SHAPE-seq counting data for Katz et al. Cell Systems (2019): "Synthetic 5’ UTRs can either up or down-regulate expression upon RBP binding"
ABSTRACT The construction of complex gene regulatory networks requires both inhibitory and up-regulatory modules. However, the vast majority of RNA-based regulatory “parts” are inhibitory. Using a synthetic biology approach combined with SHAPE-Seq, we explored the regulatory effect of RBP-RNA interactions in bacterial 5’-UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in-vivo: in the absence of RBPs, the RNA molecules can be found either in a molten state that is amenable to translation, or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene regulatory circuits at the post-transcriptional level.
Steps to reproduce
1. Alignment - paired-end reads were aligned to the reference composite sequence using Bowtie2 (--local). The composite reference was constructed from: 5s Rrna, PP7-wt mCherry sequence or PP7-Uss mCherry sequence. 2. RT Drop-off calling - RT drop-off positions were called using in house Perl script 3. Samples normalization - samples were normalized to the sum of read-ends in the maximal sample, so after normalization the sum of the read-ends in all the samples under comparison was equal. 4. Reactivity calculation - the drop-off values were used for reactivity calculation, inspired by Talkish et al. (2014). 5. The url of the fastq files: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE129163