No abstract

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5′ and 3′ nearest neighbor nucleotides (e.g., 5′ U, 3′ G). Current rational design approaches are wellsuited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA−protein interactions.

Micropeptides, encoded from small open reading frames of 300 nucleotides or less, are hidden throughout mammalian genomes, though few functional studies of micropeptides in the brain are published. Here, we describe a micropeptide known as the Plasticity–Associated Neural Transcript Short (Pants), located in the 22q11.2 region of the human genome, the microdeletion of which conveys a high risk for schizophrenia. Our data show that Pants is upregulated in early adulthood in the mossy fiber circuit of the hippocampus, where it exerts a powerful negative effect on long-term potentiation (LTP). Further, we find that Pants is secreted from neurons, where it associates with synapses but is rapidly degraded with stimulation. Pants dynamically interacts with Rtn4/Nogo-A, a well-studied regulator of adult plasticity. Pants interaction with Nogo-A augments its influence over postsynaptic AMPA receptor clustering, thus gating plasticity at adult synapses. This work shows that neural micropeptides can act as architectural modules that increase the functional diversity of the known proteome.

100-250 amino acid 'micropeptides' are hidden throughout mammalian genomes, though few functional studies of micropeptides are published. Here, we describe a micropeptide known as the Plasticity Associated Neural Transcript Short (Pants), located in the 22q11.2 region of the human genome, the microdeletion of which conveys a high risk for schizophrenia. Our data show that Pants is upregulated in early adulthood in the mossy fiber circuit of the hippocampus, where it exerts a powerful negative effect on long-term potentiation (LTP). Pants is secreted from neurons, where it associates with synapses but is rapidly degraded with stimulation. Pants dynamically interacts with Rtn4/Nogo, a well-studied regulator of adult plasticity. This study shows that neural micropeptides can act as architectural modules that increase the diversity of the known proteome.