Krishnamurthy Malathi

Small self-RNA generated by RNase L amplifies antiviral innate immunity

Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells. These RNAs activate signalling cascades that activate the genes that encode α- and β-interferon (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs. RIG-I and MDA5 interact with another CARD protein, interferon-β promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor (NF)-κB to the IFN-β gene. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L, by 2′,5′-linked oligoadenylate (2-5A) produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-β expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved 3′ of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-β expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-β during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-β in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential role in the innate antiviral immune response that relieves the requirement for direct sensing of non-self RNA.

An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors

We recently reported identification of a previously undescribed gammaretrovirus genome, xenotropic murine leukemia virus-related virus (XMRV), in prostate cancer tissue from patients homozygous for a reduced activity variant of the antiviral enzyme RNase L. Here we constructed a full-length XMRV genome from prostate tissue RNA and showed that the molecular viral clone is replication-competent. XMRV replication in the prostate cancer cell line DU145 was sensitive to inhibition by IFN-β. However, LNCaP prostate cancer cells, which are deficient in JAK1 and RNase L, were resistant to the effects of IFN-β against XMRV. Furthermore, DU145 cells rendered deficient in RNase L with siRNA were partially resistant to IFN inhibition of XMRV. Expression in hamster cells of the xenotropic and polytropic retrovirus receptor 1 allowed these cells to be infected by XMRV. XMRV provirus integration sites were mapped in DNA isolated from human prostate tumor tissue to genes for two transcription factors (NFATc3 and CREB5) and to a gene encoding a suppressor of androgen receptor transactivation (APPBP2/PAT1/ARA67). Our studies demonstrate that XMRV is a virus that has infected humans and is susceptible to inhibition by IFN and its downstream effector, RNase L.

HPC1/RNASEL Mediates Apoptosis of Prostate Cancer Cells Treated with 2′,5′-Oligoadenylates, Topoisomerase I Inhibitors, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2′,5′-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH2-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH2-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.

Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by Cdc2

Calcium (Ca2+) release from the endoplasmic reticulum (ER) controls numerous cellular functions including proliferation, and is regulated in part by inositol 1,4,5‐trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed intracellular Ca2+‐release channels found in many cell types. Although IP3R‐mediated Ca2+ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca2+ release during cell cycle progression are not known. Sequence analysis of IP3R1 reveals the presence of two putative phosphorylation sites for cyclin‐dependent kinases (cdks). In the present study, we show that cdc2/CyB, a critical regulator of eukaryotic cell cycle progression, phosphorylates IP3R1 in vitro and in vivo at both Ser421 and Thr799 and that this phosphorylation increases IP3 binding. Taken together, these results indicate that IP3R1 may be a specific target for cdc2/CyB during cell cycle progression.

Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex.

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2′,5′-oligoadenylate synthetase from prostate cancer cells

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5′-phosphorylated, 2′,5′-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2′,5′-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.

Cdc2/Cyclin B1 Interacts with and Modulates Inositol 1,4,5-Trisphosphate Receptor (Type 1) Functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.

RNase L Induces Autophagy via c-Jun N-terminal Kinase and Double-stranded RNA-dependent Protein Kinase Signaling Pathways

Background: Autophagy is induced by stress, starvation, and viral infections; however, the role of RNase L in autophagy has not been investigated. Results: Activation of RNase L induces autophagy involving JNK and PKR. Conclusion: Autophagy induced by activation of RNase L modulates viral growth. Significance: Our findings identify a novel role of RNase L in inducing autophagy affecting outcomes of viral infections. Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.

epigallocatechin-3-gallate suppresses proinflammatory cytokines and chemokines induced by Toll-like receptor 9 agonists in prostate cancer cells

Chronic inflammation of the prostate contributes to the increased risk of prostate cancer. Microbial pathogens in the prostate cause inflammation that leads to prostatitis and proliferative inflammatory atrophy frequently associated with the development of prostate cancer. Bacterial lipopolysaccharides and DNA mediate immune responses by engaging Toll-like receptor (TLR) 4 and 9, respectively. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) mimic bacterial DNA and signal through TLR9 to initiate innate immune responses. Here, we show that stimulation of DU145, PC3, or LnCap prostate cancer cells by the TLR9 agonists, CpG-ODN, induces mRNA expression of IL-6, IL-8, CXCL1, IP-10, CCL5, and TGFβ. In addition, activity of matrix metalloproteinase (MMP)-9 and -2 and cell migration increased on CpG-ODN treatment. Induction of cytokines and chemokines was mediated by NF-κB activation and translocation to the nucleus. Treatment with epigallocatechin-3-gallate (EGCG), the major constituent of green tea, prior to CpG-ODN stimulation, inhibits cytokine and chemokine gene induction, activity of MMP-9 and -2, and cell migration. EGCG treatment sequesters the p65 subunit of transcription factor NF-κB in the cytoplasm and inhibits transcriptional activity of the NF-κB-driven promoter in response to CpG-ODN. Our results suggest that the ability of the TLR9 agonists, CpG-ODN, to induce cytokines, chemokines, and MMP activity, as well as suppression by EGCG are independent of the androgen receptor and p53 status of the cells. EGCG may provide protective effects against inflammation in the prostate and benefit prostate cancer treatment.

RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1.

Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer.