Policing mammalian transcriptomes: regulation of long ncRNA synthesis by transcriptional termination, gene loops and R-loops.

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RNA polymerase II phosphorylated on CTD serine 5 interacts with the spliceosome during co-transcriptional splicing

Publication . Nojima, Takayuki; Rebelo, Kenny; Gomes, Tomás; Grosso, Ana Rita; Proudfoot, Nicholas J.; Carmo-Fonseca, Maria

The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5' splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3' splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion.

2018Journal article

Open access

Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing

Publication . Nojima, Takayuki; Gomes, Tomás; Grosso, Ana Rita; Kimura, Hiroshi; Dye, Michael J.; Dhir, Somdutta; Carmo-Fonseca, Maria; Proudfoot, Nicholas J.

Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5'splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

2015Journal article

Open access