RNase activity

Recently, the related phenomenon of RNA interference (RNAi) has attracted much attention [5]. RNAi occurs when a short (generally 21 nucleotides in length) double-stranded RNA (dsRNA) catalyticaUy represses the translation of a fully complementary mRNA sequence. The process appears to proceed via a complex formed between the antisense RNA strand and a protein with RNase activity [6]. Upon binding to the target mRNA sequence, the ribonucleoprotein complex initiates cleavage of the mRNA transcript thus preventing translation of intact protein. After dissociation from the truncated mRNAs, the ribonucleoprotein complex is free to act on other intact mRNAs. Such small interfering RNAs (siRNAs) have... 

Subsequent to DNA template linearization, the procedures for in vitro RNA transcription reaction are set up. It is essential to avoid RNase contamination by using gloves, sterile glassware, and water devoid of RNase activity [treated with 0.1% diethyl pyrocarbonate (DEPC) and autoclaved]. 

In recent years, several papers appeared in the literature concerning NMR and X-ray studies of thermostable ribonucleases. Among archaebacteria, several histone-like proteins from Sulfolobus strains have been identified and grouped into molecular mass classes. From the 7 kDa class, Sac7e from S. acidocaldarius and Sso7d from S. solfataricus, possessing DNA binding activity in combination with non-specific RNase activity, were identified. ... 

It was known from the work of Richards that RNase A could be cleaved between residues 20 and 21 by digestion with subtilisin. 29 The N-terminal 20-residue peptide (S-peptide) could be separated from the 104-residue 21-124 protein (S-protein), and each was enzymatically inactive however, when they were mixed in a 1 1 ratio, full RNase activity was regenerated even though the covalent bond between residues 20 and 21 was not re-formed. This finding was the basis for a great many structure/function studies by the synthesis of analogues of the S-peptide and their noncovalent recombination with natural S-protein.13"31 ... 

Considerable DNase but no RNase activity results if Ca-+ is replaced by Sr-+, while Fe-+ and Cu J+ cause minimal activation (3, 40). A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca-+ Hg-+, Zn2+, and Cd-+ are the most potent of these (3). Studies with synthetic substrates, to be discussed below, indicate that Ca2+ is not only required for the proper binding of substrates but also that it is required for the subsequent independent hydrolytic process. Although several divalent cations can substitute for Ca2t in the binding function, as evidenced by their competitive inhibition of enzymic activity (3) and their ability to promote nucleotide binding (62), the catalytic role of Ca2+ appears to be unique. 

The same enzyme has been highly purified from another strain (strain K) of Bacillus subtilis, and their properties have been fully investigated by Yamasaki and Arima (119, 120). They have confirmed the findings by Nishimura and Maruo and have found, moreover, that ATP and dATP strongly inhibit the enzyme. Yamasaki and Arima suggested that ATP might participate in the regulation of intracellular RNase activity. 

A new RNase activity, tentatively named RNase V, was found in cell-free extracts of E. coli. Ribonuclease V is an exoribonuclease attacking mRNA from 5 to 3 terminal producing 5 -mononucleotides. It is characterized by the requirements of ribosomes, G and T factors, tRNA, K+ or NH4+, Mg2+, GTP and a sulfhydryl compound by its specificity and by the fact that it degrades poly U, poly A, T4 phage mRNA or E. coli mRNA, but not ribosomal RNA (132). 

The probable general structure of the dimers was established in elegant experiments by Fruchter and Crestfield 381) involving alkylation with iodoacetate. The two isomeric dimers referred to above behave identically in these reactions. The two active sites in the dimers behave just like that of the monomer. Histidines 12 and 119 both react, but the reactions are mutually exclusive. The proposed structure is outlined in Fig. 19. The tail of one monomer combines with the body of the other and vice versa. The His 12 and 119 pairs are now on separate molecules. When the dimers, fully inactivated by reaction with iodoacetate, are dissociated by heating at neutral pH, the following monomers would be expected native RNase (active), CM-His-12-RNase (inactive) CM-His-119-RNase (inactive), and di-CM-His-12-His-119-RNase (inactive). These were, in fact, found. About 2b% activity reappeared from the inactive dimer. Equally important the di-CM compound was found. This material... 

To obtain a template for the RACE, RNA isolation is necessary to rewrite it into cDNA. Precautions have to be taken, as RNA is a considerably more unstable molecule than DNA and, to add to the situation, its degrading enzymes are among the most stable known. Nowadays, most suppliers provide RNAse-free buffers and water, so DEPC (diethylpyrocarbonate) treatment to destroy RNAse activity is necessary only for home-made buffers. Kits for RNA preparation, either for total RNA or for mRNA isolation, do not require any DEPC treatment... 

At both ends of the mRNA, the ribosome display construct should include stemloops, 5 - and 3 -stemloops are known to stabilize mRNA against RNases in vivo as well as in vitro. The presence of stemloops is important, especially in the E. coli ribosome display system, because the extract used for in vitro translation contains high RNase activities. To date, five of twenty E. coli RNases have been shown to contribute to mRNA degradation (Hajnsdorf et al., 1996), and they are probably all present in the S30 extract. The efficiency of ribosome display was in-... 

RNase. Ribonuclease an enzyme which degrades RNA. It is ubiquitous in living organisms and is exceptionally stable. The prevention of RNase activity is the primary problem in handling RNA. 

Diseases Associated with Elevated Levels of RNase Activity in Body Fluids or Cell Extracts... 

RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases. The level of RNases is regulated by both activators and inhibitors. The... 

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