RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. The binding of fluorescently-labeled protein is then quantified in the sequencer. We used HiTS-RAP to measure the affinity of mutagenized libraries of GFP-binding and NELF-E binding aptamers to their respective targets and thereby identified regions in both aptamers that are critical for their RNA-protein interaction. We show that mutations additively affect binding affinity of the NELF-E binding aptamer, whose interaction depends mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depends primarily on secondary structure.
Eukaryotic RNA Polymerase II has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol2 initiation, capping, and pausing genome-wide. Our analyses reveal two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation, and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, Transcription Start Sites (TSSes) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes, and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is remarkably similar after the dramatic transcriptional changes caused by heat stress. Together, our results suggest that promoter- and enhancer-associated Pol2 is a regulatory nexus for integrating information across TSS ensembles.
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