The 5S RNP and the regulation of the tumour suppressor

PhD ThesisRibosomes are vital molecular machines involved in catalysing mRNA translation into proteins. Understandably, when ribosome production is disrupted, there are mechanisms available to prevent further cell growth. The main complex involved in this process is the 5S RNP, a ribosome assembly intermediate consisting of the ribosomal proteins RPL5 and RPL11, and the 5S rRNA. The 5S RNP induces the tumour suppressor p53 by interacting with, and inhibiting, MDM2 an ubiquitin ligase responsible for maintaining low levels of p53 in the cell. The 5S RNP-p53 pathway has been implicated in diseases known as ribosomopathies, however the regulation of these pathways have not been fully characterised and there is still much that is unknown. The aim of this project was to identify the components involved in regulating 5S RNP integration into the ribosome and to determine what further components may be required for 5S RNP-mediated p53 activation. There have been many recent studies elucidating the mechanism by which the 5S RNP is incorporated into the ribosome in yeast, however this mechanism appears to vary in humans. The data presented in this thesis determined that mimicking phosphorylation of RPL5 Y30 prevented 5S RNP incorporation into the ribosome. Furthermore, depletion of RPL7, RPL18, and RPL21 resulted in large subunit biogenesis defects and abrogated 5S RNP incorporation into the ribosome. Previous studies have suggested that SRSF1 and PRAS40 may be involved in the regulation of p53 via RPL5 and RPL11 respectively. In congruence with this, the data presented here showed SRSF1 interacts with the 5S RNP. Furthermore, depletion of SRSF1 results in reduction in 5S RNP-dependent p53 activation, mis-localisation of RPL11, and reduction in 5S RNP integration. Collectively, these data suggest that SRSF1 may be important for both ribosome production and p53 regulation. The role of PRAS40, however, still remains unclear. Despite not interacting with the 5S RNP, depletion of PRAS40 appeared to abrogate 5S-RNP mediated p53 induction. In summary, this work has provided a basis for studies into how the 5S RNP is recruited into the ribosome and provides evidence to suggest that SRSF1 is a component of the 5S RNP complex to activate p53.BBSRC and the Diamond Blackfan Anaemia Foundatio

Effect of lower tidal volume ventilation facilitated by extracorporeal carbon dioxide removal vs standard care ventilation on 90-day mortality in patients with acute hypoxemic respiratory failure

Importance In patients who require mechanical ventilation for acute hypoxemic respiratory failure, further reduction in tidal volumes, compared with conventional low tidal volume ventilation, may improve outcomes. Objective To determine whether lower tidal volume mechanical ventilation using extracorporeal carbon dioxide removal improves outcomes in patients with acute hypoxemic respiratory failure. Design, Setting, and Participants This multicenter, randomized, allocation-concealed, open-label, pragmatic clinical trial enrolled 412 adult patients receiving mechanical ventilation for acute hypoxemic respiratory failure, of a planned sample size of 1120, between May 2016 and December 2019 from 51 intensive care units in the UK. Follow-up ended on March 11, 2020. Interventions Participants were randomized to receive lower tidal volume ventilation facilitated by extracorporeal carbon dioxide removal for at least 48 hours (n = 202) or standard care with conventional low tidal volume ventilation (n = 210). Main Outcomes and Measures The primary outcome was all-cause mortality 90 days after randomization. Prespecified secondary outcomes included ventilator-free days at day 28 and adverse event rates. Results Among 412 patients who were randomized (mean age, 59 years; 143 [35%] women), 405 (98%) completed the trial. The trial was stopped early because of futility and feasibility following recommendations from the data monitoring and ethics committee. The 90-day mortality rate was 41.5% in the lower tidal volume ventilation with extracorporeal carbon dioxide removal group vs 39.5% in the standard care group (risk ratio, 1.05 [95% CI, 0.83-1.33]; difference, 2.0% [95% CI, −7.6% to 11.5%]; P = .68). There were significantly fewer mean ventilator-free days in the extracorporeal carbon dioxide removal group compared with the standard care group (7.1 [95% CI, 5.9-8.3] vs 9.2 [95% CI, 7.9-10.4] days; mean difference, −2.1 [95% CI, −3.8 to −0.3]; P = .02). Serious adverse events were reported for 62 patients (31%) in the extracorporeal carbon dioxide removal group and 18 (9%) in the standard care group, including intracranial hemorrhage in 9 patients (4.5%) vs 0 (0%) and bleeding at other sites in 6 (3.0%) vs 1 (0.5%) in the extracorporeal carbon dioxide removal group vs the control group. Overall, 21 patients experienced 22 serious adverse events related to the study device. Conclusions and Relevance Among patients with acute hypoxemic respiratory failure, the use of extracorporeal carbon dioxide removal to facilitate lower tidal volume mechanical ventilation, compared with conventional low tidal volume mechanical ventilation, did not significantly reduce 90-day mortality. However, due to early termination, the study may have been underpowered to detect a clinically important difference