Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999
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enhancer or the GAL4-VP16 transactivator as used in the current work. Furthermore, this study showed that in combination with a strong transcriptional activator, the hRPE65 promoter has the capacity to result in at least similar to or even higher transgene expression than the CMV promoter.
5. ACKNOWLEDGEMENTS
We thank the Foundation Fighting Blindness for the travel award provided to ENS to attend this meeting.
6. REFERENCES
Ali RR, Reichel MB, Hunt DM and Bhattacharya SS (1997). Gene therapy for inherited retinal degeneration.
Br J Ophthalmol 81:795-801.
Auricchio A, Kobinger G, Anand V, et al. (2001). Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model. Hum Mol Genet 10:3075-81.
Bennett J and Maguire AM (2000). Gene therapy for ocular disease. Mol Ther 1:501-5.
Boulanger A, Liu S, Henningsgaard AA, et al. (2000). The upstream region of the Rpe65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites. J Biol Chem 275:31274-82.
Cavailles V, Augereau P and Rochefort H (1993). Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells. Proc Natl Acad Sci U S A 90:203- 7.
Flannery JG, Zolotukhin S, Vaquero MI, et al. (1997). Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus. Proc Natl Acad Sci U S A 94:6916-21.
Guy J, Qi X, Muzyczka N and Hauswirth WW (1999). Reporter expression persists 1 year after adeno-associated virus-mediated gene transfer to the optic nerve. Arch Ophthalmol 117:929-37.
Jomary C, Chatelain G, Michel D, et al. (1999). Effect of targeted expression of clusterin in photoreceptor cells on retinal development and differentiation. J Cell Sci 112:1455-64.
Loser P, Jennings GS, Strauss M and Sandig V (1998). Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFkappaB. J Virol 72:180-90.
Nicoletti A, Kawase K and Thompson DA (1998). Promoter analysis of RPE65, the gene encoding a 61-kDa retinal pigment epithelium-specific protein. Invest Ophthalmol Vis Sci 39:637-44.
Prosch S, Stein J, Staak K, et al. (1996). Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro. Biol Chem Hoppe Seyler 377:195-201.
Rakoczy PE, Sarks SH, Daw N and Constable IJ (1999). Distribution of cathepsin D in human eyes with or without age-related maculopathy. Exp Eye Res 69:367-74.
Rolling F, Shen WY, Tabarias H, et al. (1999). Evaluation of adeno-associated virus-mediated gene transfer into the rat retina by clinical fluorescence photography. Hum Gene Ther 10:641-8.
Sadowski I, Ma J, Triezenberg S and Ptashne M (1988). GAL4-VP16 is an unusually potent transcriptional activator. Nature 335:563-4.
Sanftner MLH, Abel H, Hauswirth WW and Flannery JG (2001). Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Ther 4:622-9.
Sheikh MS, Augereau P, Chalbos D, et al. (1996). Retinoid regulation of human cathepsin D gene expression.
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Stone D, David A, Bolognani F, et al. (2000). Viral vectors for gene delivery and gene therapy within the endocrine system. J Endocrinol 164:103-18.
Wang F, Porter W, Xing W, et al. (1997). Identification of a functional imperfect estrogen-responsive element in the 5¢-promoter region of the human cathepsin D gene. Biochemistry 36:7793-801.
Weber M, Rabinowitz J, Provost N, et al. (2003). Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery. Mol Ther 7:774-81.
CHAPTER 38
CYTOKINE-INDUCED RETINAL DEGENERATION: ROLE OF SUPPRESSORS OF CYTOKINE SIGNALING (SOCS) PROTEINS IN PROTECTION OF THE NEURORETINA
Charles E. Egwuagu, Cheng-Hong Yu, Rashid M. Mahdi, Marie Mameza, Chikezie Eseonu, Hiroshi Takase, and Samuel Ebong*
1. INTRODUCTION
The vertebrate retina is comprised of a collection of highly specialized cell types, with each subtype playing unique roles and functions in the reception, transduction and conversion of incident light rays into visual images. The photo-transduction mechanism is extremely sensitive and minute alterations in the relative abundance of any retinal cell type can severely compromise the quality of the visual image. Because ganglion cells and other retinal neurons are terminally differentiated cells, it has been argued that evolutionarily conserved mechanisms must exist to protect retinal neurons from injury or death caused by exposure to environmental toxins or toxic bi-products of intermediary metabolism. For example, neuroretinal cells require protection from infectious agents that occasionally colonize and kill them, leading to permanent loss of such cells. Although intraocular infections is rapidly cleared by inflammatory cells, prolonged secretion of inflammatory cytokines in the retina may induce cytopathic effects that can produce retinal degenerative changes and possibly retinal degeneration. Although much effort has been made in characterizing chromosomal mutations and other biochemical lesions that may underlie the development of retinal degeneration, few studies have addressed the role of inflammation or inflammatory mediators in pathogenic mechanisms of retinal degenerative diseases. In fact, inflammation and dysregulation of activities of proinflammatory cytokines have been implicated in pathogenesis of other human degenerative diseases including Alzheimer’s disease and multiple sclerosis.
In this study, expression of the proinflammatory cytokine, interferon gamma (IFNg), was targeted to the lens of transgenic (TR) rats and the lens was used to serve as a depot
* National Eye Institute, National Institutes of Health, MD, U.S.A.
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for releasing IFNg into retina. This TR rat model allows us to directly test the hypothesis that prolonged exposure of retinal cells to pro-inflammatory cytokines, as may occur during persistent chronic infection of the retina, can induce retinal disease or retinal degenerative changes. We have also investigated mechanisms that may underlie protection of retinal cells from hypoxia. Because suppressors of cytokine signaling (SOCS) proteins play important role in regulating the activation, intensity and duration of cytokineand stress-induced signals,1-3 we examined whether retinal cells respond to cytokines and oxidative stress by inducing SOCS expression.
2. RESULTS
2.1. IFNg Induces Retinal Degenerative Changes and Apoptosis of Ganglion Cells
The chimeric construct used for generating the TR rats with constitutive expression of IFNg in the eye consists of the aA-Crystallin promoter fused to the mouse IFNg coding sequence.4 Because of lens-specificity of the aA-Crystallin promoter,5,6 transgene expression occurs preferentially in the lens and its effects are initially confined to this tissue. However, after the first month of postnatal life, the lens capsule begins to disintegrate, releasing lenticular material into the posterior chamber and vitreous cavity and this coincides temporally with appearance of the effects of IFNg on the retina. Appearance of retinal infoldings is observed in adult TR rats after three months of age (Fig. 38.1A) and number and size of these folds increase with time (data not shown). In addition, growth of the ganglion cell layer is significantly inhibited and its thickness approximates one-half of that seen in WT eye (data not shown).
The inhibition of ganglion cell growth is of particular importance in view of the critical functions of these cells in the visual process. It is therefore interesting to note that In situ detection of apoptotic cells by the TUNEL assay revealed presence of apoptotic cells in TR but not in WT rat retina (Fig. 38.1B). It is even more remarkable that the apoptotic response is restricted to the ganglion cell layer, suggesting that ganglion cells are more sensitive to the effects of IFNg. In addition, the morphological changes seen in the TR rat retina correlates with upregulated expression of interferon regulatory factors 1 (IRF-1), interferon
Figure 38.1. Chronic exposure of retinal cells can induce retinal degenerative changes.
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consensus binding protein (ICSBP), RT1-Ba (equivalent to mouse MHC class II), ICAM- 1 and TNFa genes (data not shown), suggesting that pathogenic effects of IFNg are mediated, in part, by altering normal patterns of gene expression in the eye.
2.2. Retinal Response to Inflammatory Cytokines is Under Feedback-Regulation by SOCS
We next examined whether cytokine activities in the retina are under feedback regulation by SOCS proteins. To establish that SOCS proteins are expressed in the retina, we isolated total RNA from human or murine retina, prepared cDNAs and subjected them to 30 cycles of PCR amplification as reported previously.7 We found that CIS, SOCS4, SOCS5, SOCS6, SOCS7 are constitutively expressed at very high levels in human retina while SOCS3 is not detectable even after 35 cycles of RT-PCR amplification. Although SOCS1 is also detected, it is at very low levels as detection required 35 cycles.
To examine whether SOCS expression is induced in retinal cells by proinflammatory cytokines it was necessary to establish that these cytokines do indeed activate gene transcription in the retina. Human retinal pigment epithelial (hRPE) or Müller cell line was stimulated with either interleukin 4 (IL-4) (10 ng/ml) or IFNg (100 u/ml) for 15 min and transcriptional activation was assessed by gel-shift assay. Activation by IL-4 or IFNg is mediate through STAT6 or STAT1, respectively.8 As shown in Fig. 38.3, a retarded band is induced by the STAT6 probe in nuclear extracts from cells stimulated with IL-4 while IFNg-
Figure 38.2. SOCS mRNA transcripts are constitutively expressed in the retina.
Figure 38.3. Inflammatory cytokines activate JAK/STAT signaling pathways in retinal cells.
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Figure 38.4. Retinal cell response to proinflammatory cytokines is under feedback regulation by SOCS proteins.
stimulated cells induce a STAT1 band-shift. Presence of STAT1 or STAT6 in the retarded band is confirmed by super-shift analysis indicated by SS.
We then examined whether SOCS proteins are induced in human retinal cells by proinflammatory cytokines. Müller or hRPE cells was washed, starved for 2 h before stimulation with IFNg or IL-4 and then analyzed for induction of SOCS expression by real-time RT-PCR. As indicated in Fig. 38.4, expression of SOCS1 or SOCS3 is induced by both cytokines, although intensity or kinetics of induction is different. Although SOCS6 or SOCS7 are constitutively expressed in these cells, they are not induced in response to these inflammatory cytokines.
We next examined whether retina cells respond to hypoxia by inducing SOCS expression. Mouse retina explants were propagated for varying amounts of time under hypoxia condition. Induction of vascular endothelial growth factor (VEGF) or hypoxia-inducing factor 1 (HIF-1a) expression, two markers of hypoxia, was used to verify that the cells were indeed exposed to significant hypoxia. Induction of SOCS expression was analyzed by realtime RT-PCR and as indicated in Fig. 38.5, only SOCS3 is induced.
3. DISCUSSION AND CONCLUSION
Inflammatory cells that mediate host immunity to intraocular pathogens produce copious amounts of pro-inflammatory cytokines, IFNg and IL-4. In this study we show that prolonged secretion of IFNg in the neuroretina promotes formation of retinal in-foldings in the photoreceptor layer and induces apoptotic death of retinal ganglion cells. However, these results appear to be at variance with the fact that humans are constantly infected with a variety of pathogens that induce expression of this proinflammatory with no evidence of
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Figure 38.5. Retinal cell response to hypoxia is under feedback regulation by SOCS proteins.
similar clinical or histological manifestation. It is however of note that these symptoms occur only in older rats and is therefore consistent with age-dependent occurrence of retinal degenerative diseases in older humans. The data suggests the possibility that retinal degeneration is a slow and progressive pathogenic condition that is initiated or amplified, in part, by proinflammatory cytokines produced by inflammatory cells elicited by low-grade persistent infections. Thus, retinal degenerative diseases may occur only in individuals that are not able to adequately control activities of these inflammatory mediators.
Neurotrophic factors and neuregulatory cytokines, such as, CNTF, OSM, CT1, LIF, IGFs and FGFs are also produced during inflammation and have been shown to counteract deleterious effects of proinflammatory cytokines. Thus, retinal degenerative diseases may occur in individuals that are not able to adequately control activities of inflammatory mediators or activate protective mechanism that confer protection of retinal neuronal cells from cytokineor stress-induced retinal damage. In the developing CNS and during spinal cord or brain injury, the steady-state levels of neuregulatory cytokines determines whether the neural progenitors would differentiate to neurons or astrocyte pathway and is therefore an important determinant of whether healing, repair or regeneration would occur.9-11 It is of note that most proinflammatory cytokines and neurotrophic/neuregulatory factors mediate their effects through activation Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway.12,13 Homeostatic regulation of activities of these competing factors is essential to normal physiology of the retina and under stringent control by endogenous feedback regulators of the JAK/STAT signal transduction pathway.14
Recent studies on cellular mechanisms that switch off signals induced by growth factors and cytokines have uncovered existence of a family of endogenous negative feedback regulators, generically called suppressors of cytokine signaling (SOCS).1-3 The best characterized members of the 8-member SOCS family are SOCS1, SOCS3, SOCS5 and CIS (cytokine induced SH2-domain protein) and expression one or more of these proteins is transiently induced by a wide variety of inflammatory and anti-inflammatory cytokines, including interferon IFNg, IL-3, IL-4, IL-6, IL-12, IL-13, leukemia-inhibitory factor, stem cell factor, CNTF, GM-CSF and leptin.1-3 Growth factors such as IGF-1, PDGF, FGFs, EGF, prolactin, growth hormone and erythropoietin also induce their expression Inhibitory effects of SOCS proteins derive from direct interactions with cytokine receptors and/or JAK kinases, thereby preventing recruitment of STATs to the signaling complex.1-3 In addition
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to functioning in a classical feedback regulatory loop, SOCS proteins can also inhibit responses to cytokines that are different from those that induce their expression. Interest in SOCS proteins stems from the belief that SOCS may serve to integrate multiple extracellular signals that converge on a target cell or tissue. We show here that CIS, SOCS5, SOCS6, SOCS7 are constitutively expressed at very high levels in human and murine retinas and although SOCS1, SOCS2 or SOCS3 is not detectable in the normal retina, expression of these SOCS members is significantly upregulated by proinflammatory cytokines in retinal cells. We further show that retinas maintained under hypoxic conditions express elevated levels of HIF-1a and VEGF mRNAs and expression of these genes results in significant induction of SOCS expression. However, in contrast to induction of SOCS1, SOCS2 and SOCS3 by retinal cells in response of to proinflammatory cytokines, response to hypoxia is under feedback regulation by only SOCS3, suggesting a remarkable specificity of SOCSmediated regulation in the retina.
In summary, we have shown in this study that similar to other neurodegenerative diseases, apoptotic death of retinal ganglion cells and retinal degeneration may result, in part, from chronic exposure of ocular cells to pro-inflammatory cytokines, as may occur in chronic inflammatory diseases of the eye. We also show that SOCS proteins are constitutively expressed in the retina and that retinal cells respond to cytotoxic cytokines or to hypoxic conditions by upregulating SOCS expression. These results are remarkable because SOCS proteins generally have a short half-life and are not detectable in many tissues. Demonstration that retinal cells respond to exposure to cytotoxic cytokines and hypoxia by upregulating SOCS expression, suggests that SOCS proteins may mitigate injurious effects of environmental, chemical or oxidative stress and should be exploited as neuroprotective agents of the mammalian retina.
4.REFERENCES
1.W. S. Alexander, D. J. Hilton, The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol. 22:503-29 (2004).
2.M. Kubo, T. Hanada, A. Yoshimura, Suppressors of cytokine signaling and immunity. Nat Immunol. 4(12):1169-76 (2003).
3.A. Yoshimura, H. Mori, M. Ohishi, D. Aki, T. Hanada, Negative regulation of cytokine signaling influences inflammation. Curr Opin Immunol. 15(6):704-8 (2003).
4.A. B. Chepelinsky, J. S. Khillan, K. A. Mahon, P. A. Overbeek, H. Westphal, J. Piatigorsky, Crystallin genes: lens specificity of the murine alpha A-crystallin gene. Environ Health Perspect. 75:17-24 (1987).
5.C. E. Egwuagu, R. M. Mahdi, C. C. Chan, J. Sztein, W. Li, J. A. Smith, A. B. Chepelinsky, Expression of inter- feron-gamma in the lens exacerbates anterior uveitis and induces retinal degenerative changes in transgenic Lewis rats. Clin Immunol. 91(2):196-205 (1999).
6.C. E. Egwuagu, J. Sztein, R. M. Mahdi, W. Li, C. Chao-Chan, J. A. Smith, P. Charukamnoetkanok P, A. B. Chepelinsky, IFN-gamma increases the severity and accelerates the onset of experimental autoimmune uveitis in transgenic rats. J Immunol. 162(1):510-7 (1999).
7.C. E. Egwuagu, C. R. Yu, M. Zhang, R. M. Mahdi, S. J. Kim, I. Gery, Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance. J Immunol, 168:3181-7 (2002).
8.C. R. Yu, R. M. Mahdi, S. Ebong, B. P. Vistica, J. Chen, Y. Guo, I. Gery, C. E. Egwuagu, Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling. J Immunol. 173(2):737-46 (2004).
9.A. M. Turnle, C. H. Fau, R. L. Rietze, J. R. Coonan, P. F. Bartlett, Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling. Nat Neurosci. 5(11):1155-62 (2002).
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10.A. M. Turnley, R. Starr, P. F. Bartlett, SOCS1 regulates interferon-gamma mediated sensory neuron survival. Neuroreport. 12(16):3443-5 (2001).
11.G. Wong, Y. Goldshmit, A. M. Turnley, Interferon-gamma but not TNF alpha promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells. Exp Neurol. 187(1):171-7 (2004).
12.J. E. Darnell, Jr., I. M. Kerr, G. R. Stark, Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415-21 (1994).
13.A. M. Turnley, P. F. Bartlett, Cytokines that signal through the leukemia inhibitory factor receptor-beta complex in the nervous system. J Neurochem. 74(3):889-99 (2000).
14.H. Paradis, R. L. Gendron, LIF transduces contradictory signals on capillary outgrowth through induction of stat3 and (P41/43) MAP kinase. J Cell Sci. 113(23):4331-9 (2000).
CHAPTER 39
DISEASE MECHANISMS AND GENE THERAPY IN A MOUSE MODEL FOR X-LINKED RETINOSCHISIS
Laurie L. Molday, Seok-Hong Min, Mathias W. Seeliger, Winco W.H. Wu, Astra Dinculescu, Adrian M. Timmers, Andreas Janssen, Felix Tonagel, Kristiane Hudl, Bernhard H.F. Weber, William W. Hauswirth, and
Robert S. Molday*
1. INTRODUCTION
X-linked retinoschisis (RS) is an inherited recessive macular degeneration that affects between 1 in 5000 and 1 in 25,000 males early in life (George et al., 1995; Sieving, 1998; Tantri et al., 2004). It is characterized by a loss in central vision, splitting of the retina with the appearance of spoke-like cystic cavities radiating from the parafoveal region of the retina, a loss in the b-wave of the electroretinogram (ERG), and progressive atrophy of the macula. In about 50% of the cases, bilateral schisis is observed in the peripheral retina with some loss in peripheral vision. During the course of the disease, complications can arise which include retinal detachment, vitreal hemorrhage and choroidal sclerosis.
The gene responsible for RS was identified in 1997 by positional cloning and shown to encode a retinal specific, discoidin domain containing protein known as RS1 or retinoschisin (Sauer et al., 1997). RS1 is primarily expressed in photoreceptors and secreted as a disulfide-linked homo-octameric complex (Molday et al., 2001; Reid et al., 1999). It is localized along the surface of photoreceptors at the level of the inner segment, outer nuclear and outer plexiform layers of the outer retina and the surface of bipolar cells in the inner retina.
* Laurie L. Molday, University of British Columbia, Vancouver, BC V6T 1Z3. Seok-Hong Min, University of Florida, Gainsville, FL 32610. Mathias W. Seeliger, University of Tubingen, D-72076 Tubingen, Germany, Winco W.H. Wu, University of British Columbia, Vancouver, BC V6T 1Z3. Astra Dinculescu, University of Florida, Gainesville, FL 32610. Adrian M. Timmers, University of Florida, Gainesville, FL 32610. Andreas Janssen, University of Regensburg, Regensburg, D-92043 Germany, Felix Tonagel, University of Tubingen, D-72076 Tubingen, Germany, Kristiane Hudl, University of Tubingen, D-72076 Tubingen, Germany. Bernhard H.F. Weber, University of Regensburg, D-92043 Regensburg, Germany, William W. Hauswirth, University of Florida, Gainsville, FL 32610. Robert S. Molday, University of British Columbia, Vancouver, BC V6T 1Z3.
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A mouse deficient in Rs1h, the mouse ortholog of the human RS1 gene, was generated by homologous recombination to determine the role of RS1 in retina structure and function (Weber et al., 2002). The hemizygous Rs1h-/Y mouse displays a number of features found in individuals with RS including a loss in the ERG b-wave, cystic structures within the inner retina, and progressive rod and cone degeneration. Morphological studies further reveal a highly disorganized retina with displacement of photoreceptor nuclei into the outer segment layer, merging of the outer and inner nuclear layers, gaps between bipolar cells, and disruption of the photoreceptor-bipolar synapse.
In this chapter we discuss disease mechanisms responsible for RS in relation to the structure of the RS1 protein and structural and functional recovery of the Rs1h-deficient mouse following adeno-associated viral (AAV) vector mediated gene therapy.
2. STRUCTURAL FEATURES AND DISEASE MECHANISMS
The 224 amino acid RS1 polypeptide is organized in four modules: an N-terminal signal peptide, an Rs1 domain, a discoidin (DS) domain, and a short C-terminal segment (Figure 39.1). Each module plays a key role in the biosynthesis, structure, subunit assembly and function of RS1 as a putative cell adhesion protein. Disease-causing missense mutations have been identified in each module (Consortium, 1998). The effect of selected mutations on RS1 has been reported (Wang et al., 2002; Wu and Molday, 2003).
2.1. Signal Peptide
At the N-terminus, a 23 amino acids hydrophobic signal peptide directs the nascent RS1 polypeptide to the endoplasmic reticulum (ER) membrane as an initial step in the processing of RS1 for secretion from cells (Sauer et al., 1997; Wu and Molday, 2003). Upon translocation of the polypeptide through the ER membrane, a signal peptidase in the ER lumen removes the signal peptide to produce the mature 201 amino acid RS1 polypeptide.
The effect of two disease-linked mutations in the signal peptide on the processing of RS1 has been examined (Wang et al., 2002; Wu and Molday, 2003). The L12H and L13P mutations cause a mislocalization of RS1 to the cytoplasm and rapid degradation via proteosomes. These amino acid substitutions most likely disrupt the helical conformation of the peptide thereby preventing its incorporation into the ER membrane.
Figure 39.1. Schematic linear representation of RS1 showing the four modules and selected disease-linked missense mutations in each module. Modules include: the signal peptide important for insertion of the nascent polypeptide into the ER membrane; the discoidin domain implicated in cell adhesion, and the Rs1 domain and C-terminal segment both of which contribute to the octameric assembly of RS1.