Research Highlights

Consequences of excessive noncoding transcription unveiled.

Only a fraction of the human genome codes for protein-coding genes, yet these are the most studied regions. Other elements of the genome code for non-coding RNA but the regulation and functions of these other genes remains largely unknown. Transcription, the conversion of DNA to RNA, occurs across both protein-coding and RNA-coding genes.

Takayuki Nojima, Michael Tellier et al, from the Proudfoot and Murphy labs, respectively, have shown that a transcription-associated elongation factor, SPT6 plays a role in maintaining the balance of transcription between these two types of genes by modifying the chromatin. This factor can also affect the overall transcription level of long non-coding RNA genes. Higher non-coding RNA transcription can be associated with DNA damage, through the formation of R-loops, and uncontrolled transcription of non-coding RNAs can clash with the DNA replication apparatus promoting cellular senescence. This detailed molecular study highlights the importance for the cell to control the expression of non-coding regions of the genome and indicates the presence of a molecular mechanism dependent on SPT6 differentiating transcription of protein-coding and long non-coding RNA genes.

Written by Heather Jeffery @HeatherMJeffery

Nojima T, Tellier M, Foxwell J, Ribeiro de Almeida C, Tan-Wong SM, Dhir S, Dujardin G, Dhir A, Murphy S, Proudfoot NJ. (2018)
Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senes

Mol Cell. pii: S1097-2765(18)30843-8. doi: 10.1016/j.molcel.2018.10.011. 

New tools to explore the question of life or death in caspase-activating cells

Caspases are protein-cleaving enzymes best known as master regulators of programmed cell death through apoptosis. Sequential cleavage steps firstly activate initiator caspases and then downstream effector caspases, to dismantle all of the cell components during apoptosis. Unexpectedly, recent investigations also implicate this protein family in non-apoptotic functions (for example cell proliferation, differentiation, and migration). Although many sensors have successfully shown effector caspase activation in vivo, few tools are available to directly monitor initiator caspase activity. This is key to fully understand caspase functions.

Alberto Baena-Lopez and his group have developed a suitable sensor to visualise initiator caspase activation in Drosophila tissues. Live imaging of these sensors has revealed unknown events of apoptosis that precede the previously described stages of apoptosis in flies. Strikingly, they are also able to show stereotyped patterns of caspase activation unlinked to apoptosis in many Drosophila tissues. Their novel system reports on caspase function at both a cellular and organismal level, and it promises to provide further insight into the biology of these proteins in healthy and apoptotic cells.

Written by Zoe Geraghty @zoe_geraghty

Baena-Lopez LA, Arthurton L, Bischoff M, Vincent JP, Alexandre C, McGregor R (2018)
Novel initiator caspase reporters uncover unknown features of caspase-activating cells.

Development dev.170811

Secreting secrets no more

Protein secretion systems are essential for many bacteria; they play roles in communication, virulence factor delivery, protection against harmful agents and nutrient uptake. A certain group of pathogenic Gram-negative bacteria use the Type 9 secretion system (T9SS), a protein complex that spans the cell envelope. The T9SS is key to bacterial motility and is an essential determinant of pathogenicity in severe periodontal disease. However, the identity and structure of the central transport channel (translocon) was unclear – until now.

Using different biochemical and structural approaches, Lauber, Deme (Lea lab) and colleagues identified SprA as the translocon.  The structure of SprA, solved by cryo-electron microscopy, forms an unprecedented 36-stranded β-barrel in the outer membrane – the largest observed to date. Additionally, SprA forms complexes with three other accessory proteins (PPI, Plug and PorV), which control access to the SprA barrel. Taken together, this research has uncovered the structure and function of a critical part of the protein translocation T9SS machinery, which is directly involved in the transport of virulence factors in disease-causing bacteria.

Written by Lisa Gartenmann

Lauber F, Deme JC, Lea SM, Berks BC.
Type 9 secretion system structures reveal a new protein transport mechanism.

Nature. 2018 Nov 7. doi: 10.1038/s41586-018-0693-y.

Putting the pieces together: new insights into RNA splicing

Eukaryotic genes are transcribed by the RNA polymerase II (Pol II) into mRNAs, which are then translated into protein products. These genes also contain non-coding stretches of DNA called introns; these are transcribed into RNA, but must be cut out before the final functional protein is produced. To ensure this, a multi-molecular complex called the spliceosome assembles on RNAs as they are being transcribed and splices the coding regions (exons) directly together, removing the introns. How two distant sites are physically brought together to be spliced has remained unknown.

Taka Nojima (Proudfoot group) and colleagues at the University of Lisbon have previously developed a technique to sequence elongating transcripts (mNET-seq). In a recent study, they couple this with mass spectrometry to show that the active spliceosome associates with Pol II, physically tethering the cleaved upstream exon in position while transcription continues. Interestingly, the association is specific to a Pol II modification (S5P CTD) previously thought to act only in transcription initiation. They also extensively characterise co-transcriptional splicing intermediates, providing significant insight into the mechanism and coordination of mammalian co-transcriptional splicing.

Written by Zoe Geraghty @zoe_geraghty

Nojima T, Rebelo K, Gomes T, Grosso AR, Proudfoot NJ, Carmo-Fonseca M (2018).
RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing

Mol. Cell 72(2):369-379.e4.

New Rules for the Histone Modification Code

Post-translational protein modifications (PTMs) are important for the regulation of many cellular processes. Of these, ADP-ribosylation (ADPr)—the reversible addition of ADP-ribose molecules—of histones can be particularly important for DNA damage response. Previous work in Ivan Ahel’s lab found that ADPr on serine residues (Ser-ADPr) is the most prominent type of ADPr following DNA damage. Prior to this, the exact location of ADPr on many proteins, including histones, remained elusive. Furthermore, the study of ADPr and other histone PTMs had never intersected before, despite how different combinations of histone PTMs act together in a code to regulate important nuclear functions.

In this study, Bartlett et al., examined the interplay of histone marks, and introduced a simple method to detect Ser-ADPr peptides, overcoming limitations on currently available techniques. The study reveals how Ser-ADPr is mutually exclusive with proximal phosphorylation and acetylation—other PTMs—at N-terminal tails of histones. These results indicate that the context of the local histone code affects Ser-ADPr, and vice versa. Additionally, they discovered that tyrosine residues can also undergo ADPr, whose physiological relevance is still unclear.

Written by Derek Xu @derekcxu

Bartlett E, Bonfiglio JJ, Prokhorova E, Colby T, Zobel F, Ahel I, Matic I. (2018).
Interplay of Histone Marks with Serine ADP-Ribosylation.

Cell Rep. 24(13):3488-3502.e5. doi: 10.1016/j.celrep.2018.08.092.

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RIPK1 is essential for macrophage viability

To die or not to die, RIPK1 is the answer

Macrophages are important cells in our innate immune system, whose primary roles are maintaining tissue integrity through defence against pathogens, clearing cell debris and regulating stem cells. For these roles, macrophages employ two distinct strategies: inflammation and cell death, which prevents the spreading of the pathogen. Similar to powerful weapons, these can easily turn into a harmful double-edged sword, and therefore must be carefully controlled.

Previous studies suggested that RIPK1 has a crucial role in proper macrophage activation. When RIPK1 is experimentally lost, however, macrophages fail to develop and eventually die, making its role difficult to decipher. Dr Julian Buchrieser and colleagues in the William James lab have collaborated with colleagues from Universidad de Sevilla (Spain) to uncover the role of RIPK1 in macrophage activation. They generated induced pluripotent stem cells and deleted RIPK1 from their genome by using the genome-editing tool of CRISPR-Cas9. This method allowed them to control and track every step of macrophage development. Thanks to their fine-tuned dissection, they found that RIPK1’s main role is to protect macrophages from dying, while their inflammatory activity is unaffected.

Written by Shaked Ashkenazi

Buchrieser J, Oliva-Martin MJ, Moore MD, Long JCD, Cowley SA, Perez-Simón JA, James W, Venero JL (2018).
RIPK1 is a critical modulator of both tonic and TLR-responsive inflammatory and cell death pathways in human macrophage differen

Cell Death & Disease 9(10):973

Mini-viral RNA linked to deadly influenza virus

Influenza virus is a pathogen that plagues society in the form of flu. 2018 marks 100 years since the ‘Spanish flu’ pandemic that, according to the World Health Organisation, infected a third of the human population worldwide and led to more deaths than World War I. The influenza virus contains its genetic information in the form of RNA instead of DNA and relies on infecting a host cell in order to replicate.

Researchers in the Fodor lab have identified a link between influenza virus infection and the infected cell response. This takes the form of mini-viral RNA (mvRNA) and is produced by dysregulated viral RNA replication. These mvRNA molecules bind to and activate retinoic acid-inducible gene 1 (RIG-1), which starts a signalling cascade resulting in cytokine production and cell death. The most harmful influenza virus strains, including the 1918 pandemic strain and the H5N1 bird flu, produce high levels of mvRNA. Understanding the mechanisms of mvRNA production and their contribution to virulence is important for developing ways to combat future viral strains.

Written by Heather Jeffery @HeatherMJeffery

Velthuis AJWt, Long JC, Bauer, DLV, Fan, RLY, Yen HL, Sharps J, Siegers JY, Killip MJ, French H, Oliva-Martín MJ, Randall RE, Wit Ed, Riel Dv, Poon LLM, Fodor E (2018)
Mini viral RNAs act as innate immune agonists during influenza virus infection

Nature Microbiology 10.1038/s41564-018-0240-5

Determining the fate of cells during embryonic differentiation

During embryonic development, key signalling cues dictate cell fates and allow specification of different cell populations. Three of these cues are Nodal, an extracellular ligand of a family of growth factors (TGFb), and Smad2 and Smad3, the downstream effectors inside the cell responsible for determining the head-to-tail axis of the embryo. Previous genetic studies showed that Smad2/3 double-mutant mouse embryos die shortly after implantation, yet the underlying molecular mechanisms remained unknown.

Senft and colleagues in the Robertson and Bikoff labs have recently characterised the effects of Smad2/3-deficiency in mouse embryonic stem cells (ESCs) using in vitro differentiation protocols. They found that Smad2/3 signalling is required to maintain embryonic and extra-embryonic cell identities, and to undergo key cell fate allocation steps during embryonic germ layers differentiation, in the pre-implantation mammalian embryo. Additionally, the balance of signalling pathways during neural precursor differentiation was not properly maintained in these mutant ESCs, with Nodal target genes being downregulated and Bmp upregulated. This detailed molecular study of the signalling in early mammalian development widens our comprehension of the genetic programs involved in embryonic post-implantation development.

Written by Anna Caballe @caballe_anna

Senft AD, Costello I, King HW, Mould AW, Bikoff EK, Robertson EJ (2018)
Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage

Cell Rep 24(8):1977-1985.e7.

Don’t stop the beat: helping parasites change direction

Flagella are tail-like structures that beat to propel single-celled organisms along. The moving flagellum resembles a wave. Some parasites, such as Trypanosoma and Leishmania, can change their direction by adjusting where this wave starts.

Researchers from the Gull Lab have found that this ability is afforded by breaking the flagellum’s symmetry. They showed that a class of proteins, known as ODA-docking complexes (DCs), are unevenly distributed along trypanosome flagella. Some (dDCs) sit at the flagellum tip, while others (pDCs) sit nearer the cell body. Mathematical modelling suggested that this pattern is achieved by a process called intraflagellar transport (IFT) that constantly moves pDC towards the base. This frees up binding sites at the opposite end, which dDCs can fill.

This asymmetry is important for wave initiation; the localisation of a novel protein (‘LC4-like’), which acts as a molecular switch to change where the wave starts from, is dependent on dDC. This study explores why parasites are such expert movers and shakers and may also have implications for human health; DC mutations can cause ciliary dyskinesia, a disease characterised by inefficient ciliary movement.

 

Written by Laura Hankins

Edwards BFL, Wheeler RJ, Barker AR, Moreira-Leite FF, Gull K, Sunter JD
Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry

PNAS 115(31): E7341-E7350

A common birthplace for distinct organelles

Membrane-bound subcellular compartments (or organelles) are the hallmark of eukaryotic cells. Lipid droplets (LDs) and peroxisomes are distinct organelles at the centre of cellular metabolism; their dysfunction is linked to devastating diseases in humans, including diabetes and neurodevelopmental disorders. Both are thought to ‘bud off’ from the endoplasmic reticulum (ER), the cell’s largest organelle. However, how their assembly sites are defined and the mechanism by which they bud from the ER remained mysterious.

A recent study by Sihui Wang from the Carvalho lab and colleagues has uncovered that these structurally distinct organelles share their birthplace at specific ER domains. Using lipidomics, microscopy and genetic strategies in yeast, Wang et al. explore the cooperation of the protein complex Seipin and the membrane-shaping protein Pex30. They show that these factors facilitate LD and peroxisome formation by organising membrane regions of a lipid composition that favour budding. Their data advocate a new model for LD and peroxisome biogenesis, which paves the way for understanding the mechanisms of organelle formation at the ER.

 

Written by Zoë Geraghty @zoe_geraghty

Wang, Idrissi FZ, Hermansson M, Grippa A, Ejsing CS, Carvalho P.
Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum

Nat Commun. 9(1):2939. doi: 10.1038/s41467-018-05278-2

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