Point Technology Illuminates The Processing Of Polymerase-Associated Intact Nascent Transcripts
Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing. HIGHLIGHTSO_LIPOINT methodology dissects intact nascent RNA processing C_LIO_LISpecificity of Xrn2 exonuclease in co-transcriptional RNA degradation C_LIO_LISplicing suppresses Xrn2-dependent premature termination C_LIO_LIDifferent kinetic classes of co-transcriptional splicing in human genes C_LI