PT  - JOURNAL ARTICLE
AU  - Noa Katz
AU  - Roni Cohen
AU  - Oz Solomon
AU  - Beate Kaufmann
AU  - Noa Eden
AU  - Orna Atar
AU  - Zohar Yakhini
AU  - Sarah Goldberg
AU  - Roee Amit
TI  - An <em>in vivo</em> binding assay for RNA-binding proteins based on repression of a reporter gene
AID  - 10.1101/168625
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 168625
4099  - http://biorxiv.org/content/early/2018/04/13/168625.short
4100  - http://biorxiv.org/content/early/2018/04/13/168625.full
AB  - We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wide-swath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation. Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo.