Research Highlights

Signalling before the exit: a tight checkpoint at the mitotic spindle

Cell division requires highly-controlled events to ensure faithful separation of DNA. Many of the pivotal signals governing mitosis are kinases and phosphatases that activate/inactivate protein substrates via phosphorylation or dephosphorylation. Prior to mitotic exit, cells monitor the attachment of chromosomes, via the kinetochores, to the mitotic spindle using the spindle checkpoint, which can trigger cell cycle arrest when errors are detected.

Spindle checkpoint signalling is initiated by the recruitment of MPS1 kinase to unattached kinetochores, yet how the spindle checkpoint is kept responsive to late-stage spindle defects is unknown. A study by Hayward and colleagues from the Gruneberg lab, in collaboration with the Barr and Novak labs (Biochemistry), has revealed the mechanism by which cells restrict MPS1 localisation to a defined time during mitosis. In particular, they show that CDK1-CyclinB1 promotes MPS1 recruitment and is opposed by the phosphatase PP2A-B55, which terminates signalling before mitotic exit. They nailed down the key residue in MPS1’s kinetochore binding domain (Ser281) targeted by this regulatory mechanism that allows cells to localise MPS1 and sustain the checkpoint-responsive window until chromosome segregation but not beyond, thus avoiding fatal cell division errors.

Written by Anna Caballe @caballe_anna

Hayward D, Alfonso-Pérez T, Cundell MJ, Hopkins m, Holder J, Bancroft J, Hutter LH, Novak B, Barr FA, Gruneberg U. (2019).
CDK1-CCNB1 creates a spindle checkpoint permissive state by enabling MPS1 kinetochore localisation

J Cell Biol DOI: 10.1083/jcb.201808014 |

Cell division throttle is the brakes as well: CDK1 is also a spindle checkpoint protein

Cell division is regulated by the protein Cyclin B-dependent kinase (CDK1-CCNB1). A master cell cycle regulator, CDK1 promotes entry into mitosis, and also activates the proteins that prevent mitotic exit. These proteins, such as MPS1 and MAD1, are known as spindle checkpoint proteins. They function by localising to unattached kinetochores, preventing the completion of mitosis until the kinetochores of all chromosomes are properly attached to the spindle apparatus through microtubules. This ensures that daughter cells are free from errors in chromosome number.

In a joint study, Daniel Hayward of Ulrike Gruneberg’s lab and Tatiana Alfonso-Pérez of Francis Barr’s lab (Biochemistry) and colleagues show that in addition to promoting mitosis, CDK1-CCNB1/CycB1 surprisingly functions as a bona fide spindle checkpoint protein. They demonstrate that CCNB1 (CycB1) is recruited to unattached kinetochores by a direct interaction with the spindle checkpoint protein MAD1. They additionally show that CCNB1 is necessary for a positive-feedback loop that recruits the key spindle checkpoint regulator MPS1 in a timely manner upon mitotic entry and sustains spindle checkpoint arrest (see RH on Hayward et al. 2019 for further details).

Written by Derek Xu @derekcxu

Alfonso-Pérez T, Hayward D, Holder J, Gruneberg U, Barr FA. (2019).  
MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

J Cell. Biol. DOI: 10.1083/jcb.201808015 

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Protein Data Bank Europe

Lessons from ancient fish on the selectivity and inhibition of protein modifications

ADP-ribosylation is a post-translational modification characterised by the addition of ADP-ribose on target proteins, a modification that is placed on a variety of residues. The specific nature of the modification determines the effect on many important processes, including DNA damage responses and the regulation of programmed cell death.

In vertebrates, two of the enzymes that remove ADP-ribose are ARH1 (cleaves it from arginine residues) and ARH3 (from serine residues). Rack and colleagues from Ivan Ahel’s lab set out to investigate the underlying differences between these enzymes. To that end, they solved the 3D structures of the ARH1/3 enzymes and their substrates to atomic level. Since human ARH3 was reluctant to crystalise, they scanned known genomes of alternative species and found an ancient orthologue: coelacanth (Latimeria chalumnae), a “living fossil” that has barely changed in 400 million years. The resulting structures revealed the structural basis for the differences. Together with supporting experiments, they represent an important step in our understanding of how these enzymes select their targets. This will be useful in designing selective drugs to target these essential enzymes

Written by Shaked Ashkanazi

Rack JGM, Ariza A, Drown BS, Henfrey C, Bartlett E, Shirai T, Hergenrother PJ, Ahel I (2018).
(ADP-ribosyl)hydrolases: Structural Basis for Differential Substrate Recognition and Inhibition

Cell Chem. Biol. 25(12):1533-1546.e12. doi: 10.1016/j.chembiol.2018.11.001. Epub 2018 Nov 21.

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

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