Size heterogeneity of the largest subunit of nuclear RNA polymerase II. An immunological analysis

Antibodies raised against the 180-kDa subunit of cauliflower RNA polymerase II bind selectively to the largest subunit of RNA polymerase II purified from a variety of plant species. The selective binding of this antibody to the largest RNA polymerase II subunit has allowed us to probe for the size of this subunit in crude cell extracts, in fractions containing partially purified RNA polymerase II, and in isolated nuclei. Fractions containing RNA polymerase II were subjected to electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose, and blots were probed with antibody. Immunoglobulin complexes were revealed with 125I-Protein A. Published purification procedures result in rapid conversion of a 220-kDa subunit to a 180-kDa polypeptide, but purification at high pH (pH 9.0) retards this proteolysis. RNA polymerase II associated with isolated nuclei is largely protected from proteolytic degradation, and a 240-kDa polypeptide as well as a 220-kDa polypeptide can be detected. These results suggest that the 180-kDa subunit of RNA polymerase II arises artificially during cell lysis and enzyme purification, and that even the 220-kDa polypeptide may be a degradation product of a 240-kDa polypeptide in plants.     

Restricted degradation of U6 RNA in a nuclear extract of Ehrlich ascites tumor cells

A new nucleolytic activity that causes restricted digestion of U6 RNA was found in a nuclear extract of Ehrlich ascites tumor cells. This nucleolytic activity specifically degrades U6 RNA in the vicinity of its 3'-end with accumulation of a discrete sized degradation product of RNA of 90-95 nucleotides. Since this degradation product was not digested further by the nuclease under these conditions, this trimming of U6 RNA is supposed to be a biologically meaningful reaction. This nucleolytic activity required Mg2+, and was inhibited by Zn2+ or Ca2+.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro.

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.     

The preparative synthesis of oligodeoxyribonucleotides using RNA ligase.

The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated. Reacting using from 18 to 200 nmol of substrates in which a single 2'-deoxyribonucleoside 3',5'-bisphosphate was added to an oligodeoxyribonucleotide resulted in yields from 13 to 95%. When two oligodeoxyribonucleotides were similarly joined using RNA ligase, the yields ranged from 10 to 50%. Although the reactions contained high concentrations of enzyme and were incubated from 5 to 21 days, there was little degradation of either substrates or products. We have also characterized an unusual product which arises when 3'-phosphate terminated oligodeoxyribonucleotides are incubated with RNA ligase and high concentrations of ATP. This product has an adenylyl group linked to the 3'-phosphate by an anhydride bond. The mechanistic and synthetic implications of forming this product are discussed.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.     

Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis.

We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell.     

Discrimination between RNA circles, interlocked RNA circles and lariats using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis can be used to identify structural forms of RNA such as linear RNA, circular RNA, interlocked circles and lariats. The procedure is based upon the characteristic migration behaviour of the degradation products derived from the intact structures present already before the start of the experiment or formed during or after electrophoresis in the first dimension. After autoradiography to detect the positions of the radiolabeled RNA molecules, circles broken during electrophoresis of the first dimension give rise to horizontal lines touching the diagonal formed by linear RNAs at a point corresponding to the length of the RNA circle from which it was derived. Products derived from interlocked RNA circles by breakage after completion of the first dimension appear on a vertical line underneath the intact complex and consist of free RNA circles and their linear derivatives. Broken lariats give rise to two lines depending on the location of the break. Lariats with broken tails are present on a line to a position that corresponds to the length of their tail and that runs parallel to the diagonal formed by linear products. Lariats with a broken eye form a line running from the position of the intact product to the diagonal formed by the linear RNAs.     

Uptake and degradation of cytoplasmic RNA by hepatic lysosomes. Quantitative relationship to RNA turnover.

Past evidence has suggested that the lysosomal pathway is an important site of cytoplasmic RNA degradation in the hepatic parenchymal cell (Lardeux, B. R., Heydrick, S. J., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14507-14519). We now provide additional support for this notion by quantitating degradable RNA in lysosomes and correlating its pool size with hepatic RNA degradation. Rat livers, previously labeled with [6-14C]orotic acid, were perfused with graded levels of amino acids over the full range of induced autophagy; RNA degradation was determined from [14C]cytidine release. Close correspondence between the marker beta-acetylglucosaminidase and the breakdown of RNA to cytidine in subcellular fractions indicated that the lysosome was the main site of catabolism, a conclusion supported by the fact that degradation was enhanced when external pH was lowered from 7 to 6. Although [14C]cytidine was also released in homogenates by the action of natural ribonucleases on cytosolic RNA, this source was eliminated by unlabeled exogenous RNA. The size of the degradable RNA pool in lysosomes, determined from the total release of cytidine in homogenates, correlated directly with rates of hepatic RNA degradation over the full range of basal and induced degradation. A direct correlation was also seen between RNA degradation and cytidine pools within lysosomal particles. Because cytosolic cytidine was not taken up by lysosomes under these conditions, the pool could only have arisen from the breakdown of intralysosomal RNA. As determined by cytidine production, these findings support the view that the lysosomal-vacuolar system is the main, if not sole, site of induced and basal RNA degradation in liver.     

Specific endonucleolytic cleavage of the mRNA for ribosomal protein S20 of Escherichia coli requires the product of the ams gene in vivo and in vitro.

Endonucleolytic cleavage is believed to initiate the degradation of most bacterial mRNAs, but with several exceptions, the enzymes responsible have yet to be identified. Crude (S-30) or partially fractionated extracts of Escherichia coli strains with reduced exonuclease activities catalyze the cleavage of a 372-residue RNA substrate containing the sequences coding for ribosomal protein S20 to yield a number of discrete products. The major product of 147 residues is obtained in 60 to 70% yield, is coterminal with the 3' end of the substrate, and is identical to an mRNA fragment previously characterized in vivo (G. A. Mackie, J. Bacteriol. 171:4112-4120, 1989). A number of other products of 150 to 340 residues are also formed, and the cleavage sites, typically N decreases AU sequences, have been identified in the S20 mRNA substrate by Northern (RNA) blotting and primer extension. All cleavages required a native rather than a denatured RNA substrate. The rate of cutting of the S20 mRNA substrate at the site yielding the prominent 147-residue product appears to be independent of cleavages at other sites. In addition, the activity of the putative endonuclease(s) depends strongly, both in vivo and in vitro, on the product of the ams gene, which is known to influence mRNA lifetimes in vivo. Taken together, the data show that the fractionated extract described here reproduces steps in the degradation of some mRNAs which occur in living cells.     

Faithful degradation of soybean rbcS mRNA in vitro.

The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.     

Translation of honeybee promelittin messenger RNA. Formation of a larger product in a mammalian cell-free system.

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell-free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell-free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro. The larger polypeptide detected in vitro may represent a precursor of promelittin.     

Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.     

Consequences of RNase E scarcity in Escherichia coli

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10-20% of normal and that the feedback mechanism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.     

Expression of srnB gene of F plasmid by altered RNA polymerase in Escherichia coli

Degradation of otherwise stable rRNA and tRNA takes place in the presence of rifampin, dependent on the F plasmid srnB gene. We have reported that a protein newly synthesized in the presence of rifampin might be a product of the srnB gene required for stable RNA degradation (Ito, R. and Ohnishi, Y. (1983) Biochim. Biophys. Acta 739, 27–34). Here we have further studied the mechanism of srnB expression. Among eighteen mutants with altered RNA polymerase, two (TJ2470 (rpoC4) and TJ302 (rpoC56)) showed RNA degradation at high temperature (42°C) when the srnB gene was present. Labeling proteins at 42°C in strain TJ2470 indicated that a protein of molecular weight 12 000 was a product of the srnB gene, and that expression of the srnB gene provoked RNA degradation. Using plasmid pTK4, in which the srnB gene is inserted downstream of the promoter of lacZ, lac promoter-dependent expression of the srnB gene, with production of the putative protein product, also induced RNA degradation at 42°C, with no requirement for added rifampin or altered RNA polymerase. RNA degradation in these conditions was quite similar to that in the case of the addition of rifampin; e.g., it showed some responses to Mg²⁺, temperature and RNAase I content of the cells. Expression of the srnB gene dependent on lac promoter was also observed in minicells. Thus, it is inferred that the srnB gene is probably repressed under normal conditions with its own promoter; its expression initiates RNA turnover.