Challenging the widespread belief that mammalian SINEs are neutral genomic parasites, we find that human Alu repeats regulate translation. The abundance of pol III directed Alu transcripts increases in response to various cellular insults; Alu RNA inhibits activation of PKR (the eIF2 kinase that is regulated by double stranded RNA), increasing protein synthesis. The Alus and regions within Alu that bind PKR will be identified by sequencing PKR bound Alu transcripts and by footprinting PKR complexes (Aim 1A). The functional importance of this binding site(s) will be determined by comparing the effects of selected Alu transcripts on PKR binding and inhibition (Aim 1A). The specificity of PKR-Alu complexes seemingly resides in their kinetic rather than thermodynamic stability; this stability may significantly enhance Alu RNA's PKR inhibitory activity. The stability both in vitro and in vivo of PKR complexes formed with divergent Alu transcripts will be compared and correlated with their differential effects on protein expression and PKR antagonism in vivo (Aim 1B). Alus are not amenable to the usual genetic tests for function so that alternative approaches are required. Using a controlled Pol III promoter in stably-transfected cells, the direct, immediate and specific responses of PKR activity and protein synthesis to increased levels of Alu RNA will be tested (Aim 1C). The evolution of this proposed translational regulatory role for mammalian SINE transcripts will be tested in rodent and rabbit cells by observing the effects of their SINE transcripts upon protein expression and PKR activity (Aim 2A). Human Alu RNA may be a highly specialized PKR inhibitor. Functional coevolution of mammalian SINE sequences with PKR will be investigated by comparing the effects of rodent, rabbit and human SINE transcripts on PKR activity and protein expression in homologous and heterologous systems and by comparing the kinetic stability of homologous and heterologous PKR-RNA complexes (Aim 2B).
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