing the activity in few minutes (t1/2 at 46°C, about 5 min vs 2 hours of the wild-type enzyme). Therefore, although mutations targeted different regions of the P5′N-1 structure, unexpectedly they produced similar aberrant effects on the molecular properties of the enzyme. Gly157 is a conserved amino acid, located close to the substrate binding site. Very likely, position 157 cannot tolerate the large and charged arginine side-chain introduced by c.469G>C mutation. Thus, it is conceivable that the drastic G157R substitution not only indirectly affects the binding of the substrate(s), but also weakens the protein stability. Cys63 and Ile247 are located far away from the catalytic site. Nevertheless, our biochemical data indicate that they are functionally and structurally important for preserving the enzyme activity. Thus, as in other cases, the decreased catalytic efficiency of C63R and I247T enzymes seems to result from secondary effects related to propagating conformational changes.

Stability of RNA isolated from post-mortem tissues of Atlantic salmon (Salmo salar L.)

Studies of post-mortem interval on the stability of RNA from a number of various mammals have shown RNA to be stable for between 24 and 48 h following death. As yet there have been no studies looking at RNA stability in post-mortem tissues of poikilothermic fish. Brain, kidney, liver and muscle were collected from Atlantic salmon (Salmo salar) parr and samples of each tissue were placed into RNAlatertrade mark after 0-24 h post-mortem storage at room temperature. Electrophoretic analysis of the total RNA showed degradation of ribosomal RNA only in muscle from 8 h onwards. Probing of northern blots with beta-actin showed that, in the brain, beta-actin mRNA was stable for 24 h post-mortem but degradation of mRNA was observed after 8 h with the kidney and liver and after 4 h with the muscle. Expression of the weakly expressed thyroid hormone receptor beta (TRbeta) was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in all tissues up to 24 h post-mortem although a reduction in PCR product was observed after 8 h with muscle and 24 h with kidney. Analysis with an Agilent 2100 Bioanalyzer showed that the RNA integrity number (RIN) of brain total RNA remained constant for 8 h post-mortem with only a small fall at 24 h post-mortem. The RINs of the remaining tissues indicated degradation at 8 h post-mortem with kidney and muscle and at 24 hours post-mortem with liver. Taken together these findings show that degradation of Atlantic salmon RNA is tissue dependent but stable for at least one hour post-mortem.

The exosome, plugged.

The correct processing, quality control and turnover of cellular RNA molecules is crucial to many aspects of cell physiology. The exosome—a large molecular assembly with exoribonuclease activity—has emerged as a crucial component in many, if not most, stages of RNA metabolism, including degradation and maturation of ribosomal RNA, RNA quality control and turnover of cytosolic messenger RNA (reviewed by Houseley et al , 2006). Exosomes are found in eukaryotes and archaea. The eukaryotic core exosome consists of six polypeptides with sequence similarity to the phosphate‐dependent 3‘→5’ exoribonuclease RNase PH and three protein K homology (KH) and/or S1‐domain‐containing RNA‐binding proteins (Fig 1A; Mitchell et al , 1997). Additional subunits include the RNase R homologue Rrp44/Dis3 (yeast exosome) and the RNase D homologue Rrp6 (nuclear isoform). The archaeal (a) exosome exhibits a simplified subunit composition and is assembled from three copies each of two RNase PH‐like proteins (aRrp41 and aRrp42), aRrp4 or aCsl4 (Evguenieva‐Hackenberg et al , 2003). Figure 1. Comparison of exosomes in archaea and eukaryotes. ( A ) Schematic comparison of subunit composition of archaeal, human and yeast exosomes (viewed from the caps of Rrp4/Rrp40/Csl4). Identified active sites are indicated by black (phosphorolytic) or white (hydrolytic) asterisks. Inactive phosphorolytic subunit pairs are shaded grey. The position of Rrp6 is inferred from native mass spectrometry (Hernandez et al , 2006). Archaeal (a) exosomes might have additional subunits and mixed aRrp4/aCsl4 trimers …

Apoptosis induced by a cytopathic hepatitis A virus is dependent on caspase activation following ribosomal RNA degradation but occurs in the absence of 2′–5′ oligoadenylate synthetase

We have presented previously evidence that the cytopathogenic 18f strain of hepatitis A virus (HAV) induced degradation of ribosomal RNA (rRNA) in infected cells [Arch. Virol. 148 (2003) 1275–1300]. In contrast, the non-cytopathogenic parent virus HM175 clone 1 had no effect on rRNA integrity. We present here data showing that rRNA degradation is followed by apoptosis accompanied by characteristic DNA laddering in the cytoplasm of 18f infected cells. The DNA laddering coincided with the detection of caspase 3 and PARP-1 cleavage and was dependent upon activation of the caspase pathway, since treatment with Z-VAD-FMK, a pan-caspase inhibitor, inhibited both events. RNase L mRNA was present in both virus-infected and uninfected cells. Messenger RNA for the interferon inducible enzyme 2′–5′ oligoadenylate synthetase (2′–5′ OAS), which polymerizes ATP into 2′–5′ oligo adenylate (2–5A, the activator of RNase L) in the presence of double-stranded RNA, was not detected following virus infection. 2′–5′ OAS mRNA was induced by treatment of the cells with interferon-β (IFN-β). IFN-β mRNA was marginally induced following infection. However, phosphorylated STAT 1, a key regulator of interferon-stimulated gene transcription was not detected in virus infected cells. STAT 1 phosphorylation in response to IFN treatment was lower in virus-infected cells, compared to uninfected cells treated with interferon, suggesting that 18f virus infection interferes with interferon signaling. The results suggest that 18f infection causes the induction of a 2–5A independent RNase L like activity.

Studies on the compaction of isolated nucleoids from Escherichia coli

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine–spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (∼3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 °C. The incubation at 37 °C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine–DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.

Erythroid pyrimidine 5′-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia

AbstractA characteristic process of terminal erythroid differentiation is the degradation of ribosomal RNA into mononucleotides. The pyrimidine mononucleotides can be dephosphorylated by pyrimidine 5′-nucleotidase (P5N-I). In humans, a lack of this enzyme causes hemolytic anemia with ribosomal structures and trinucleotides retained in the red blood cells (RBCs). Although the protein/nucleotide sequence of P5N-I is known in mammals, the onset and regulation of P5N-I during erythroid maturation is unknown. However, in circulating chicken embryonic RBCs, the enzyme is induced together with carbonic anhydrase (CAII) and 2,3-bisphosphoglycerate (2,3-BPG) by norepinephrine (NE) and adenosine, which are released by the embryo under hypoxic conditions. Here, we present the chicken P5N-I sequence and the gene expression of P5N-I during RBC maturation; the profile of gene expression follows the enzyme activity with a rise between days 13 and 16 of embryonic development. Thep5n-I expression is induced (1) in definitive but not primitive RBCs by stimulation of β-adrenergic/adenosine receptors, and (2) in definitive RBCs by hypoxic incubation of the chicken embryo. Since embryonic RBCs increase their hemoglobin-oxygen affinity by degradation of nucleotides such as uridine triphosphate (UTP) and cytidine triphosphate (CTP), the induction ofp5n-I expression can be seen as an adaptive response to hypoxia. (Blood. 2003;102:4198-4205)

The cytopathic 18f strain of Hepatitis A virus induces RNA degradation in FrhK4 cells

Summary. The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for β-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

Electrophoretic characterization of ribosomal subunits and proteins in apoptosis: specific downregulation of S11 in staurosporine-treated human breast carcinoma cells.

Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits.

Ribosomes in incompatible pollen tubes in the Solanaceae

Some members of the Solanaceae have a self‐incompatibility mechanism preventing self‐fertilization. Stylar ribonucleases (S‐RNases) are responsible for growth inhibition of self‐pollen tubes. A prevalent model postulates that the S‐RNases act as intracellular cytotoxins that degrade ribosomal RNA, and possibly also messenger RNA, in the incompatible pollen tubes. Since ribosomes and polysomes are easily noticed with the electron microscope, it should be possible to confirm disintegration of these structures. However, our inspection by electron microscopy revealed the presence of ribosomes and polysomes in pollen tubes formed after self‐pollination of the self‐sterile species Brugmansia (Datura) suaveolens and Nicotiana alata. There was no decrease over time in the number of bound ribosomes per unit of rough endoplasmic reticulum (RER) membrane. The results indicate that the inhibition of tube growth is not due to a general degradation of ribosomal and messenger RNA. Therefore, the substrate for S‐RNases presumably is very specific.

16S Mitochondrial Ribosomal RNA Degradation Is Associated with Apoptosis

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 μmol H 2O 2 per 10 7 cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis. © 1997 Elsevier Science Inc.

A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces.

Role of ribosome degradation in the death of heat-stressed Salmonella typhimurium.

Heat treatment of Salmonella typhimurium results in cell death, which coincides with a significant reduction of the cellular content of 16S ribosomal RNA. It is suggested that the degradation of ribosomal RNA is a direct cause of cell death. This conclusion is based on the observation of carbon-starved and magnesium-supplemented cells, which survive heat treatment much better, and which also maintain stable levels of ribosomal RNA.

Role of ribosome degradation in the death of heat-stressed Salmonella typhimurium

Heat treatment of Salmonella typhimurium results in cell death, which coincides with a significant reduction of the cellular content of 16S ribosomal RNA. It is suggested that the degradation of ribosomal RNA is a direct cause of cell death. This conclusion is based on the observations of carbon-starved and magnesium-supplemented cells, which survive heat treatment much better, and which also maintain stable levels of ribosomal RNA.

Expression of Mammalian Antiviral Enzymes from the 2–5A System in Transgenic Plants

The 2–5A system is an interferon-regulated antiviral RNA decay pathway present in cells of higher vertebrates. Two enzymes are essential, a 2–5A synthetase which produces 5′-phosphorylated, 2′,5′-linked oligoadenylates (2–5A) in response to doublestranded RNA, and the 2–5A-dependent RNase L. To determine if these human proteins would be functional in plants, we expressed the human cDNAs for a 2–5A synthetase and RNase L in separate tobacco plants. Both proteins were enzymatically active in extracts of transgenic plants while such activities were not detected in the control plants. Furthermore, activation by 2–5A of RNase L in the transgenic plant leaves was shown to cause degradation of ribosomal RNA. The requirement for both the synthetase and RNase L for antiviral activity was underscored by the observations that expression of human RNase L alone or 2–5A-synthetase alone was insufficient to protect plants against either tobacco etch virus or tobacco mosaic virus.

Large-Scale Preparation of a Ribonuclease fromRana catesbeiana(Bullfrog) Oocytes and Characterization of Its Specific Cytotoxic Activity against Tumor Cells

Rana catesbeianaribonuclease (RC-RNase) is a pyrimidine–guanine sequence-specific ribonuclease found only inR. catesbeiana(bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09MNaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.