Uncovering the dynamics and consequences of RNA isoform changes during neuronal differentiation.

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.

High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription.

Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes.

Illuminating Enhancer Transcription at Nucleotide Resolution with Native Elongating Transcript Sequencing (NET-Seq).

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.

A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3'-processing and termination.

Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3'-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3'-RNA processing factors at the 5'-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5'-elongation control and 3'-RNA processing.

Conserved DNA sequence features underlie pervasive RNA polymerase pausing.

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression.

Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD-mimicking region in Sen1.

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

Structure and dynamics of the RNAPII CTDsome with Rtt103.

RNA polymerase II contains a long C-terminal domain (CTD) that regulates interactions at the site of transcription. The CTD architecture remains poorly understood due to its low sequence complexity, dynamic phosphorylation patterns, and structural variability. We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3'-end RNA-processing and transcription termination factor. Rtt103 forms homodimers via its long coiled-coil domain and associates densely on the repetitive sequence of the phosphorylated CTD via its N-terminal CTD-interacting domain. The CTD-Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins.

Structural insight into recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by Rtt103p.

Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.

The CTD code of RNA polymerase II: a structural view.

RNA polymerase II (RNA pol II) is not only the fundamental enzyme for gene expression but also the central coordinator of co-transcriptional processing. RNA pol II associates with a large number of enzymes and protein/RNA-binding factors through its C-terminal domain (CTD) that consists of tandem repeats of the heptapeptide consensus Y(1)S(2)P(3) T(4)S(5)P(6)S(7). The CTD is posttranslationally modified, yielding specific patterns (often called the CTD code) that are recognized by appropriate factors in coordination with the transcription cycle. Serine phosphorylations are currently the best characterized elements of the CTD code; however, the roles of the proline isomerization and other modifications of the CTD remain poorly understood. The dynamic remodeling of the CTD modifications by kinases, phosphatases, isomerases, and other enzymes introduce changes in the CTD structure and dynamics. These changes serve as structural switches that spatially and temporally regulate the binding of processing factors. Recent structural studies of the CTD bound to various proteins have revealed the basic rules that govern the recognition of these switches and shed light on the roles of these protein factors in the assemblies of the processing machineries.

C-terminal extension of the yeast mitochondrial DNA polymerase determines the balance between synthesis and degradation.

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.