What Is RNase? The Basics of Ribonuclease

What Is RNase?

RNases (or ribonucleases) are a class of hydrolytic enzymes that catalyzes both the in vivo and in vitro degradation of ribonucleic acid (RNA) molecules into smaller components. The nuclease operates at the level of transcription and translation and breaks down the RNA by cleaving the phosphorus-oxygen bonds.

RNase enzymes are categorized into two groups:

• Exoribonucleases: The exoribonuclease is an exonuclease ribonuclease that degrades RNA by removing terminal nucleotides from either the 5′ end or the 3′ end of the RNA molecule. It has six families of nucleases, including members such as RNase R, RNase T, and RNase D.
• Endoribonucleases: The endonuclease ribonuclease cleaves RNA molecules internally. It can cleave either single-stranded or double-stranded RNA, depending on the enzyme. It has several forms that structurally consist of either single proteins and or a complex of proteins with RNA.

Examples of single proteins are RNase III, RNase A, RNase T1, RNase T2, and RNase H. There are also complexes of the ribonuclease protein and RNA, including RNase P and the RNA-induced silencing complex.

Among all of these RNases, RNase A (or ribonuclease A) is the most commonly studied. It was first isolated from the pancreata of cattle and is also the first enzyme in which a complete amino acid sequence was determined.

The bovine pancreatic ribonuclease (or Ribonuclease A) is also known as a digestive enzyme. It specifically “digests” or hydrolyzes RNA polymers by endonuclease cleavage of the phosphodiester bonds. It leads to the formation of covalent links between adjacent ribonucleotide residues in RNA molecules.

The Importance of RNase in the Body

RNase is involved in the regulation of many catalytic functions of the body. Its normal function is in vivo enzymatic RNA degradation into smaller oligonucleotides.

Why & Where Do We Produce RNase?

RNase is ubiquitous. It’s found almost everywhere in every organism. But, for most studies, they are isolated from different mammalian tissues, such as the brain, liver, pancreas, skin cells, eosinophil, and several other somatic cells.

All three types of RNA—mRNA, tRNA, and rRNA—play a major role in gene expression and protein synthesis. Some other RNAs like siRNA and miRNA also have roles in regulating gene expression.

Sometimes, some RNAs are left unused. In these cases, the RNase enzyme causes the catalysis and degradation of the unused RNAs and clears the cell.

Why Do We Need It & What happens When We Don't Have Enough RNase?

Different RNases are involved in various functions in organisms. Functions range from clearing unused and unprocessed cellular RNA, to affecting biological processes such as self-incompatibility, plant flowering, and angiogenesis.

Some of the RNase functions based on their types are given below:

• RNase III is actively involved in the regulation of transcription and mRNA lifetime.
• RNase L is an interferon-induced RNase that destroys all RNA within a cell.
• RNase T is the major contributor to the 3′-to-5′ maturation of many stable RNAs.
• RNase R can degrade RNA with secondary structures without any help from accessory factors.
• RNase H cleaves 3’-O-P RNA bonds in an RNA/DNA hybrid duplex to form 3’- hydroxyl and 5’- phosphate terminated products.

The lack of RNases or any mutations in the protein subunits of the enzyme prevents our bodies from responding to the above-mentioned metabolic functions and causes several syndromes and genetic disorders. For example, mutations in any subunit of human RNase H2 cause Aicardi-Goutières syndrome (AGS), an autosomal recessive genetic disorder.

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What are the Sources of RNase?

RNase has been isolated and biochemically characterized from many organisms, including parasites, bacteria, fungi, plants, and a variety of tissues from mammals. In E.coli, nine ribonucleases have been found, including RNase G, RNase E, RNase III, RNase I, and RNase  P.

How Does RNase Work?

Typically, all RNase works by breaking the bonds between nucleotides, making nucleic acids shorter and more accessible to other enzymes. However, all of them are structurally different, facilitating metabolic function using different metabolic pathways. Below, we’ve covered the working mechanism of the most commonly studied RNase among the rest-ribonuclease A.

• RNase A is a small protein having 124 amino acid residues with no attached carbohydrate. Its structure involves 19 out of 20 amino acids, leaving tryptophan. It cleaves the phosphodiester linkage between the 5′-ribose of a nucleotide and the phosphate group attached to the 3′-ribose of an adjacent pyrimidine nucleotide. The resulting 2′- 3′-cyclic phosphate is hydrolyzed to the corresponding 3′-nucleoside phosphate.
• The His12 of ribonuclease acts as a base after accepting the 2′-OH proton of the sessile ribonucleic sugar ring.
• The reaction promotes a nucleophilic attack by the 2′-O on the more positively charged phosphorus (P) atom, thus creating a 2′-3′-cyclic ribonucleotide phosphate intermediate.
• The intermediate formation is also facilitated by the imidazole of His119. It acts as a general acid by donating its proton to the oxygen atom in the susceptible P-O-R’ bond.
• Then, the acid-base activities of the side-chains of both histidines are reversed.
• His12 acts as a general acid and donates its newly acquired proton to the 2′-3′-cyclic ribonucleotide phosphate intermediate. Conversely, His119 acts as a general base and accepts a proton from water to promote hydroxyl attacks on the same 2′-3′-cyclic intermediate.

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Application of RNase In Labs

RNA is the most common contamination in labs during DNA extraction and is very difficult to remove. But, the use of commercially available ribonuclease A makes it easier to cleave RNA contaminants and purify the DNA samples. It disrupts the phosphodiester bond between the adjacent nucleotides of RNA and facilitates the cleavage process.

RNase is an essential substrate in reaction mixtures including other reagents like polymerases, SDS, and tris buffers. The DNase and protease-free RNase solutions are used in several in vitro assays of molecular biology and biochem labs, including in vitro transcription and RT-PCR.

Some of the other main applications of RNases in labs are given below:

• DNA extraction processes
• Plasmid purification procedure
• Plasmid and genomic DNA preparation
• Mapping single-base mutations in DNA or RNA
• Removal of RNA from recombinant protein preparations
• cDNA synthesis

However, RNase may not be useful in every lab application. In RNA extraction procedures, for example, RNases are a huge problem. In this process, DNases are used to remove DNA, but complete elimination of RNase is difficult. For this purpose, RNA inhibitors are used to stop the catalytic activity of the ribonuclease.

RNase inhibitors are larger, acidic, and leucine amino acid-rich recombinant enzymes that form a tight complex with RNases for their inhibition in the given samples.

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RNase or ribonucleases are a group of enzymes that are involved in the degradation of RNA molecules. They are ubiquitous and are isolated and studied from different cells of different organisms. The variant types of RNases perform several metabolic functions in the body, including cleaning up unused RNA and angiogenesis.

These enzymes are used in labs for DNA extraction, plasmid purification, and many other workflows. It is precisely why high-quality and lab-grade RNases are necessary to get excellent end products. Besides these reagents, high-tech equipment is necessary to ensure the accuracy, precision, and reliability of obtained results.

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