Michael J. Dye

Integrating mRNA Processing with Transcription

The messenger RNA processing reactions of capping, splicing, and polyadenylation occur cotranscriptionally. They not only influence one anothers efficiency and specificity, but are also coordinated by transcription. The phosphorylated CTD of RNA polymerase II provides key molecular contacts with these mRNA processing reactions throughout transcriptional elongation and termination.

Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing

Summary 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.

Multiple Transcript Cleavage Precedes Polymerase Release in Termination by RNA Polymerase II

The requirement of poly(A) signals to elicit transcription termination of RNA polymerase II (pol II) is firmly established. However, little else is known about the actual process of pol II transcription termination. Evidence presented in this paper, based on analysis of nascent transcripts of the human beta- and epsilon-globin genes, demonstrates that pol II transcription termination involves two distinct and temporally separate events. The first event, termed pretermination cleavage (PTC), is mediated by sequence tracts located downstream of the poly(A) site which appear to promote heterogeneous cleavage of the nascent transcript. The second event, in which pol II disengages from the DNA template, requires that polymerase has transcribed both a PTC sequence tract and a functional poly(A) site.

Molecular Dissection of Mammalian RNA Polymerase II Transcriptional Termination

Summary Transcriptional termination of mammalian RNA polymerase II (Pol II) is an essential but little-understood step in protein-coding gene expression. Mechanistically, termination by all DNA-dependent RNA polymerases can be reduced to two steps, namely release of the RNA transcript and release of the DNA template. Using a simple nuclear fractionation procedure, we have monitored transcript and template release in the context of both natural and artificial Pol II terminator sequences. We describe the timing and relationship between these events and in so doing establish the roles of the poly(A) signal, cotranscriptional RNA cleavage events, and 5′-3′ exonucleolytic RNA degradation in the mammalian Pol II termination process.

Transcription of the human U2 snRNA genes continues beyond the 3′ box in vivo

The 3′ box of the human class II snRNA genes is required for proper 3′ processing of transcripts, but how it functions is unclear. Several lines of evidence suggest that termination of transcription occurs at the 3′ box and the terminated transcript is then a substrate for processing. However, using nuclear run‐on analysis of endogenous genes, we demonstrate that transcription continues for at least 250 nucleotides beyond the 3′ box of the U2 genes. Although in vivo footprinting analysis of both the U1 and U2 genes detects no protein–DNA contacts directly over the 3′ box, a series of G residues immediately downstream from the 3′ box of the U1 gene are clearly protected from methylation by dimethylsulfate. In conjunction with the 3′ box of the U1 gene, this in vivo footprinted region causes termination of transcription of transiently transfected U2 constructs, whereas a 3′ box alone does not. Taken together, these results indicate that the 3′ box is not an efficient transcriptional terminator but may act as a processing element that is functional in the nascent RNA.

Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Gγ-globin gene

ABSTRACT The human γ-globin genes form part of a 5-kb tandem duplication within the β-globin gene cluster on chromosome 11. Despite a high degree of identity between the two genes, we show that while the upstream Gγ-globin gene terminates transcription efficiently, termination in the Aγ gene is inefficient. This is primarily due to the different strengths of the poly(A) signals of the two genes; Gγ-globin has a functionally stronger poly(A) signal than the Aγ gene. The probable cause of this difference in poly(A) efficiency characteristics lies with a number of base changes which reduce the G/U content of the GU/U-rich region of the Aγ poly(A) signal relative to that of Gγ. The 3′ flanking regions of the two γ-globin genes have similar abilities to promote transcription termination. We found no evidence to suggest a cotranscriptional cleavage event, such as that seen in the human β-globin gene, occurs in either γ-globin 3′ flank. Instead we find evidence that the 3′ flank of the Gγ-globin gene contains multiple weak pause elements which, combined with the strong poly(A) signal the gene possesses, are likely to cause gradual termination across the 3′ flank.

Definition of RNA polymerase II CoTC terminator elements in the human genome.

Summary Mammalian RNA polymerase II (Pol II) transcription termination is an essential step in protein-coding gene expression that is mediated by pre-mRNA processing activities and DNA-encoded terminator elements. Although much is known about the role of pre-mRNA processing in termination, our understanding of the characteristics and generality of terminator elements is limited. Whereas promoter databases list up to 40,000 known and potential Pol II promoter sequences, fewer than ten Pol II terminator sequences have been described. Using our knowledge of the human β-globin terminator mechanism, we have developed a selection strategy for mapping mammalian Pol II terminator elements. We report the identification of 78 cotranscriptional cleavage (CoTC)-type terminator elements at endogenous gene loci. The results of this analysis pave the way for the full understanding of Pol II termination pathways and their roles in gene expression.

AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination

RNA Polymerase II (Pol II) termination is dependent on RNA processing signals as well as specific terminator elements located downstream of the poly(A) site. One of the two major terminator classes described so far is the Co-Transcriptional Cleavage (CoTC) element. We show that homopolymer A/T tracts within the human β-globin CoTC-mediated terminator element play a critical role in Pol II termination. These short A/T tracts, dispersed within seemingly random sequences, are strong terminator elements, and bioinformatics analysis confirms the presence of such sequences in 70% of the putative terminator regions (PTRs) genome-wide.