Ribonuclease L Explained

Ribonuclease L or RNase L (for latent), known sometimes as ribonuclease 4 or 2'-5' oligoadenylate synthetase-dependent ribonuclease, is an interferon (IFN)-induced ribonuclease which, upon activation, destroys all RNA within the cell (both cellular and viral). RNase L is an enzyme that in humans is encoded by the RNASEL gene.^[1]

This gene encodes a component of the interferon-regulated 2'-5'oligoadenylate (2'-5'A) system that functions in the antiviral and antiproliferative roles of interferons. RNase L is activated by dimerization, which occurs upon 2'-5'A binding, and results in cleavage of all RNA in the cell. This can lead to activation of MDA5, an RNA helicase involved in the production of interferons.

Synthesis and activation

RNase L is present in very minute quantities during the normal cell cycle. When interferon binds to cell receptors, it activates transcription of around 300 genes to bring about the antiviral state. Among the enzymes produced is RNase L, which is initially in an inactive form. A set of transcribed genes codes for 2'-5' Oligoadenylate Synthetase (OAS).^[2] The transcribed RNA is then spliced and modified in the nucleus before reaching the cytoplasm and being translated into an inactive form of OAS. The location of OAS in the cell and the length of the 2'-5' oligoadenylate depends on the post-transcriptional and post-translational modifications of OAS.^[2]

OAS is only activated under a viral infection, when a tight binding of the inactive form of the protein with a viral dsRNA, consisting of the retrovirus' ssRNA and its complementary strand, takes place. Once active, OAS converts ATP to pyrophosphate and 2'-5'-linked oligoadenylates (2-5A), which are 5' end phosphorylated.^[3] 2-5 A molecules then bind to RNase L, promoting its activation by dimerization. In its activated form RNase L cleaves all RNA molecules in the cell leading to autophagy and apoptosis. Some of the resulting RNA fragments can also further induce the production of IFN-β as noted in the Significance section.^[4]

This dimerization and activation of RNase L can be recognized using Fluorescence Resonance Energy Transfer (FRET), as oligoribonucleotides containing a quencher and a fluorophore on opposite sites are added to a solution with inactive RNase L. The FRET signal is then recorded as the quencher and the fluorophore are very close to each other. Upon the addition of 2-5A molecules, RNase L becomes active, cleaving the oligoribonucleotides and interfering in the FRET signal.^[5]

In vitro, RNase L can be inhibited by curcumin.^[6]

Significance

RNase L is part of the body's innate immune defense, namely the antiviral state of the cell. When a cell is in the antiviral state, it is highly resistant to viral attacks and is also ready to undergo apoptosis upon successful viral infection. Degradation of all RNA within the cell (which usually occurs with cessation of translation activity caused by protein kinase R) is the cell's last stand against a virus before it attempts apoptosis.

Interferon beta (IFN-β), a type I interferon responsible for antiviral activity, is induced by RNase L and melanoma differentiation-associated protein 5 (MDA5) in the infected cell. The relationship between RNase L and MDA5 in the production of IFNs has been confirmed with siRNA tests silencing the expression of either molecule and noting a marked decline in IFN production.^[7] MDA5, an RNA helicase, is known to be activated by complex high molecular weight dsRNA transcribed from the viral genome.^{[7] [8]} In a cell with RNase L, MDA5 activity may be further enhanced.^[7] When active, RNase L cleaves and identifies viral RNA and feeds it into MDA5 activation sites, enhancing the production of IFN-β. The RNA fragments produced by RNase L have double stranded regions, as well as specific markers, that allow them to be identified by the RNase L and MDA5.^[4] Some studies have suggested that high levels of RNase L may actually inhibit IFN-β production, but a clear linkage still exists between RNase L activity and IFN-β production.^[4]

Furthermore, it has been shown that RNase L is involved in many diseases. In 2002, the "hereditary prostate cancer 1" locus (HPC1) was mapped to the RNASEL gene, indicating that mutations in this gene cause a predisposition to prostate cancer.^{[9] [10] [11]} Impairments of the OAS/RNase L pathway in chronic fatigue syndrome (CFS) have been investigated.^{[12] [13]}

Further reading

• Chakrabarti A, Jha BK, Silverman RH . New insights into the role of RNase L in innate immunity . Journal of Interferon & Cytokine Research . 31 . 1 . 49–57 . January 2011 . 21190483 . 3021357 . 10.1089/jir.2010.0120 .
• Castelli J, Wood KA, Youle RJ . The 2-5A system in viral infection and apoptosis . Biomedicine & Pharmacotherapy . 52 . 9 . 386–90 . 1999 . 9856285 . 10.1016/S0753-3322(99)80006-7 .
• Leaman DW, Cramer H . Controlling gene expression with 2-5A antisense . Methods . 18 . 3 . 252–65 . July 1999 . 10454983 . 10.1006/meth.1999.0782 .
• Silverman RH . Implications for RNase L in prostate cancer biology . Biochemistry . 42 . 7 . 1805–12 . February 2003 . 12590567 . 10.1021/bi027147i .
• Kieffer N, Schmitz M, Scheiden R, Nathan M, Faber JC . Involvement of the RNAse L gene in prostate cancer . Bulletin de la Société des Sciences Médicales du Grand-Duché de Luxembourg . 1 . 21–8 . 2006 . 16869093 .
• Bisbal C, Silverman RH . Diverse functions of RNase L and implications in pathology . Biochimie . 89 . 6–7 . 789–98 . 2007 . 17400356 . 2706398 . 10.1016/j.biochi.2007.02.006 .
• Carter BS, Beaty TH, Steinberg GD, Childs B, Walsh PC . Mendelian inheritance of familial prostate cancer . Proceedings of the National Academy of Sciences of the United States of America . 89 . 8 . 3367–71 . April 1992 . 1565627 . 48868 . 10.1073/pnas.89.8.3367 . 1992PNAS...89.3367C . free .
• Dong B, Xu L, Zhou A, Hassel BA, Lee X, Torrence PF, Silverman RH . Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase . The Journal of Biological Chemistry . 269 . 19 . 14153–8 . May 1994 . 7514601 . 10.1016/S0021-9258(17)36767-4 . free .
• Bisbal C, Martinand C, Silhol M, Lebleu B, Salehzada T . Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway . The Journal of Biological Chemistry . 270 . 22 . 13308–17 . June 1995 . 7539425 . 10.1074/jbc.270.22.13308 . free .
• Zhou A, Hassel BA, Silverman RH . Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action . Cell . 72 . 5 . 753–65 . March 1993 . 7680958 . 10.1016/0092-8674(93)90403-D . free .
• Hassel BA, Zhou A, Sotomayor C, Maran A, Silverman RH . A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon . The EMBO Journal . 12 . 8 . 3297–304 . August 1993 . 7688298 . 413597 . 10.1002/j.1460-2075.1993.tb05999.x .
• Smith JR, Freije D, Carpten JD, Grönberg H, Xu J, Isaacs SD, Brownstein MJ, Bova GS, Guo H, Bujnovszky P, Nusskern DR, Damber JE, Bergh A, Emanuelsson M, Kallioniemi OP, Walker-Daniels J, Bailey-Wilson JE, Beaty TH, Meyers DA, Walsh PC, Collins FS, Trent JM, Isaacs WB . 6 . Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search . Science . 274 . 5291 . 1371–4 . November 1996 . 8910276 . 10.1126/science.274.5291.1371 . 1996Sci...274.1371S . 42684655 .
• Egesten A, Dyer KD, Batten D, Domachowske JB, Rosenberg HF . Ribonucleases and host defense: identification, localization and gene expression in adherent monocytes in vitro . Biochimica et Biophysica Acta (BBA) - Molecular Cell Research . 1358 . 3 . 255–60 . October 1997 . 9366257 . 10.1016/S0167-4889(97)00081-5 .
• Eeles RA, Durocher F, Edwards S, Teare D, Badzioch M, Hamoudi R, Gill S, Biggs P, Dearnaley D, Ardern-Jones A, Dowe A, Shearer R, McLellan DL, McLennan DL, Norman RL, Ghadirian P, Aprikian A, Ford D, Amos C, King TM, Labrie F, Simard J, Narod SA, Easton D, Foulkes WD . 6 . Linkage analysis of chromosome 1q markers in 136 prostate cancer families. The Cancer Research Campaign/British Prostate Group U.K. Familial Prostate Cancer Study Collaborators . American Journal of Human Genetics . 62 . 3 . 653–8 . March 1998 . 9497242 . 1376940 . 10.1086/301745 .
• Dong B, Silverman RH . Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L . Nucleic Acids Research . 27 . 2 . 439–45 . January 1999 . 9862963 . 148198 . 10.1093/nar/27.2.439 .
• Carpten JD, Makalowska I, Robbins CM, Scott N, Sood R, Connors TD, Bonner TI, Smith JR, Faruque MU, Stephan DA, Pinkett H, Morgenbesser SD, Su K, Graham C, Gregory SG, Williams H, McDonald L, Baxevanis AD, Klingler KW, Landes GM, Trent JM . 6 . A 6-Mb high-resolution physical and transcription map encompassing the hereditary prostate cancer 1 (HPC1) region . Genomics . 64 . 1 . 1–14 . February 2000 . 10708513 . 10.1006/geno.1999.6051 .
• Zhou A, Nie H, Silverman RH . Analysis and origins of the human and mouse RNase L genes: mediators of interferon action . Mammalian Genome . 11 . 11 . 989–92 . November 2000 . 11063255 . 10.1007/s003350010194 . 35650613 .
• Dong B, Niwa M, Walter P, Silverman RH . Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p . RNA . 7 . 3 . 361–73 . March 2001 . 11333017 . 1370093 . 10.1017/S1355838201002230 .

Notes and References

1. Squire J, Zhou A, Hassel BA, Nie H, Silverman RH . Localization of the interferon-induced, 2-5A-dependent RNase gene (RNS4) to human chromosome 1q25 . Genomics . 19 . 1 . 174–5 . January 1994 . 7514564 . 10.1006/geno.1994.1033 .
2. Book: Sarkar SN, Pandey M, Sen GC . Interferon Methods and Protocols . Assays for the Interferon-Induced Enzyme 2′,5′ Oligoadenylate Synthetases . Methods in Molecular Medicine . 116 . 81–101 . 2005 . 16000856 . 10.1385/1-59259-939-7:081 . Human Press Inc. . 978-1-58829-418-0 .
3. Liang SL, Quirk D, Zhou A . RNase L: its biological roles and regulation . IUBMB Life . 58 . 9 . 508–14 . September 2006 . 17002978 . 10.1080/15216540600838232 . free .
4. Banerjee S, Chakrabarti A, Jha BK, Weiss SR, Silverman RH . Cell-type-specific effects of RNase L on viral induction of beta interferon . mBio . 5 . 2 . e00856-14 . February 2014 . 24570368 . 3940032 . 10.1128/mBio.00856-14 .
5. Book: Thakur CS, Xu Z, Wang Z, Novince Z, Silverman RH . Interferon Methods and Protocols . A Convenient and Sensitive Fluorescence Resonance Energy Transfer Assay for RNase L and 2′,5′ Oligoadenylates . Methods in Molecular Medicine . 116 . 103–13 . 2005 . 16000857 . 10.1385/1-59259-939-7:103 . Human Press Inc. . 978-1-58829-418-0 .
6. Gupta A, Rath PC . Curcumin, a natural antioxidant, acts as a noncompetitive inhibitor of human RNase L in presence of its cofactor 2-5A in vitro . BioMed Research International . 2014 . 817024 . 2014 . 25254215 . 4165196 . 10.1155/2014/817024 . free .
7. Luthra P, Sun D, Silverman RH, He B . Activation of IFN-β expression by a viral mRNA through RNase L and MDA5 . Proceedings of the National Academy of Sciences of the United States of America . 108 . 5 . 2118–23 . February 2011 . 21245317 . 3033319 . 10.1073/pnas.1012409108 . 2011PNAS..108.2118L . free .
8. Pichlmair A, Schulz O, Tan CP, Rehwinkel J, Kato H, Takeuchi O, Akira S, Way M, Schiavo G, Reis e Sousa C . 6 . Activation of MDA5 requires higher-order RNA structures generated during virus infection . Journal of Virology . 83 . 20 . 10761–9 . October 2009 . 19656871 . 2753146 . 10.1128/JVI.00770-09 .
9. Smith JR, Freije D, Carpten J, Gronberg H, Xu J, Isaacs S, Brownstein M, Bova S, Guo H, Bujnovszky P, Nusskern D, Damber J, Bergh A, Emanuelsson M, Kallioniemi O, Walker-Daniels J, Bailey-Wilson J, Beaty T, Meyers D, Walsh PC, Collins FS, Trent J, Isaacs W . 6 . Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search . Science . 274 . 5291 . 1371–4 . Nov 1996 . 8910276 . 10.1126/science.274.5291.1371 . 1996Sci...274.1371S . 42684655 .
10. Web site: Entrez Gene: RNASEL ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent).
11. Carpten J, Nupponen N, Isaacs S, Sood R, Robbins C, Xu J, Faruque M, Moses T, Ewing C, Gillanders E, Hu P, Bujnovszky P, Makalowska I, Baffoe-Bonnie A, Faith D, Smith J, Stephan D, Wiley K, Brownstein M, Gildea D, Kelly B, Jenkins R, Hostetter G, Matikainen M, Schleutker J, Klinger K, Connors T, Xiang Y, Wang Z, De Marzo A, Papadopoulos N, Kallioniemi OP, Burk R, Meyers D, Grönberg H, Meltzer P, Silverman R, Bailey-Wilson J, Walsh P, Isaacs W, Trent J . 6 . Germline mutations in the ribonuclease L gene in families showing linkage with HPC1 . Nature Genetics . 30 . 2 . 181–4 . February 2002 . 11799394 . 10.1038/ng823 . 2922306 .
12. Nijs J, De Meirleir K . Impairments of the 2-5A synthetase/RNase L pathway in chronic fatigue syndrome . In Vivo . 19 . 6 . 1013–21 . Nov–Dec 2005 . 16277015 .
13. Suhadolnik RJ, Peterson DL, O'Brien K, Cheney PR, Herst CV, Reichenbach NL, Kon N, Horvath SE, Iacono KT, Adelson ME, De Meirleir K, De Becker P, Charubala R, Pfleiderer W . 6 . Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome . Journal of Interferon & Cytokine Research . 17 . 7 . 377–85 . July 1997 . 9243369 . 10.1089/jir.1997.17.377 .