The various steps of RNA polymerase II transcription, including transcription initiation, splicing, and termination, are interlinked and tightly coordinated. Efficient 3'end processing is defined by sequence motifs emerging in the nascent-transcribed RNA strand and the cotranscriptional binding of regulatory proteins. The processing of a mature 3'end consists of cleavage and polyadenylation and is coupled with RNA polymerase II transcription termination and the dissociation of the nascent RNA transcript from the chromatin-associated transcriptional template. The subcellular and subnuclear topological specificity of the various RNA species is important for their functions. For instance, the formation of RNA-binding protein interactions, critical for the final outcome of gene expression, may require the nucleoplasmic fully spliced and polyadenylated form of an RNA transcript. Thus, interfering with the critical step of transcription termination and 3'end formation provides a means for assaying the functional potential of a given RNA of interest.In this protocol, we describe a method for blocking 3'end processing of the nascent RNA transcript, by using RNase H-inactive antisense oligonucleotides targeting cleavage and polyadenylation, delivered via transient transfection in cell culture.
Keywords: 2-O-methyl phosphorothioate; 2′OMePS; 3′end formation; Antisense oligonucleotide; Nascent RNA transcript; Polyadenylation signal; Transcription termination; Transcriptional readthrough.