RNases and their inhibitors

  • Published: 20 January 2022
We don't hear much about RNase inhibitors. They are these small things that professors like to go over quickly during classes and later you don’t really hear about them until you get more into cDNA synthesis or RT-qPCR. Maybe not even then. So why do we need to talk about them? RNases fulfil a broad range of biological roles, and they are among the most commonly secreted enzymes by cell. If that didn’t convince you, then also these small proteins might be the only things standing between a ruined assay and a successful first strand cDNA synthesis, RT-PCR, RT-qPCR, in vitro transcription, or translation experiment. And who wouldn’t want to know how to have a successful experiment. Right?
 
What are RNases?
RNases, also known as ribonucleases, are enzymes that degrade RNA into smaller components. They are also part of RNA processing, turnover, and gene regulation [1][2][3][4]. All organisms contain RNases, which shows that they belong to a very ancient process that is vital for life [1][5]. In fact, RNases are so abundant in organisms, that they often create a risk of contamination for any kind of RNA related science work, like cDNA synthesis, RT-PCR, RT-qPCR etc. There are also many different types of RNases, which are divided into two big groups: endoribonucleases and exoribonucleases [1].
 
Endoribonucleases are enzymes that cleave RNA molecules internally - they cut the RNA from the middle to degrade it [1][6][7]. They can further be divided into 3 groups: specificity for single-stranded RNA (e.g. RNase A), specificity for double-stranded RNA (e.g. RNase C) and specificity for other substrates (e.g. RNase H) [8]. Here is a short list of RNases that seemed the most interesting to our author.
Crystal structure of wild-type bovine pancreatic ribonuclease A. From the Protein Data Bank.

Crystal structure of wild-type bovine pancreatic ribonuclease A. From the Protein Data Bank.

  • RNase A is the most commonly used RNase in research. Most RNase inhibitors on the market also target specifically this RNase. Ribonuclease A belongs to the pancreatic ribonuclease family because it is found in high quantities in the pancreas of mammals and some reptiles [9]. RNase A specifically degrades single-stranded RNA by cleaving ssRNA after pyrimidine residues [8]. It is mostly found outside the cell, being secreted by the pancreas [10]. Fun fact: RNase A was the first artificially synthesised enzyme, which proved that biological molecules can be constructed artificially [11]. This discovery was rewarded with a Nobel prize [12].  
  • RNase B is also very often used in research or as a target for RNase inhibitors. It belongs to the same family of RNases as RNase A [9][13]. The two are similar, only differing by one residual sugar molecule, making RNase B glycosylated unlike RNase A, which is nonglycosylated [13]. Because of that RNase B is known to be more stable of the two [14].
  • RNase C (RNase III) is a Mg2+ -dependent hydrolytic endoribonuclease that specifically targets double-stranded rRNA by cleaving it and transforming the previous dsRNA into a mature RNA [1][2][8][15]. This is necessary because in case of stable RNAs the pre-RNAs need to be cut so that they would take their functional structure and start translating [1]. Therefore, RNase C is actively involved in the regulation of transcription and mRNA lifetime [1][2]. It also has a role in rRNA processing, posttranscriptional gene expression control, and defence against viral infection [2][15].
  • RNase H, like RNase C, is Mg2+-dependent [1]. It cleaves RNA in a DNA/RNA duplex during replication and repair [16]. It’s non-specific and catalyses the cleavage of RNA by a hydrolytic mechanism together with Mg2+ [1]. Because of its special RNA cleaving abilities, RNase H is important for cDNA synthesis and helps prevent aberrant chromosome replication [1][16].   
  • RNase P is interesting because it is one of the two known multiple turnover ribozymes in nature [5][17][18] - also a discovery that earned a Nobel prize [18][19]. One of its best-known functions is to cleave off a leader sequence from the 5' end of pre-tRNA to create mature tRNAs [1]. Another is regulating the transcription of various small non-coding RNAs [20]. Fun fact: While RNase P enzymes generally are composed of RNA plus one or more proteins, there has been discovered a form of RNase P that is a protein and does not contain any RNA - human mtRNase P [21]. It’s also used in COVID-19 tests as an internal control to prove that the test was done correctly.
  • RNase E is a bacterial ribonuclease. It’s responsible for stable RNA maturation (rRNA and tRNA) and most aspects of RNA processing and degradation (key player in mRNA decay) [1][22]. It also plays an important role in SOS responses in bacteria when it comes to stress from DNA damage [23].
  • RNase G is a paralogue of RNase E and important in chromosome separation and cell division [1][24]. It is involved in processing 5’end of 16S rRNA and is considered as one of the regulating components of cytoplasmic axial filament bundles [1][24]. Like RNase E it is responsible for the maturation of rRNAs, but unlike RNase E it is not necessary for viability [1][24].
 
Exoribonucleases are enzymes that cleave RNA molecules externally - they degrade starting from the 5’ or 3’ end [1][25]. It is not uncommon that first an endoribonuclease cuts the RNA from the middle and then exoribonuclease will finish the job of degrading the RNA completely [6]. This is because oftentimes RNAs are protected from 3’ by hairpin structure and 5’ end by CAP structure, so they first have to be cut in the middle for the exoribonuclease to approach some end that they can start degrading [6]. Here are some examples of exoribonucleases.
 
  • PNPase belongs to the PDX family [1]. It starts degrading RNA from the 3’ end, is highly conserved and participates in maturation and quality control of stable RNA in bacteria, plants and humans [1][26][27]. PNPase also plays a very important role in global mRNA decay [1][26].
  • RNase T is very important for the 3'-to-5' maturation of stable RNAs (like rRNA and tRNA), because unlike other RNases it can efficiently remove residues near a double-stranded stem [28][29]. RNase T is also capable of cleaving both DNA and RNA and has an unusual substrate specificity discriminating against cytosine residues [29][30].
  • Oligoribonuclease starts working after other RNases have already degraded RNA and left behind fragments of 2–5 nucleotides. Oligoribonuclease then degrades these short oligonucleotides to mononucleotides [1][31]. 
  • Exoribonuclease I (Xrn 1) is a highly conserved eukaryotic exoribonuclease [32]. It’s responsible for cytoplasmic mRNA degradation degrading single-stranded RNA from 5'-to-3' [32][33].
 
Where can RNases be found?
As previously mentioned all prokaryotic and eukaryotic organisms contain RNases, but not all organisms have the same RNases – there are homologues, but also completely different ones [1][5]. For example, Exoribonuclease I is only present among eukaryotes [32][33]. RNases are present in most of the cells of the body, some of them are also secreted outside the cell [1][10]. For example, in humans these secreted RNases help to protect our body from RNA viruses [1][34]. 
 
Why are RNases important?
While RNases may ruin your experiment by contaminating and destroying the RNA you are working with, they are still essential for life in general. A lot of the different functions and roles that RNases have has been mentioned already, so here is a short list just to sum up.
 
  • They play key roles in the maturation of RNA molecules (both mRNAs and non-coding RNAs) [1][2][20]. 
  • They are important for cell metabolism, gene expression, cell growth and differentiation [1].
  • They have a part in the induction of apoptosis [35].
  • They are the first defence against RNA viruses and are also part of more advanced cellular immune strategies (e.g RNA interference) [1][34].
  • They are part of stress-response toxins in prokaryotes [36]. 
  • They also play an important role in stress-response in prokaryotes [23] and eukaryotes [37]
  • They have an important role in angiogenesis [38].
  • They are included in the self-incompatibility mechanisms of plants [39]. 
  • Etc.
 
 What are RNase inhibitors?
As the name refers, RNase inhibitors inhibit RNase’s activity. More precisely they are proteins that bind with very high affinity to corresponding RNases [40]. Since RNase inhibitors usually occur in complex with the RNases they are inhibiting, they are important in all the same processes that were mentioned above [40]. One thing, that is specifically RNase inhibitor function, and is crucial for research and diagnostics, is protecting RNA research samples from degradation by RNases [41].
 
Porcine ribonuclease inhibitor. From the Protein Data Bank.
You may think that after purifying RNA there are no RNases that could cause problems for the experiment, but RNases can easily enter the sample from the lab. For example, if you put your bare hand against the sample (That’s never happened to anyone, right?) or if you haven’t properly cleaned your pipet or even from the air conditioner. 
 
While RNases are widespread, their inhibitors are not so commonly available in nature as synthetic options. RNase A, B and C are targeted by inhibitors in a laboratory environment. For other RNases there really aren’t many inhibitors on the market. Why is that? There is a simple reason - so far very few inhibitors have been properly characterised. One, that is well characterised though, is human placental RNase inhibitor, which is known to inhibit RNase A and its homologues in other mammals [40].
 
Like RNases, their inhibitors also tend to be species or at least class specific [41][42]. For example, mammalian RNase inhibitors can’t bind to certain types of RNases from other non-mammalian species [42]. This knowledge has been used in cancer research, where amphibian RNases were used to kill cancer cells [40][42]. Another application for this is RNase inhibitor therapy for allergies [40]. 
 
There are many other methods as well how RNase inhibitors could be used to cure cancer and other diseases, which continues to prove how important these little molecules are [42]. 
 
Currently the biggest application for RNase inhibitors is COVID-19 testing, since different types of PCR play a big role in getting accurate test results. As we have established by now, the only way to get excellent results, avoid RNase contamination and RNA degradation is by using RNase inhibitors while doing the PCR.
 
What is RiboGrip®?
RiboGrip® is Solis BioDyne’s very own in silico designed protein-based ribonuclease inhibitor, which inactivates RNases A and B. It inhibits the activity of these ribonucleases by noncovalently binding in a non-competitive mode at a ration 1:1. RiboGrip® is active at least for 1h at 60°C. It also has an enhanced stability at room temperature, thanks to Stability TAG technology, with no activity loss for up to 1 month. 
 
Where does RiboGrip® come from?
It is purified from an E. coli strain that carries an overproducing plasmid containing a RiboGrip® RNase Inhibitor gene. 
 
Where can I find it?
For your convenience RiboGrip® is already included in all the Solis BioDyne’s RT-qPCR   products and in the SOLIScript® 1-step CoV Kit, so you can be sure that there is no RNase contamination in your experiment, and you don’t have to make any extra purchases. You can of course get it from our e-shop as well.
 
Why RiboGrip®?
  • Great value 
  • Contains Stability TAG (so reaction set-up and shipment without ice)
  • Active at least for 1h at 60 °C
  • Protects RNA from RNase A
  • Reduces your carbon footprint and costs
 
Thanks to the incredible work we can now manufacture RiboGrip® on an industrial scale. For more information contact sales@solisbiodyne.com or buy directly from our e-shop.
References

[1] Arraiano, C. M., Andrade, J. M., Domingues, S., Guinote, I. B., Malecki, M., Matos, R. G, Moreira, R. N., Pobre, V., Reis, F. P., Saramago, M.; Silva, I. J., Viegas, S. C. (2010). The critical role of RNA processing and degradation in the control of gene expression. FEMS Microbiology Reviews, 34(5), 883–923, https://doi.org/10.1111/j.1574-6976.2010.00242.x

[2] Snow, S., Bacon, E., Bergeron, J., Katzman, D., Wilhelm, A., Lewis, O., Syangtan, D., Calkins, A.; Archambault, L., Anacker, M. L., Schlax, P. J. (2020). Transcript decay mediated by RNase III in Borrelia burgdorferi. Biochemical and Biophysical Research Communications, 529(2), 386–391. doi:10.1016/j.bbrc.2020.05.201

[3] Wellner, K., Betat, H., Mörl, M. (2018). A tRNA's fate is decided at its 3′ end: Collaborative actions of CCA-adding enzyme and RNases involved in tRNA processing and degradation. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1861(4), 433-441. doi: 10.1016/j.bbagrm.2018.01.012.

[4] Kang, S. O., Caparon, M. G., Cho, K. H. (2010). Virulence Gene Regulation by CvfA, a Putative RNase: the CvfA-Enolase Complex in Streptococcus pyogenes Links Nutritional Stress, Growth-Phase Control, and Virulence Gene Expression. Infection and Immunity, 78(6), 2754–2767. doi:10.1128/IAI.01370-09

[5] Hartmann, E., Hartmann, R.K. (2003). The enigma of ribonuclease P evolution. Trends in Genetics, 19(10), 561-569. https://doi.org/10.1016/j.tig.2003.08.007

[6] Tomecki, R. & Dziembowski, A. (2010). Novel endoribonucleases as central players in various pathways of eukaryotic RNA metabolism. RNA, 16(9), 1692–1724. https://doi.org/10.1261/rna.2237610

[7] Ming Li, W., Barnes, T., Lee, C. H. (2010). Endoribonucleases – enzymes gaining spotlight in mRNA metabolism., 277(3), 627–641. doi:10.1111/j.1742-4658.2009.07488.x

[8] Murchison, E.P. (2013). RNAases. In S. Maloy, K. Huges (Ed.). Brenner's Encyclopedia of Genetics (Second Edition, pp. 270). Academic Press. https://doi.org/10.1016/B978-0-12-374984-0.01355-3.

[9] Beintema J. J. & van der Laan J. M. (1986). Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases. FEBS Lett, 194(2), 338-42. doi: 10.1016/0014-5793(86)80113-2. 

[10] Gotte, G. & Menegazzi, M. (2019). Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs. Frontiers in immunology, 10, 2626. https://doi.org/10.3389/fimmu.2019.02626.

[11] Gutte, B. & Merrifield, R. B. (1971), "The Synthesis of Ribonuclease A", The Journal of Biological Chemistry, 246 (6), 1922–1941. https://doi.org/10.1016/S0021-9258(18)62396-8.

[12] Kresge, N., Simoni, R.D., Hill, R.L. (2006). The Solid Phase Synthesis of Ribonuclease A by Robert Bruce Merrifield. Journal of Biological Chemistry, 281(26): e21-e23. https://doi.org/10.1016/S0021-9258(20)55702-5.

[13] Rudd, P. M., Joao, H. C., Coghill, E., Fiten, P., Saunders, M. R., Opdenakker, G., Dwek, R. A. (1994). Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry, 33(1), 17–22. doi:10.1021/bi00167a003

[14] Arnold U., Schierhorn A., Ulbrich-Hofmann R. (1999). Modification of the unfolding region in bovine pancreatic ribonuclease and its influence on the thermal stability and proteolytic fragmentation. Eur J Biochem, 259(1-2), 470-5. doi: 10.1046/j.1432-1327.1999.00059.x. 

[15] Court, D. L., Gan, J., Liang, Y. H., Shaw, G. X., Tropea, J. E., Costantino, N., Waugh, D. S., & Ji, X. (2013). RNase III: Genetics and function; structure and mechanism. Annual review of genetics, 47, 405–431. https://doi.org/10.1146/annurev-genet-110711-155618

[16] Cerritelli, S. M. & Crouch, R. J. (2009). Ribonuclease H: the enzymes in eukaryotes. The FEBS journal, 276(6), 1494–1505. https://doi.org/10.1111/j.1742-4658.2009.06908.x

[17] Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell, 3 Pt 2, 849-57. doi: 10.1016/0092-8674(83)90117-4. 

[18] Robertson H. D., Altman S., Smith J. D. (1972). Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid presursor. J Biol Chem, 247(16), 5243-51. 

[19] The Nobel Prize in Chemistry 1989. The Nobel Prize. Retrieved 16 January 2022, from https://www.nobelprize.org/prizes/chemistry/1989/summary/

[20] Reiner, R., Ben-Asouli, Y., Krilovetzky, I., Jarrous, N. (2006). A role for the catalytic ribonucleoprotein RNase P in RNA polymerase III transcription. Genes & development, 20(12), 1621–1635. https://doi.org/10.1101/gad.386706

[21] Holzmann J., Frank P., Löffler E., Bennett K. L., Gerner C., Rossmanith, W. (2008). RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. Cell, 135(3), 462-74. doi: 10.1016/j.cell.2008.09.013. PMID: 18984158.

[22] Mackie, G. (2013). RNase E: at the interface of bacterial RNA processing and decay. Nat Rev Microbiol 11, 45–57. https://doi.org/10.1038/nrmicro2930

[23] Manasherob, R., Miller, C., Kim, K., Cohen, S. N. (2012). Ribonuclease E Modulation of the Bacterial SOS Response. PLoS ONE, 7(6), e38426. doi:10.1371/journal.pone.0038426

[24] Wachi, M., Umitsuki, G., Shimizu, M., Takada, A., Nagai, K. (1999). Escherichia coli cafA Gene Encodes a Novel RNase, Designated as RNase G, Involved in Processing of the 5′ End of 16S rRNA. Biochemical and Biophysical Research Communications, 259(2), 0–488. doi:10.1006/bbrc.1999.0806

[25] Zuo, Y. & Deutscher, M. P. (2001). Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic acids research, 29(5), 1017–1026. https://doi.org/10.1093/nar/29.5.1017

[26] Briani F., Carzaniga T., Dehò G. (2016). Regulation and functions of bacterial PNPase. Wiley Interdiscip Rev RNA, 7(2):241-58. doi: 10.1002/wrna.1328. 

[27] Portnoy, V., Palnizky, G., Yehudai-Resheff, S., Glaser, F., Schuster, G. (2008). Analysis of the human polynucleotide phosphorylase (PNPase) reveals differences in RNA binding and response to phosphate compared to its bacterial and chloroplast counterparts. RNA, 14(2), 297–309. https://doi.org/10.1261/rna.698108

[28] Zuo, Y. & Deutscher, M. P. (1999). The DNase activity of RNase T and its application to DNA cloning, Nucleic Acids Research, 27(20), 4077–4082, https://doi.org/10.1093/nar/27.20.4077

[29] Zuo, Y., Deutscher, M. P. (2002). The Physiological Role of RNase T Can Be Explained by Its Unusual Substrate Specificity. Journal of Biological Chemistry, 277(33), 29654–29661. doi:10.1074/jbc.m204252200

[30] Hsiao, Y. Y., Duh, Y., Chen, Y. P., Wang, Y. T., & Yuan, H. S. (2012). How an exonuclease decides where to stop in trimming of nucleic acids: crystal structures of RNase T-product complexes. Nucleic acids research, 40(16), 8144–8154. https://doi.org/10.1093/nar/gks548

[31] Ghosh, S. & Deutscher, M. P. (1999). Oligoribonuclease is an essential component of the mRNA decay pathway. Proceedings of the National Academy of Sciences, 96(8), 4372–4377. doi:10.1073/pnas.96.8.4372

[32] Nagarajan, V. K., Jones, C. I., Newbury, S. F., & Green, P. J. (2013). XRN 5'→3' exoribonucleases: structure, mechanisms and functions. Biochimica et biophysica acta, 1829(6-7), 590–603. https://doi.org/10.1016/j.bbagrm.2013.03.005

[33] Andrew M. P., Kristina D., Catherine M., Richard D. K., Arlen W. J. (1998). Mutational analysis of exoribonuclease I from Saccharomyces cerevisiae, Nucleic Acids Research, 26(16), 3707–3716. https://doi.org/10.1093/nar/26.16.3707

[34] Ilinskaya, O. N., & Mahmud, R. S. (2014). Ribonucleases as antiviral agents. Molecular biology, 48(5), 615–623. https://doi.org/10.1134/S0026893314040050

[35] Sato, A., Naito, T., Hiramoto, A., Goda, K., Omi, T., Kitade, Y., Sasaki, T., Matsuda, A., Fukushima, M., Wataya, Y., Kim, H.-S. (2010). Association of RNase L with a Ras GTPase-activating-like protein IQGAP1 in mediating the apoptosis of a human cancer cell-line, 277(21), 4464–4473. doi:10.1111/j.1742-4658.2010.07833.x

[36] Ramage, H. R., Connolly, L. E., Cox, J. S. (2009). Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: implications for pathogenesis, stress responses, and evolution. PLoS Genet, 5(12):e1000767. doi: 10.1371/journal.pgen.1000767. 

[37] Thompson, D. M. & Parker, R (2009). The RNase Rny1p cleaves tRNAs and promotes cell death during oxidative stress in Saccharomyces cerevisiae. J Cell Biol, 185 (1): 43–50. doi: https://doi.org/10.1083/jcb.200811119

[38] Lasch, M., Kumaraswami, K., Nasiscionyte, S., Kircher, S., van den Heuvel, D., Meister, S., Ishikawa-Ankerhold, H., & Deindl, E. (2020). RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue. Frontiers in physiology, 11, 576736. https://doi.org/10.3389/fphys.2020.576736

[39] Williams, J. S., Wu, L., Li, S., Sun, P., Kao, T.-H. (2015). Insight into S-RNase-based self-incompatibility in Petunia: recent findings and future directions. Frontiers in Plant Science, 6(), –. doi:10.3389/fpls.2015.00041

[40] Yakovlev, G. I., Mitkevich, V. A., Makarov, A. A. (2006). Ribonuclease Inhibitors. Molecular Biology, 40(6): 867-874. DOI: 10.1134/S0026893306060045

[41] Dickson, K. A., Haigis, M. C., Raines, R. T. (2005). Ribonuclease inhibitor: structure and function. Progress in nucleic acid research and molecular biology, 80, 349–374. https://doi.org/10.1016/S0079-6603(05)80009-1

[42] Ardelt, W., Shogen, K., Darzynkiewicz, Z. (2008). Onconase and amphinase, the antitumor ribonucleases from Rana pipiens oocytes. Current pharmaceutical biotechnology, 9(3), 215–225. https://doi.org/10.2174/138920108784567245