Abstract
Defining the in vivo folding pathway of cellular RNAs is essential to understand how they reach their final native conformation. We here introduce a novel method, named Structural Probing of Elongating Transcripts (SPET-seq), that permits single-base resolution analysis of transcription intermediates' secondary structures on a transcriptome-wide scale, enabling base-resolution analysis of the RNA folding events. Our results suggest that cotranscriptional RNA folding in vivo is a mixture of cooperative folding events, in which local RNA secondary structure elements are formed as they get transcribed, and non-cooperative events, in which 5'-halves of long-range helices get sequestered into transient non-native interactions until their 3' counterparts have been transcribed. Together our work provides the first transcriptome-scale overview of RNA cotranscriptional folding in a living organism.