Identification of Rho-dependent termination site in vivo using synthetic sRNA

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region at the 3'-end of mRNA without secondary structure. Determining the exact RDT site in vivo is challenging because the 3'-end of mRNA is rapidly removed by 3'- to 5'-exoribonuclease digestion after RDT. Here, we applied synthetic sRNA (sysRNA) to pinpoint RDT sites in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined assays of rapid amplification of cDNA 3'-ends, primer extension, and capillary electrophoresis, we could precisely locate and quantify mRNA 3'-ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA seems to protect the cleaved RNA 3'-ends from rapid degradation by 3'- to 5'-exonuclease, thereby stabilizing the mRNA 3'-end. We further verified that the signal intensity at the 3'-end was positively correlated with amounts of mRNA. By constructing a series of sysRNAs with target sites in close proximity, and comparing the difference in signal intensity at the 3'-end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within 21 bp range, which was determined as RDT site. Our results demonstrated the ability to use sysRNA as a novel tool to precisely localize RDTs in vivo and expanded the range of sysRNA applications. IMPORTANCEWith the emergence of more new tools for inhibiting gene expression, sysRNA, which was once widely used, has gradually faded out of people's attention due to its unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new function to identify RDT sites in vivo using sysRNA. This new feature is important because, since the discovery of the Rho protein in 1969, it has been difficult to specifically identify RDT sites in vivo due to the rapid processing of RNA 3'-ends by exonucleases, and sysRNA might provide a new way to address this challenge.