RNA Aptamers Targeting the Cell Death Inhibitor CED-9 Induce Cell Killing in Caenorhabditis elegans

Abstract

Bcl-2 family proteins include anti-and proapoptotic factors that play important roles in regulating apoptosis in diverse species. Identification of compounds that can modulate the activities of Bcl-2 family proteins will facilitate development of drugs for treatment of apoptosis-related human diseases. We used an in vitro selection method named systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that bind the Caenorhabditis elegans Bcl-2 homolog CED-9 with high affinity and specificity and tested whether these aptamers modulate programmed cell death in C. elegans. Five CED-9 aptamers were isolated and classified into three groups based on their predicted secondary structures. Biochemical analyses indicated that two of these aptamers, R9-2 and R9-7, and EGL-1, an endogenous CED-9-binding proapoptotic protein, bound to distinct regions of CED-9. However, these two aptamers shared overlapping CED-9 binding sites with CED-4, another CED-9-binding proapoptotic factor. Importantly ectopic expression of these two aptamers in touch receptor neurons induced efficient killing of these neurons largely in a CED-3 caspase-dependent manner. These findings suggest that RNA aptamers can be used to modulate programmed cell death in vivo and can potentially be used to develop drugs to treat human diseases caused by abnormal apoptosis.Apoptosis is an essential cellular process that is critical for tissue homeostasis and animal development in metazoans. Abnormal inactivation of apoptosis can result in uncontrolled cell growth, leading to development of cancer and autoimmune disorders. By contrast, inappropriate activation of apoptosis can cause too much cell death, leading to neurodegenerative diseases and immunodeficiency (1, 2). Development of effective therapeutic methods that can correct or reverse inappropriate apoptosis is thus a critical issue in clinical medicine.Apoptosis is controlled and executed by an evolutionarily conserved cell death pathway (3, 4). At the center of this pathway is a family of conserved cell death regulators first defined by the human proto-oncogene bcl-2, which promotes cell survival and was identified by virtue of its overexpression in a number of B-cell lymphomas (5-8). Subsequently a family of Bcl-2-related proteins, characterized by the presence of at least one of four conserved Bcl-2 homology (BH) 3 domains, has been discovered and found in organisms as distantly related as Caenorhabditis elegans and humans (8). Members of this family can be either antiapoptotic or proapoptotic and can form heterodimers with selected family members to affect apoptosis. The mechanisms by which Bcl-2 family proteins regulate cell death appear to be quite complicated but likely involve modulation of the mitochondrial permeability and the release of crucial apoptogenic factors such as cytochrome c, apoptosisinducing factor, and endonuclease G, which promote activation of caspases, the cell death executors, and other cell death events such as chromosome fragmentation (8, 9). In ...

Clathrin and AP2 Are Required for Phagocytic Receptor-Mediated Apoptotic Cell Clearance in Caenorhabditis elegans

“…Finally, we tested whether CHC-1 and individual AP2 subunits could directly interact with LST-4 and/or DYN-1. As shown in Figure 7D, 35 S-labeled APA-2, APB-1, and DPY-23 interacted with GST-fused DYN-1 and LST-4, but not GST or GST-fused CED-9, an anti-apoptotic protein acting at the cell-killing stage [43]. Similarly, purified recombinant CHC-1C interacted with GST-DYN-1 and GST-LST-4 but not GST or GST-fused CED-9.…”

RNA aptamers interfering with nucleophosmin oligomerization induce apoptosis of cancer cells

“…Briefly, an RNA library was generated by in vitro transcription of an oligonucleotide library containing a central region of 49 randomized nucleotides flanked by constant sequences at both ends and a bacterial T7 promoter at the 5 0 end (Yang et al, 2006). For each round of selection, recombinant NPM (NPMHis 6 ; Figure 1a) was incubated with 32 P-labeled RNA library to yield RNA/ protein complexes, which were subsequently isolated by either EMSA or dot-blot assay.…”

“…SELEX was performed by using EMSA for the first five rounds and dot-blot assay for the last six rounds. The EMSA steps of SELEX were essentially performed as previously described (Yang et al, 2006). For dotblot assay, different amount of NPMHis 6 was dotted on nitrocellulose membrane and incubated with 32 P-labeled RNA.…”

CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics

“…Aptamers have been successfully generated to antagonize the prosurvival activity of CED-9 and kill cells through the normally programmed cell death pathway. These results suggest that RNA aptamers for key cell death regulators can yield, or serve as leads, to generate potent compounds that modulate apoptosis in vivo [29–31]. The transcription factor signal transducer and activator of transcription (Stat3) plays an essential role in embryonic development and assumes specialized tasks in many differentiated tissues.…”