Transcription elongation rate affects nascent histone pre-mRNA folding and 3’ end processing

We report that slow transcription by mutant RNA pol II caused accumulation of polyadenylated histone mRNAs that extend past the stem loop processing site. UV irradiation, which decelerates pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3’ processing by slow pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem loop fails to fold in transcripts made by slow pol II, thereby explaining the absence of SLBP and failure to process 3’ ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure, and suggest a mechanism by which alternative processing could be controlled The effect of transcription elongation rate on Stem Loop Binding Protein (SLBP), Negative Elongation Factor (Nelf) and Cleavage and polyadenylation factor 77 (CSTF77) was analyzed by ChIP-seq in isogenic human HEK293 cell lines that inducibly express alpha-amanitin resistant mutants of the RNA polymerase II large subunit with slow elongation rates. Anti-pol II total nascent RNA isolation followed by targeted structural probing with DMS and 1M7 was employed to determine the nascent structure of replicative histone 3' stem loop. .