Seminars

Calvin Tiengwe, Department of Life Sciences, Imperial College London

Iron-dependent control of transferrin receptor expression in Trypanosoma brucei

Iron is an essential co-factor for many enzymatic reactions, but also potentially very destructive at high concentrations. Consequently, cells exert a tight control on intracellular iron levels. A prominent control nexus involves iron regulatory RNA binding proteins (RBP) binding iron responsive elements to modulate stability of mRNAs encoding major components of the iron-acquisition pathway, such as the transferrin receptor. This process has been studied in many systems revealing unique mechanisms in each, yet remains relatively poorly understood in T. brucei, a “neglected” pathogen that causes African sleeping sickness in humans and livestock. To understand how T. brucei controls expression of its transferrin receptor (TbTfR), we performed genome-wide transcriptomic and proteomic studies to identify parasite-specific iron-responsive factors.

The first half of this talk will cover functional characterisation of an RBP as a novel post-transcriptional regulator of TbTfR expression. In the second half, I discuss our initial attempts to use orthogonal organic phase separation (OOPS), a method first described by the group of Kathryn Lilley, to comprehensively capture the RBPome of bloodstream stage trypanosomes. Our ultimate goal is to characterise RNA-protein dynamic interactions involved in iron homeostasis in T. brucei.

Host: Atlanta Cook

Camilla Björkegren, Karolinska Institutet, Sweden

Wellcome Centre Seminar - The Smc5/6 complex, DNA supercoiling and chromosome segregation

Cohesin, condensin and the Smc5/6 complex (Smc5/6) belong to the family of Structural Maintenance of Chromosome (SMC) protein complexes. By regulating chromosome structure and dynamics, these complexes influence chromosome replication, segregation, repair, and transcription. Cohesin is most well-known for its essential role in sister chromatid cohesion, condensin for its function in chromosome condensation, and Smc5/6 was first shown to control DNA repair and recombination. Chromosome segregation also fails in unchallenged cells lacking Smc5/6, but the reason for this remains mostly unknown.

To reveal the function of Smc5/6, we analyze the Saccharomyces cerevisiae complex in vivo and in vitro. The investigations show that the chromosomal association of Smc5/6 is dynamic and regulated by multiple factors, including chromosome length, sister chromatid entanglement, and transcription of convergently oriented genes. We also find that the purified complex promotes DNA decatenation by Top2, and compacts supercoiled DNA in a process that depends on ATP hydrolysis. Taken together, the results suggest that Smc5/6 function is linked to the level of supercoiling in chromosomal DNA. Results from these investigations, and their implications for the understanding of Smc5/6 function and supercoiling of the replicated genome, will be presented and discussed.

Host: Adele Marston

Claude Prigent, French National Centre for Scientific Research, Institute of Genetics and Development of Rennes

Wellcome Centre Seminar - Aurora-A : much more than a mitotic kinase

Aurora-A has been discovered as a centrosome protein kinase. Its activity is necessary for mitosis, it controls the maturation of centrosomes, the G2 / M transition, the assembly of the bipolar spindle, the spindle assembly checkpoint, the assembly of the central spindle, etc. Aurora-A is also an oncogene and is overexpressed in many epithelial cancers. This over-expression is correlated with the aggressiveness of cancer. Claude Prigent is currently investigating how overexpression of Aurora-A confers an advantage on cancer cells.

Host: Bill Earnshaw

Michael Emanuele, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, USA

Ubiquitin in normal and cancer cell cycles

My lab studies the role of ubiquitin signaling in cell cycle, genome stability and cancer. A major focus is on mechanisms underlying cell cycle transitions, including exit from mitosis and entry into S-phase. Both are governed by the APC/C, an essential cell cycle ubiquitin ligase that both promotes mitotic exit and restrains S-phase entry. We recently identified a deubiquitinase that antagonizes APC/C substrate degradation, and which is recurrently amplified in breast cancer. Ongoing studies to diagnostically map substrates using computational methods have revealed unforeseen contributions to cell physiology, and potential consequences of its dysfunction in disease.

Emanuele lab website:  www.emanuelelab.com

Recent review article

1. Who guards the guardian? Mechanisms that restrain APC/C during the cell cycle. Kernan J, Bonacci T, Emanuele MJ. BBA - Molecular Cell Research. Biochim Biophys Acta Mol Cell Res. 2018 Dec;1865(12):1924-1933. PMID: 30290241

Primary research highlighting our interests

2. Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates. Bonacci T, Suzuki A, Grant G, Cook JG, Brown NG, Emanuele MJ. EMBO Journal.  2018 Jul 4. PMID: 29973362

3. Choudhury R, Bonacci T, Wang W, Truong A, Arceci A, Zhang Y, Mills, CA, Kernan JL, Liu P, Emanuele MJ. The E3 ubiquitin ligase SCF(Cyclin F) transmits AKT signaling to the cell cycle machinery. Cell Reports. 2017 Sep 26;20(13):3212-3222. PMID: 28954236.

4. APC/C and SCF(cyclin F) constitute a reciprocal feedback circuit controlling S-phase entry. Choudhury C, Bonacci T, Arceci A, Lahiri D, Mills CA, Kernan JL, Branigan TB, DeCaprio JA, Burke D, Emanuele MJ. Cell Reports. 2016 Sep 20;16(12):3359-72. PMID: 27653696

5. Global identification of modular cullin-RING ligase substrates. Emanuele MJ, Elia EH, Xu Q, Thoma CR, Izhar L, Guo A, Rush J, Hsu PW, Yen HS, Elledge SJ. Cell. 2011 Oct 14;147(2):459-74. PMID: 21963094

Host: Tony Ly

Alex Dammermann, Max F. Perutz Laboratories, University of Vienna, Austria

Defining Centriole Function in Centrosome and Cilium Biogenesis

Centrioles are small cytoplasmic structures essential for the formation of two cellular organelles, centrosomes that function in cell division and cilia, cellular projections that perform a variety of sensory and motile roles. In my talk I will be presenting recent work from the lab in which we have sought to use the nematode C. elegans as an experimental model to define the contribution of centrioles to centrosome and cilium biogenesis. I will also be presenting the results of an ongoing phylogenetic-profiling-based screen in C. elegans and the fruit fly Drosophila to identify novel ciliogenesis genes.

Host: David Tollervey

Luisa Cochella, IMP Research Institute of Molecular Pathology, Vienna, Austria

Transcriptional and post-transcriptional mechanisms for cellular diversification

Multicellular organisms rely on the segregation of multiple functions across vastly diverse cellular types. Distinct cellular morphologies and physiologies are defined by cell-type-specific gene expression, which in turn is controlled by specific regulators. Our goals are to uncover the basic regulatory principles that instruct cellular diversity during development, and to illuminate how cell types have diversified during evolution. Using C. elegans as our experimental model, we have uncovered a novel mechanism for cellular diversification that relies on the temporal integration of transcriptional inputs that a cell receives during its developmental path. We have also generated a framework to understand the contribution of post-transcriptional repression by microRNAs to the generation of cellular diversity during animal development, and have uncovered new principles in miRNA-mediated regulation.

Host: David Tollervey

Professor Dr Karsten Weis, Institute of Biochemistry, ETH, Zurich

Wellcome Centre Seminar - The life of an mRNA - from birth to death

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation (LLPS) has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalise their components into membraneless organelles such as the nucleolus, processing bodies or stress granules. How the assembly and turnover of these organelles is controlled, and how these biological condensates selectively recruit or release components is poorly understood.  

I present results demonstrating that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs) are global regulators of RNA-containing phase-separated organelles in pro- and eukaryotes. Using in vitro reconstitution and in vivo experiments we can show that DDXs promote phase separation in their ATP-bound form, and ATP hydrolysis induces compartment turnover and RNA release. This mechanism of membraneless organelle regulation reveals a novel principle of cellular organisation that is conserved from bacteria to man. We further show that DDXs control RNA flux between phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that affords cells spatial and temporal control of various RNA processing steps regulating the composition and fate of ribonucleoprotein particles.

Host: Vadim Shchepachev and David Tollervey

Dr Tim Nott - Department of Biochemistry, University of Oxford

Wellcome Centre Seminar - Emergent properties of liquid-like membraneless organelles

Condensation of cellular material into phase-separated liquid-like droplets has emerged as a fundamental new organising principle in cell biology. The dynamic and membraneless compartments formed in this way are predominantly associated with processing nucleic acids and are indispensable for cellular function. Surprisingly, we know little about the solvent environment inside these and other cellular bodies, yet it is likely to have a significant influence on the biochemical reactions that take place within them.

One important class of enzymes that are biochemically active inside membraneless organelles are DNA and RNA helicases, which remodel the structures of nucleic acids. In addition to ATP-dependent catalytic domains, several helicases possess intrinsically disordered regions that readily undergo liquid-liquid phase separation in cells and in vitro. We have recently found that model membraneless organelles reconstituted from only the disordered tails of the DEADbox helicase Ddx4 display emergent biochemical properties. Among these are the ability to selectively absorb RNAs based on their structure, and the destabilisation of nucleic acid duplexes. We hypothesise that in the context of a membraneless organelle, these properties could complement the catalytic activity of the helicase domain.

Here we show that the emergent biochemical properties of membraneless organelles formed from only the disordered tails of DEADbox RNA helicases can be tuned by their amino acid sequence, and subtle changes to their surrounding environment. These results suggest novel ways in which cells could modulate the intrinsic properties of membraneless organelle interiors to achieve specific biochemical outcomes.

Host: Georg Kustatscher and Juri Rappsilber

Arshad Desai, Ludwig Institute for Cancer Research, San Diego Branch, USA

Cell division: from C. elegans to a potential cancer therapeutic

Functional genomic and proteomic approaches in models such as the early C. elegans embryo helped uncover conserved molecular machinery involved in cell division.  In my talk, I will focus on centrosomes, the major microtubule-organizing centers of animal cells that promote spindle formation during cell division.  Functional genomic screens in C. elegans identified the components involved in centrosome duplication and in their function as scaffolds for microtubule generation.  Following the initial discovery and characterization phase, we initiated a collaborative effort to target the key regulator of centrosome assembly - the kinase Plk4 - using small molecule inhibitors in human cells.  This effort culminated in the development of centrinone, a potent, specific and cellularly active Plk4 inhibitor which enables routine generation of centrosome-less human cells. Using centrinone, we have identified specific cancer contexts where cell division is highly sensitive to centrosome loss.  I will discuss our efforts to understand the reasons for this sensitivity, which is revealing new mechanisms acting during cell division and is helping identify contexts where Plk4 inhibition may provide a therapeutic benefit.

Host: JP Arulanandam/Dhanya Cheerambathur

Dr Jeroen Krijgsveld - German Cancer Research Centre, Heidelberg

Wellcome Centre Seminar - Protein networks with RNA and chromatin: encounters that open up a new world

Proteins rarely act alone, and they are well-known to assemble into complexes to fulfill their function. In addition, they interact with DNA and RNA in intricate ways to shape the epigenetic machinery, regulate gene expression, and control translation, however studying this in a systematic manner has remained a challenge. In this presentation I will describe novel proteomic methodologies that we developed to examine protein interactions in the context of chromatin, and to chart protein-RNA interactions across all RNA biotypes. Furthermore, I will show novel insights that we gained by investigating how the identified networks change in the face of stress and cell state transition.

Host: Stefania del Prete and Philipp Voigt

Ana Boskovic, Rando Lab, UMass Medical School, Worcester MA, USA

Molecular mechanism of MERV-L regulation via sperm-borne tRNA fragments

It is becoming increasingly clear that epigenetic information can be transmitted through gametes from parents to offspring in mammals. However, the molecular details of how this is achieved are almost completely lacking. In our dietary paradigm, protein restriction of male mice modulates levels of various small RNAs in mature sperm. We have previously shown that one such RNA, 5’ fragment of tRNA Glycine-GCC (5’tRF-GG) is consistently upregulated in response to low-protein diet. Functional studies in both embryonic stem cells and in preimplantation embryos revealed that this regulatory RNA can modulate expression of genes associated with LTRs of the endogenous retroelement MERV-L. We set out to elucidate the mechanism of tRF-based regulation of MERV-L-associated transcripts.  We show that tRF-GG enhances the production of noncoding RNAs normally synthesized in Cajal bodies. Among these noncoding RNAs, tRF-GG regulation of U7 snRNA modulates heterochromatin-mediated transcriptional repression of MERV-L elements by supporting an adequate supply of histone proteins. Importantly, effects of inhibiting tRF-GG on histone mRNAs, activity of histone 3’UTR reporters, and ultimately on MERV-L regulation could all be suppressed by U7 RNA. We show that the RNA-binding proteins hnRNPF and H bind directly to tRF-GG, and are required for Cajal body biogenesis. Together, our data reveal a conserved mechanism for 5’ tRNA fragment-based control of noncoding RNA biogenesis and, consequently, global chromatin organization. Our work provides central molecular details of transgenerational epigenetic inheritance in mammals, functionally linking tRNA fragments as dietary sensors in sperm to chromatin changes that affect early development. 

Host: Robin Allshire

Minhaj Sirajuddin, Institute for Stem Cell Science and Regenerative Medicine, Bangalore

Nanoengineering for discovery and mechanism related to motility systems

Eukaryotic biological motions across scales and orders of magnitude involve cytoskeleton elements. Because of their importance in cell division, motility and muscle contraction, mutations in cytoskeleton are frequently associated with human pathology e.g., cardiomyopathies, neurological syndromes and ciliopathies. Our lab is focused on understanding how cytoskeleton assemblies coordinate during physiological and their deregulation during disease conditions. In this talk I will highlight work from our lab, which utilizes the power of nanoengineering (protein and DNA engineering) and in vitro reconstitution to uncover new findings in motility systems mediated by cytoskeleton elements.

Host: Julie Welburn & JP Arulanandam

Dr Emmanuel Derivery - MRC LMB, Cambridge

Wellcome Centre Seminar - Polarized trafficking during asymmetric cell division

Asymmetric cell division gives rise to two daughter cells, which inherit different determinants, thereby acquiring different fates. The asymmetric sorting of signalling endosomes from the central spindle has recently emerged as an important feature of asymmetric cell division contributing to differential cell fate assignation. However, this phenomenon is poorly understood at the molecular level, notably in terms of cytoskeleton.  We have identified a new player, the Elongator complex, which is a multi-subunit complex localized at the central spindle that binds microtubules in vivo, controlling central spindle asymmetry and Sara endosomes asymmetric segregation in Drosophila Sensory Organ Precursors. Since these endosomes contain the cell fate determinants Notch and its receptor Delta, this contributes to asymmetric signalling and thereby cell fate. Moreover, the in vitro experiments show that Elongator complex has a direct effect on microtubule is able to directly bind to microtubules, modulating its dynamics and stability in vitro, and that Elp4 is the subunit responsible for this. Together, this data suggests that that the Elongator complex asymmetrically stabilizes microtubules on one side of the central spindle, playing a major role in linking the signalling complexes polarity signals present at the membrane with the cytosol, asymmetrically stabilizing the central spindle, and thus controlling cell fate during in Drosophila Sensory Organ Precursors development.

Host: Ben Craske and Julie Welburn