Rnase

Ribonuclease, commonly abbreviated as RNase, is a type of nuclease catalyzing the degradation of RNA into smaller components. RNases can be generally classified into endoribonucleases and exoribonucleases, and encompass a few sub-classes within the EC 2.7 (for the phosphorolytic enzymes) and 3.1 (for the hydrolytic enzymes) types of enzymes. The active site of RNases looks like a rift valley, where all the active side residues construct the wall and bottom of the valley. Small substrate could fit perfectly in the middle of the active site, thus allowing for perfect interaction with the residues. Actually the site has a little curvature, which also happens in the substrate. Nevertheless, most exoribonucleases and endoribonucleases are usually not sequence specific. Animal organs are rich in RNase, especially in the pancreas.

Classification

Ribonuclease can be mainly groped into endoribonucleases and exoribonucleases. Similar to restriction enzymes that cleave highly specific sequences of double-stranded DNA, a variety of endoribonucleases recognizing and splitting specific sequences of single-stranded RNA have been recently classified, which include RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, and RNase V. RNase A is one of the first enzymes attained in large amounts in a purified, crystalline form and is predominantly hardy in common laboratory usage. Herein, it is usually isolated by boiling a crude cellular extract until all enzymes other than RNase A are denatured. RNase A has specificity for single-stranded RNAs and cleaves the 3'-end of unpaired C and U residues, finally generating a 3'-phosphorylated product via a 2',3'-cyclic monophosphate intermediate without the requirement for any cofactors. RNase H is also a frequently-used endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism to leave a 5'-phosphorylated product, which is assisted by an enzyme-bound divalent metal ion. Major types of exoribonucleases consist of polynucleotide phosphorylase (PNPase), RNase PH, RNase R, RNase D, RNase T, oligoribonuclease, exoribonuclease I, and exoribonuclease II, among which the PNPase and RNase PH also function as a nucleotidyltransferase. RNase R as a close homolog of RNase II could degrade RNA with secondary structures without the help of accessory factors.

Figure 1. Transphosphorylation and hydrolysis reactions catalyzed by RNase A. (Cuchillo C M; et al. 2011)

Function

Almost all studied organisms contain two different kinds of RNases, which indicated that the degradation of RNA is a very ancient and important process. RNases occupy an important position in many biological processes, like angiogenesis and self-incompatibility in flowering plants. Many stress-response toxins in prokaryotic toxin-antitoxin systems have been demonstrated to display RNase activity and homology. RNases play pivotal roles in the maturation of both messenger RNAs carrying genetic material for making proteins and non-coding RNAs that function in varied cellular processes. RNases could also clean cellular RNA that is no longer required. Additionally, active RNA degradation systems as the first defense against RNA viruses provide underlying machinery for more advanced cellular immune strategies such as RNAi. RNases are extremely common and usually result in very short lifespans for any RNA that is not in a protected environment since some cells secrete copious non-specific RNases such as RNase A and RNase T1. It is of noteworthy that all intracellular RNAs are protected from RNase activity through many strategies including 5' end capping, 3' end polyadenylation, as well as folding within an RNA protein complex. Another protection method is the application of RNase inhibitor that contains a relatively large fraction of cellular protein in some cell types, and could bind to certain RNases with the highest affinity of any protein-protein interaction. The dissociation constant for the RNase inhibitor-RNase A complex is about 20 fM under physiological conditions. Most laboratories exploit RNase inhibitor to protect their samples against degradation from environmental RNases.

RNase Contamination

The extraction of RNA in molecular biology experiments is critically complicated due to the existence of ubiquitous and hardy RNases that could degrade RNA samples. Certain RNases are extremely hardy and their inactivation is quite difficult in comparison with neutralizing DNases. Apart from the cellular released RNases, there are several RNases present in the environment. RNases have evolved to exhibit many extracellular functions in various organisms. For example, RNase 7 secreted by human skin, is a member of the RNase A superfamily and could function as a defense against potent antipathogen. The enzymatic RNase activity of these secreted RNases may not even be necessary for its new, extended function.

Inactivation of RNase

RNase is very stable and can be temporarily deactivated in some extreme conditions, but can be rapidly reactivated after the removal of restriction factor. Neither conventional high temperature and high pressure steam sterilization nor protein inhibitors can completely devitalize the activity of RNase. It is widely distributed in human skin, gloves therefore must be used in molecular biology experiments related to the preparation of RNA. Under normal circumstances, the internal and external surface of liquid dispenser is scrubbed with the 70% ethanol prepared by DEPC to basically meet the requirements. Glassware is usually baked in a 140 degree oven for 3 hours.

Reference

1. Cuchillo C M, Nogués M V, Raines R T. Bovine pancreatic ribonuclease: fifty years of the first enzymatic reaction mechanism. Biochemistry, 2011, 50(37):7835–7641.