David L.V. Bauer

Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence.

RNA Polymerase Pausing during Initial Transcription

Summary In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

DNA Catenation Maintains Structure of Human Metaphase Chromosomes

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.

New Technologies for DNA analysis-A review of the READNA Project

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription

Summary Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription.

Mini viral RNAs act as innate immune agonists during influenza virus infection

The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors1. However, it is generally accepted that a strong innate immune dysregulation known as ‘cytokine storm’ contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype2–4. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression5. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-β. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.Aberrant mini viral RNAs, which are produced by erroneous RNA polymerase activity during the replication of the viral RNA genome, act as the main agonists of RIG-I during influenza virus infection.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.RNA synthesis by bacterial RNA polymerase is interrupted by pauses but their role in RNA synthesis is poorly understood. Here the authors use single-molecule FRET and biochemical analysis to show that pausing regulates branching between the abortive and productive outcomes of initial transcription.

Visualization, mapping and sequencing of megabase lengths of DNA

Prior to replication, a chromosome comprises a single length of DNA. We report on new methods for handling tens of kilobase to megabase lengths of single DNA molecules. We further report on the direct visualization of sequence organisation and the action of processive enzymatic activity along the molecules. We show how individual molecules can be captured and processed on single molecule microarrays [1] using a ligation base sequencing biochemistry that we have developed [2]. Finally, we describe how our methods will complement next generation DNA sequencing.

Pausing controls branching between productive and non-productive pathways during initial transcription

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

The structure of the influenza A virus genome

Influenza A viruses (IAVs) are segmented single-stranded negative sense RNA viruses that constitute a major threat to human health. The IAV genome consists of eight RNA segments contained in separate viral ribonucleoprotein complexes (vRNPs) that are packaged together into a single virus particle1,2. While IAVs are generally considered to have an unstructured single-stranded genome, it has also been suggested that secondary RNA structures are required for selective packaging of the eight vRNPs into each virus particle3,4. Here, we employ high-throughput sequencing approaches to map both the intra and intersegment RNA interactions inside influenza virions. Our data demonstrate that a redundant network of RNA-RNA interactions is required for vRNP packaging and virus growth. Furthermore, the data demonstrate that IAVs have a much more structured genome than previously thought and the redundancy of RNA interactions between the different vRNPs explains how IAVs maintain the potential for reassortment between different strains, while also retaining packaging selectivity. Our study establishes a framework towards further work into IAV RNA structure and vRNP packaging, which will lead to better models for predicting the emergence of new pandemic influenza strains and will facilitate the development of antivirals specifically targeting genome assembly.