Research Highlights

RNA as a target for ADP-ribosylation

The human genome encodes several poly(ADP-ribose) polymerases (PARPs), which are enzymes involved in ADP-ribosylation, a reversible chemical modification, of macromolecules such as proteins and DNA. ADP-ribosylation occurs in both prokaryotes and eukaryotes and is linked to vital cellular processes namely stress responses, DNA repair, host-virus interactions, etc. This modification is mostly associated with proteins and to some extent, DNA. It was recently proposed that RNA molecules could also be cellular targets of ADP-ribosylation.

Deeksha Munnur and colleagues from Ivan Ahel’s lab, have now shown that ADP-ribosylation of RNA molecules indeed happens and is a more common phenomenon than previously believed. They found that ADP-ribosylation occurs at the terminal phosphate of RNA forming a non-canonical RNA cap and can be regulated by both PARP-like proteins from bacteria and by some human PARPs. Furthermore, they provide the first evidence that ADP-ribosylation of RNA is a reversible process that can be controlled by ADP-ribosylhydrolases from human as well as VEEV and SARS viruses. This study shines a light on a new regulatory mechanism of RNA molecules with potential therapeutic relevance.

Written by Iqbal Dulloo

Munnur D, Bartlett E, Mikolčević P, Kirby IT, Matthias Rack JG, Mikoč A, Cohen MS, Ahel I. Reversible ADP-ribosylation of RNA.

Nucleic Acids Res. 47(11): 5658-5669

Understanding Pandemic Flu

Influenza A viruses are responsible for both seasonal outbreaks of respiratory disease and occasional pandemics. Their genome is made of eight RNA segments. If two different virus strains of infect the same cell, they can “shuffle” their segments instead of packaging just their own, and generate a new strain. This can result in the generation of a pandemic, since people may lack the immunity necessary to defeat the new virus. This happened in 1957 and 1968, when pandemic viruses killed millions worldwide. While we can detect this genomic “shuffling”, little is known about how they occur. Therefore, predicting them is still a challenge.

Research led by David Bauer in Ervin Fodor’s group addressed the question of the structure of the viral RNA genome. Collaborating with groups in Australia (Lorena Brown) and in the US (Alain Laederach), they employed high-throughput analysis techniques to reveal specific interactions that control packaging of RNA segments. Rarely, these interactions facilitate inter-strain “shuffling”. This work sheds light on influenza virus biology and is a major step in our understanding of influenza pandemic generation.

Written by Shaked Ashkanazi

Dadonaite B, Gilbertson B, Knight ML, Trifkovic S, Rockman S, Laederach A, Brown LE, Fodor E, Bauer DLV. The structure of the influenza A virus genome.

Nature Microbiology (2019).

The role of sugars in bacterial adhesion

Bacteria display molecules on their outside to facilitate adhesion and interaction with surfaces. This is particularly important during the initial stages of infection and biofilm formation for pathogenic bacteria. This adhesive ability allows bacteria to move on surfaces, both living and non-living, and eventually adhere irreversibly to form biofilms.

The Bharat lab uses a commonly studied bacterium, Caulobacter crescentus, to investigate this adhesion process. There are two main methods of adhesion, via proteins or polysaccharides (sugars). The focus of this paper is polysaccharide-mediated adhesion, in which a specialized sugar called holdfast plays a key role. In this paper, Sulkowski et al use a range of structural biology techniques, including cryo-electron microscopy, to identify the proteins which anchor and display holdfast on the cell surface. By looking at the mechanism of positioning holdfast at the membrane and maintenance of its cellular position, the role of sugars in bacterial adhesion can be better understood.

Written by Heather Jeffery @HeatherMJeffery

Sulkowski NI, Hardy GG, Brun YV, Bharat TAM A multi-protein complex anchors adhesive holdfast at the outer membrane of Caulobacter crescentus.

J. Bacteriol pii: JB.00112-19. doi: 10.1128/JB.00112-19. [Epub ahead of print]

DNA replication unfazed by disruption of cohesin-dependent DNA organisation

DNA replication is a process fundamental to all known organisms on earth. This highly regulated process is linked to chromatin structure − the way DNA is organised in the cell. It has been hypothesised that chromatin structure determines the accessibility of DNA by the replication machinery and hence dictates DNA replication timing.

Cohesin, well-known for its function in separating sister chromatids during cell division, has been shown to help form loop-based chromatin structures called topologically associating domains (TADs). Increasing evidence showed that TADs correlate with replication domains, but a causative relationship has not been established.

Phoebe Oldach, a DPhil student from the Nieuduzynski Lab, studied the relationship between TADs and DNA replication timing by using a system that can rapidly cut down levels of cohesin. After confirming the efficiency and efficacy of the system, she showed that replication timing patterns are not impacted by the absence of cohesin in either G1 or S phases of the cell cycle, during which the cell prepares for and executes DNA replication, respectively. This indicates that replication timing is not driven by cohesin-dependent TADs.

Written by Sheng Pong

Oldach P. and Nieduszynski C. A. (2019). Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains.

Genes, 10(3). pii: E196. doi: 10.3390/genes10030196.

Balancing act — How Nodal and Bmp signaling impact the emergence of primordial germ cells in mice

Multicellular organisms consist of two fundamental types of cells: somatic cells and germ cells. While somatic cells make up most of the organism, germ cells are a small group of cells in the gonads that undergo meiosis to produce haploid gametes—either the oocyte or the spermatocyte. These cells are capable of generating a new organism after fertilization, thus transmitting genetic information from one generation to another. Early in development, the primordial germ cells (PGCs) are set aside as the precursors of sperm and eggs. We still do not have a complete understanding of exactly how these cell-fate decisions are made in development.

Dr. Anna Senft and colleagues in the Robertson lab have used transgenic mouse embryos to determine that two signaling pathways—Nodal and Bmp—and their downstream effectors, the Smad proteins, cooperate to regulate the numbers and development of PGCs. The balance of Nodal/Bmp signaling in embryonic and extra-embryonic tissues of the early embryo provide early instructions for the specification of the PGC lineage, while protecting PGCs from a somatic fate.

Written by Derek Xu @derekcxu

Senft AD, Bikoff EK, Robertson EJ, Costello I. Genetic dissection of Nodal and Bmp signalling requirements during primordial germ cell development in mouse.

Nat Commun10(1):1089. doi: 10.1038/s41467-019-09052-w.

Macrophage Cannibalism – a Double-Edged Sword

Macrophages are essential players in innate immunity and in tissue homeostasis. Their primary function is phagocytosis, by which they eliminate pathogens, components of dying cells, etc. Another macrophage function, which is far less characterised, is efferocytosis, where macrophages phagocytose apoptotic cells. Sometimes macrophages will phagocytose apoptotic macrophages, a process we call “cannibalistic efferocytosis”. By doing so, the “predators” consume their “prey” with all its content.

The Greaves group has collaborated with colleagues from the Department of Zoology and the Mathematical Institute in Oxford, as well as from the School of Mathematics and Statistics from the University of Sydney to better understand cannibalistic efferocytosis. They established a mathematical model and calculated that indeed if a latex bead is present in the “prey”, it would be passed to its “predator”, which will be consumed by another “predator”, etc, until many latex beads would ultimately be concentrated in very few cells. Using sophisticated image analysis software, the Dunn School researchers demonstrated that the model accurately describes macrophage biology. This is an important step in our understanding the role of macrophages in tissue inflammation.

Written by Shaked Ashkanazi

Ford HZ, Zeboudj L, Purvis GSD, Ten Bokum A, Zarebski AE, Bull JA, Byrne HM, Myerscough MR, Greaves DR. Efferocytosis perpetuates substance accumulation inside macrophage populations.

Proc Biol Sci.286(1904):20190730. doi: 10.1098/rspb.2019.0730.

Key proteins for parasite motility and infectivity uncovered

Two recent papers from the Gull lab have highlighted the role of the flagellum attachment zone and the flagellum basal plate in the kinetoplastid parasites, Leishmania mexicana and Trypanosoma brucei. One of the first authors, Jack Sunter, now leads his own research group at Oxford Brookes University.

The Sunter et al paper investigated the structure of the flagellar pocket, where the flagellum exits the cell body, by looking at the role of a FAZ (flagellum attachment zone) protein in both cell cultures and in vivo. They found that correct establishment of the flagellar pocket is essential for subsequent infection of sand flies and mice.

The Dean et al paper investigated the role of basalin, a protein that is responsible for building the ‘basal plate’, a structure that is found at the base of the flagellum. They found that knockdown of basalin produced immotile flagella caused by defects in the central pair of microtubules.

These papers both link individual proteins with flagellar structures. Two key functions of the flagellum, movement and attachment, are both vital to the kinetoplastid lifecycle and infection ability. Therefore, understanding these processes gives an insight into how these could be targeted in future treatments.

Dean, S. Moreira-Leite F, Gull K. Basalin is an evolutionarily unconstrained protein revealed via a conserved role in flagellum basal plate function. ELife, 8 pii:e42282

Sunter JD, Yanase R, Wang Z, Catta-Preta CMC, Moreira-Leite F, Myskova J, Pruzinova K, Volf P, Mottram JC, Gull K (2019). Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host. Proc Natl Acad Sci USA 116(13) 6351-6530 doi: 10.1073/pnas.1812462116

Written by Heather Jeffery @HeatherMJeffery

DNA Replication – Singled Out

DNA replication is tightly regulated, since errors in replication would be passed on to daughter cells and essential information could be lost. In eukaryotic cells, an important regulatory step is the activation of replication origins, which leads to the formation of two replication forks that move away from the origin. Interestingly, the order by which replication origins activate is tightly regulated, as evident from the reproducible replication time of each chromosomal region. However, the information about this order was obtained from large populations of cells, therefore overlooking rare replication initiation events. 

Researchers from Conrad Nieduszynski’s group have developed a high-resolution method to study DNA replication at a single cell level. They treated budding yeast cells with low concentrations of a nucleotide analogue and used nanopore sequencing to detect the incorporation of the analogue, thus identifying nascent DNA. Based on the distribution of the analogue, they could identify replication origins and replication fork movement genome-wide. This is the first time that genome replication dynamics have been characterised by single-molecule sequencing.

Written by Shaked Ashkanazi

Müller CA, Boemo MA, Spingardi P, Kessler BM, Kriaucionis S, Simpson JT, Nieduszynski CA (2019). Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads.

Nature Methods

Dual functions of PCF11 – all done by halting RNA production

Transcription, the process by which DNA is copied into RNA, needs to be stopped at the right time to ensure the production of accurate RNA molecules. Transcription termination is coupled with the processing of the newly generated RNA’s tail: first it is cleaved and then a string of adenine bases is appended to it. This is the achieved by the cleavage and polyadenylation (CPA) complex. It was known from yeast studies that a component of the CPA complex, PCF11, participates in both RNA tail processing and transcription termination.

Kinga Kamieniarz-Gdula (Proudfoot Lab) and her colleagues characterised PCF11 in human and zebrafish by using state-of-the-art sequencing techniques. They found that PCF11 enhances both CPA and transcription termination in human cells, and is indispensable for zebrafish development. PCF11 increases the expression of genes that are close to each other by stopping RNA polymerase II from transcribing aberrantly into the neighbouring gene. Conversely, PCF11 lowers the expression of many transcriptional regulators by inducing premature termination. This research shows that transcription termination not only puts finishing touches on the RNA, but also regulates gene expression.

Written by Sheng Kai Pong

Kamieniarz-Gdula K, Gdula MR., Panser K, Nojima T, Monks J, Wisniewski JR, Riepsaame J, Brockdorff N, Pauli A, Proudfoot NJ (2019)  Selective Roles of Vertebrate PCR11 in Premature and Full-Length Transcript Termination

Molecular Cell. doi: 10.1016/j.molcel.2019.01.027.

Lon protease maintains virulence plasmid in Shigella

Shigella flexneri is a bacterium that can infect the human gut and cause diarrhoea in humans. Shigella infections lead to more than 1 million deaths every year. These infections can be treated by antibiotics but a large number of deaths occur in developing countries where these antibiotics may not be available. With rising antibiotic resistance, new treatments will be needed in the future.

The Tang lab, including Gareth McVicker who has since moved to Nottingham Trent University, have investigated the interactions between protease degradation and toxin-antitoxin systems in bacteria. Shigella has a large invasion plasmid, pINV, which contains many toxin-antitoxin systems. The plasmid needs to be transmitted from parent to daughter cells to maintain characteristics essential to cause disease. This new research identified a link between a protease named Lon and acetyltransferase family toxin-antitoxin systems. Lon enhances the stability of pINV, therefore ensuring that the bacteria can infect the human host. In addition, they identified two partitioning systems that further develop the current understanding of the virulence of Shigella

Written by Heather Jeffery @HeatherMJeffery

McVicker, G, Hollingshead, S, Pilla, G, Tang CM (2019) Maintenance of the virulence plasmid in Shigella flexneri is influenced by Lon and two functional partitioning systems.

Mol. Microbiol. doi: 10.1111/mmi.14225.