Interactions between double-stranded RNA regulators and the protein kinase DAI.

Abstract

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The In mammalian cells, a regulatory mechanism involving an RNA-activated protein kinase and the eukaryotic initiation factor 2 (eIF-2) has been intensively studied. This initiation factor forms a ternary complex with GTP and Met-tRNAF and delivers the initiator tRNA to the ribosomal site of protein synthesis initiation. Discharged eIF-2 is subsequently released as a complex with GDP which must be replaced with GTP to permit the formation of another ternary complex in preparation for a further round of initiation. The factor is composed of three dissimilar subunits, cx, I, and -y. Phosphorylation of a single residue, serine-51 of the a subunit, inhibits translation by trapping a second initiation factor, the guanosine nucleotide exchange factor (or eIF-2B), which is required to catalyze the substitution of GTP for GDP in the discharged eIF-2 complex. Phosphorylation of sufficient eIF-2 can sequester all of the guanosine nucleotide exchange factor, thereby preventing eIF-2 recycling and halting the initiation pathway.In mammals, two protein kinases are capable of phosphorylating the a subunit of eIF-2 in this way (reviewed in references 20, 37, and 46). One of them, the heme-controlled repressor, is found chiefly in reticulocytes. It is activated by the absence of hemin, as well as by other stimuli, and serves to prevent the accumulation of globin in the absence of iron or heme. A second kinase, the double-stranded RNA-activated inhibitor (DAI; also referred to as P1 kinase, p68 kinase, P1/eIF-2a kinase, and PKdS, etc.) is present in a wide range of tissues. DAI is an important element in the host antiviral response, and its synthesis is induced at the transcriptional level by interferon (reviewed in references 21, 54, 56, and 59). The enzyme is ribosome associated (11,34) (8,24,26,36,47,60). It has also been implicated in cellular differentiation (23,52), in the inhibition of cell proliferation (6, 51), in the heat shock response (10), and possibly in transcriptional induction (61, 64). Moreover, in yeast cells, the related protein kinase GCN2 mediates the growth response to amino acid starvation (9). As its name implies, DAI is activated by doublestranded RNA (dsRNA). Other polyanions such as heparin can also activate it, while small, highly structured RNA molecules such as adenovirus VA RNA suppress its activation (38). Thus, DAI is a pivotal cellular regulatory enzyme whose level and activity are modulated by factors of both viral and cellular origin.The interactions between DAI and its RNA effectors are complicated and incompletely understood. The kinase is activated by dsRNA but not by DNA or DNA-RNA hybrids (22,32,35,58). Single-stranded RNA, either synthetic or natur...

PKR; a sentinel kinase for cellular stress

“…This allowed a model of the interaction between dsRBD and dsRNA (Nanduri et al, 1998) where conserved residues on the surface of each dsRBM make hydrogen bond contacts with the 2'-OH groups, as well as electrostatic interactions with the oxygens of the phosphate backbone (Nanduri et al, 1998). The¯exible linker region allows the protein to wrap around the minor groove of the dsRNA helix, covering about 11 bp, consistent with biochemical studies (Bevilacqua and Cech, 1996;Manche et al, 1992). The accuracy of this model is supported by the crystal structure of a complex between dsRNA and the second dsRBM of Xenopus laevis RNA-binding protein A (Ryter and Schultz, 1998).…”

“…DsRNA binding causes a major conformational change in PKR as evidenced by gel analyses of protein-RNA complexes (Manche et al, 1992), or by biophysical techniques using tryptophan¯uorescence quenching and neutron scattering (Carpick et al, 1997). The conformational change likely serves to uncover a catalytic site(s) for autophosphorylation, or to shift domains from di erent parts of the same PKR molecule (or from di erent PKR molecules within a protein-protein complex) into an active conformation.…”

Induction of stable RNA interference in mammalian cells

“…Indeed, recent preliminary data analyzing RNAi induced by siRNA duplexes that differ only in whether they form a perfect duplex or instead contain a central, singlenucleotide mismatch shows that, while both knock down their specific mRNA target with equal efficiency, the siRNA duplexes bearing the mismatch cause far fewer nonspecific effects (F Neipel, personal communication). Therefore, although it has been previously reported that efficient activation of the interferon response requires perfect dsRNAs of X30 bp in length, 31 I strongly recommend the inclusion of at least one additional bulge within the shRNA design, as seen in native miRNAs, instead of designing a perfect RNA helix. This is, of course, again achieved by introducing a mutation into the passenger (sense) strand of the shRNA (Figure 2), not into the guide strand.…”

High‐throughput RNAi screening to dissect cellular pathways: A how‐to guide

“…In mammalian cells, the application of long dsR-NA induces a strong interferon response [20,21]. Therefore, other strategies for RNA-induced gene silencing have to be applied.…”