mRNA caps are methylated structures that form on the 5’ end of RNA pol II transcripts, which recruit RNA processing and translation factors. Our research investigates how mRNA cap formation is regulated and its impact on pre-mRNA transcription, processing and translation. We investigate how cellular signalling pathways influence the expression or activity of the mRNA cap methyltransferases, resulting in changes in gene expression, cell function and cell physiology. We have found that the different signals which the cell encounters (developmental, immunological, oncogenic) can alter the rate and extent to which the mRNA cap forms, either across the transcriptome or on specific transcripts. Thus the mRNA cap is an integrator of cellular signalling information, which directs reshaping of the cellular proteome in response to external and internal signals. Our recent research investigates the impact of the mRNA cap in vivo, with the finding that the mRNA cap has both core functions and organ-specific impact. The influence of mRNA cap regulation on embryonic stem cell differentiation and T cell activation will be discussed.

Long non-coding RNAs have emerged as players in development in mammalian systems. Fundamental questions regarding the structure of lncRNAs have yet to be addressed, including, (1) are lncRNAs organized into modular sub-domains or linear chains of stem loops?, and (2) what do structure-function relations look like in the case of lncRNA systems? We have developed an experimental strategy, called Shotgun Secondary Structure (3S) determination, to derive secondary structures of intact, individual lncRNAs. We chemically probe the entire lncRNA transcript and repeat on overlapping fragments of the transcript. Matching profiles suggest the fragment folds into a modular sub-domain or modular secondary motif, eliminating a large number of other possible folds. We applied this method to the mouse Braveheart lncRNA and plant COOLAIR lncRNA, both of which possess clear phenotypes. Braveheart and COOLAIR each show highly structured sub-domains and share a unique expansive internal loop (r-turn motif). In the case of Braveheart, CRISPR-Cas9 analysis demonstrates that the expansive loop motif is necessary for cardiomyocyte lineage commitment. This loop was found to interact with a zinc finger transcription factor (CNBP). In the case of COOLAIR, we used the 3S-generated secondary structure for A. thaliana to correctly predict the existence of COOLAIR in several other species. We then performed 3S on COOLAIR in B. rapa and demonstrated that the structure of COOLAIR is conserved across ~20 million years of evolution. We will also present preliminary SAXS data on Braveheart.
Regarding translocation of tRNAs through the ribosome, we propose a new translocational intermediate consistent with recent single molecule FRET data called superswivel. This conformation extends the amplitude of 30S head swivel, delivers tRNAs to their fully translocated positions. Here, we integrate molecular dynamics simulations of the 70S ribosome translocation complex with single-molecule FRET studies to characterize the conformational changes in the ribosome accompanying translocation in atomistic detail, and the transit of key intermediates late in translocation that have recently been identified by structural and smFRET investigations. Placing our findings from simulation and single-molecule FRET in the context of available crystallographic and cryo-EM data on translocation, we find that the super-swivel state achieves correct positioning of the peptidyl- and deacyl-tRNAs in the P/P and E/E states, where the deacyl-tRNA can then disengage from the E site, facilitating backwards head swivel and the completion of translocation.




Structural architecture of the human long non-coding RNA, steroid receptor RNA activator. Novikova, IV, Hennelly, SP, and Sanbonmatsu, KY. Nucleic acids research, 40 (11), 5034-5051, 2012.

A G-Rich Motif in the LncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage. Xue, Z, Hennelly, S.P, Doyle, B, Gulati, A.A, Novikova, I.V, Sanbonmatsu, K.Y, and Boyer, L.A. Molecular Cell, 64 (1), 37-50, 2016.

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structure. Hawkes, E.J, Hennelly, S.P, Novikova, I.V, Irwin, J.A, Dean, C, and Sanbonmatsu, K.Y. Cell Reports, 16 (12), 3087-3096, 2016.

Long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission change synaptic pathways in an activity-dependent manner. Although such changes seem to be stable over days, it is less clear how plasticity affects individual synapses over time. We showed previously that LTD preferably leads to elimination of low release probability synapses, suggesting that weight adjustments affect synaptic lifetime. However, we do not know how ongoing activity, potentiation and depression are integrated over time at individual synapses to regulate their persistence.

We combine optogenetic and chemogenetic tools to tightly control activity at identified Schaffer collateral synapses in organotypic hippocampal slice cultures. All-optical induction of LTD and LTP in combination with 2-photon calcium imaging allowed us to measure the strength of individual synapses and to follow their fate over 7 days. Although potentiated synapses typically persisted for the next 7 days, induction of plasticity of opposite polarity (LTP followed by LTD) completely reversed this effect. Conversely, LTP induction overwrote the destabilizing effect of LTD. Thus, multiple weight adjustments are not linearly integrated but rather synapse survival is dominated by the most recent plasticity event. In addition, we tested whether chronic chemogenetic reduction of activity at identified synapses has an impact on their lifetime. Unexpectedly, shutdown of evoked synaptic transmission for 7 days did not alter the lifetime of native synapses, suggesting that reduced transmission alone could not account for increased synapse elimination. In summary, plasticity is required to set the lifetime of synapses, while reduced transmission alone has no effect.

 “Evidence that sub-cellular oscillators may time and execute organelle biogenesis”

The accurate timing and execution of organelle biogenesis is crucial for cell physiology, yet we have little idea about how these processes are regulated. In order to study this question, we recently established the Drosophila centriole as a model, as its linear structure makes its biogenesis easy to define and measure. Centriole biogenesis occurs only in S-phase and it is strictly regulated by Polo-like kinase 4 (Plk4). Unexpectedly, we find that centriole growth in early Drosophila embryos is homeostatic: When centrioles grow slowly, they grow for a longer period; when centrioles grow quickly, they grow for a shorter period. This ensures that centrioles grow to a consistent size. Plk4 is localized at the base of the growing daughter centriole and it sets both the rate and period of daughter centriole growth. The activity of Plk4 kinase controls the growth rate, but how Plk4 determines the growth period is unclear. I will discuss our recent data that suggests that Plk4 forms an adaptive oscillator at the base of the growing centriole, whose propagation times and sets the duration of centriole biogenesis in Drosophila embryos. The Plk4 oscillator appears to be free-running of, but entrained and calibrated by, the core Cdk/Cyclin cell-cycle oscillator. Mathematical modelling and experimental evidence indicate that Plk4 oscillations are generated by a time-delayed negative-feedback loop. We speculate that such free-running oscillators could regulate the timing and execution of organelle biogenesis more generally.

TBC

Ca2+-channel blockers, such as the dihydropyridines (DHPs) amlodipine and isradipine, are clinically used for first-line treatment of hypertension and angina by inhibiting ion permeation through voltage-gated L-type Ca2+-channels (LTCCs, Cav1) in arterial smooth muscle and the heart. LTCCs are also key regulators of Ca2+-dependent signaling processes in the brain. In neurons Cav1.2 and Cav1.3 LTCC isoforms are located postsynaptically at dendrites and at the cell soma. They control neuronal excitability, synaptic morphology and couple synaptic activity to gene transcription. Through their distinct biophysical properties Cav1.2 and Cav1.3 contribute in different ways to various forms of learning, memory, emotional and drug-taking behaviours. Cav1.3 also appears to play a major role for the high Ca2+ -dependent vulnerability of Substantia nigra dopamine neurons in Parkinsons Disease.

We have recently characterized three human disease-relevant de novo missense mutations in the pore-forming a1-subunit of Cav1.3 channels (CACNA1D), including gain-of-function mutations in three patients with sporadic autism spectrum disorder (ASD) with or without intellectual disability and neurological symptoms. Our recent finding of a fourth mutation in a patient with a severe, undiagnosed developmental disorder (DECIPHER DD cohort) adds CACNA1D to the list of major risk genes for neurodevelopmental disorders, including ASD. We have successfully generated a mouse model carrying one of the human ASD mutations (A749G). First results obtained with this mouse model confirm behavioral abnormalities consistent with behaviors observed in ASD patients. Brain-permeable LTCC inhibitors already in clinical use (e.g. isradipine, nimodipine) may therefore serve as promising therapeutics in individuals carrying such Cav1.3 gain-of-function mutations, a hypothesis that can be tested in A749G mice. However, we provide experimental evidence that the therapeutic window of DHPs may be limited, because isradipine inhibits Cav1.3 channels during simulated neuronal activity patterns with an order of magnitude lower potency than smooth muscle Cav1.2, its peripheral target for blood pressure lowering. Therefore, inhibition of neuronal LTCCs Ca2+ channels with existing LTCCblockers could be a novel option for the treatment of neuropsychiatric disease and perhaps also as a neuroprotectant in Parkinsons Disease. However, chronic treatment may require high maintenance doses likely to cause side effects in some patients.

Register at https://www2.mrc-lmb.cam.ac.uk/groups/nextgen/

There will be 12 talks from speakers covering topics from fundamental technology and method development to the use of cutting edge biophysics in the pharmaceutical industry. There will be four main themes; Nucleic Acid Enzymes & Machines, Protein Conformation, New Technologies and Signaling. There will also be a keynote lecture from the LMB’s Richard Henderson in the area of cryoEM.

Please register here https://www.eventbrite.co.uk/e/3rs-replication-repair-recombination-meeting-october-2018-tickets-49955268553

External speaker: Frank Uhimann, Francis Crick Institute, London