Human Topoisomerase 1 (TOP1) is a dynamic enzyme that functions to overcome topological constraints on the DNA that are caused by strand separation. In this study, we demonstrate that TOP1 is an RNA binding protein (RBP) as revealed by ultraviolet-crosslinked RNA immunoprecipitation (UV-RIP) analyses at intergenic and promoter regions in colon cancer cells treated with acute doses of tumor necrosis alpha (TNF-. Through biochemical methods, we found that TOP1 directly interacts with different RNAs. Furthermore, proteomic analyses reveal that RNAs add an additional layer of regulation by contributing to the maintenance of TOP1 ribonucleoprotein complexes.