Integrated workflow for discovery of microprotein-coding small open reading frames.

Small open reading frame (smORF)-encoded microproteins, proteins containing less than 100-150 amino acids, are an emerging class of functional biomolecules. Here, we present a protocol for identifying translated smORFs in mammalian systems genome wide. We describe steps for generation of ribosome profiling (Ribo-seq) data, in silico translation of a transcriptome assembly to create an ORF database, and computational analysis of Ribo-seq to score individual smORFs for translation. Identification of translated smORFs is the first step to studying the functions of microproteins. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.

Sex- and Genotype-Dependent Nicotine-Induced Behaviors in Adolescent Rats with a Human Polymorphism (rs2304297) in the 3'-UTR of the CHRNA6 Gene.

In human adolescents, a single nucleotide polymorphism (SNP), rs2304297, in the 3'-UTR of the nicotinic receptor subunit gene, CHRNA6, has been associated with increased smoking. To study the effects of the human CHRNA6 3'-UTR SNP, our lab generated knock-in rodent lines with either C or G SNP alleles. The objective of this study was to determine if the CHRNA6 3'-UTR SNP is functional in the knock-in rat lines. We hypothesized that the human CHRNA6 3'-UTR SNP knock-in does not impact baseline but enhances nicotine-induced behaviors. For baseline behaviors, rats underwent food self-administration at escalating schedules of reinforcement followed by a locomotor assay and a series of anxiety tests (postnatal day (PN) 25-39). In separate cohorts, adolescent rats underwent 1- or 4-day nicotine pretreatment (2×, 30 µg/kg/0.1 mL, i.v.). After the last nicotine injection (PN 31), animals were assessed behaviorally in an open-field chamber, and brain tissue was collected. We show the human CHRNA6 3'-UTR SNP knock-in does not affect food reinforcement, locomotor activity, or anxiety. Further, 4-day, but not 1-day, nicotine exposure enhances locomotion and anxiolytic behavior in a genotype- and sex-specific manner. These findings demonstrate that the human CHRNA6 3'-UTR SNP is functional in our in vivo model.