Ceg1 depletion reveals mechanisms governing degradation of non-capped RNAs in Saccharomyces cerevisiae.

Most functional eukaryotic mRNAs contain a 5' 7-methylguanosine (m7G) cap. Although capping is essential for many biological processes including mRNA processing, export and translation, the fate of uncapped transcripts has not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that although the degradation of cap-deficient mRNA is dominant, the levels of hundreds of non-capped mRNAs increase upon depletion of the capping enzymes. Overall, the abundance of non-capped mRNAs is inversely correlated to the expression levels, altogether resembling the effects observed in cells lacking the cytoplasmic 5'-3' exonuclease Xrn1 and indicating differential degradation fates of non-capped mRNAs. The inactivation of the nuclear 5'-3' exonuclease Rat1 does not rescue the non-capped mRNA levels indicating that Rat1 is not involved in their degradation and consequently, the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the cap presence is essential to initiate the Xrn1-dependent degradation of mRNAs underpinning the role of 5' cap in the Xrn1-dependent buffering of the cellular mRNA levels.

The induction of p53 correlates with defects in the production, but not the levels, of the small ribosomal subunit and stalled large ribosomal subunit biogenesis.

Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.

RPS27a and RPL40, Which Are Produced as Ubiquitin Fusion Proteins, Are Not Essential for p53 Signalling.

Two of the four human ubiquitin-encoding genes express ubiquitin as an N-terminal fusion precursor polypeptide, with either ribosomal protein (RP) RPS27a or RPL40 at the C-terminus. RPS27a and RPL40 have been proposed to be important for the induction of the tumour suppressor p53 in response to defects in ribosome biogenesis, suggesting that they may play a role in the coordination of ribosome production, ubiquitin levels and p53 signalling. Here, we report that RPS27a is cleaved from the ubiquitin-RP precursor in a process that appears independent of ribosome biogenesis. In contrast to other RPs, the knockdown of either RPS27a or RPL40 did not stabilise the tumour suppressor p53 in U2OS cells. Knockdown of neither protein blocked p53 stabilisation following inhibition of ribosome biogenesis by actinomycin D, indicating that they are not needed for p53 signalling in these cells. However, the knockdown of both RPS27a and RPL40 in MCF7 and LNCaP cells robustly induced p53, consistent with observations made with the majority of other RPs. Importantly, RPS27a and RPL40 are needed for rRNA production in all cell lines tested. Our data suggest that the role of RPS27a and RPL40 in p53 signalling, but not their importance in ribosome biogenesis, differs between cell types.

National Ambulance Surveillance System: A novel method using coded Australian ambulance clinical records to monitor self-harm and mental health-related morbidity.

Self-harm and mental health are inter-related issues that substantially contribute to the global burden of disease. However, measurement of these issues at the population level is problematic. Statistics on suicide can be captured in national cause of death data collected as part of the coroner's review process, however, there is a significant time-lag in the availability of such data, and by definition, these sources do not include non-fatal incidents. Although survey, emergency department, and hospitalisation data present alternative information sources to measure self-harm, such data do not include the richness of information available at the point of incident. This paper describes the mental health and self-harm modules within the National Ambulance Surveillance System (NASS), a unique Australian system for monitoring and mapping mental health and self-harm. Data are sourced from paramedic electronic patient care records provided by Australian state and territory-based ambulance services. A team of specialised research assistants use a purpose-built system to manually scrutinise and code these records. Specific details of each incident are coded, including mental health symptoms and relevant risk indicators, as well as the type, intent, and method of self-harm. NASS provides almost 90 output variables related to self-harm (i.e., type of behaviour, self-injurious intent, and method) and mental health (e.g., mental health symptoms) in the 24 hours preceding each attendance, as well as demographics, temporal and geospatial characteristics, clinical outcomes, co-occurring substance use, and self-reported medical and psychiatric history. NASS provides internationally unique data on self-harm and mental health, with direct implications for translational research, public policy, and clinical practice. This methodology could be replicated in other countries with universal ambulance service provision to inform health policy and service planning.

The National Ambulance Surveillance System: A novel method for monitoring acute alcohol, illicit and pharmaceutical drug related-harms using coded Australian ambulance clinical records.

Although harmful consumption of alcohol and other drugs (both illicit and pharmaceutical) significantly contribute to global burden of disease, not all harms are captured within existing morbidity data sources. Indeed, harms occurring in the community may be missed or under-reported. This paper describes the National Ambulance Surveillance System, a unique Australian system for monitoring and mapping acute harms related to alcohol and other drug consumption. Data are sourced from paramedic electronic patient care records provided by ambulance services from across Australia. Coding occurs in a purpose-built system, by a team of specialised research assistants. Alcohol, and specific illicit and pharmaceutical drugs, rather than broad drug classes, are manually coded and the dataset is reviewed and cleaned prior to analysis. The National Ambulance Surveillance System is an ongoing, dynamic surveillance system of alcohol and other drug-related harms across Australia. The data includes more than 140 output variables per attendance, including individual substances, demographics, temporal, geospatial, and clinical data (e.g., Glasgow Coma Scale score, naloxone provision and response, outcome of attendance). The National Ambulance Surveillance System is an internationally unique population-level surveillance system of acute harms arising from alcohol and other drug consumption. Dissemination of National Ambulance Surveillance System data has been used to inform and evaluate policy approaches and potential points of intervention, as well as guide workforce development needs and clinical practice at the local and national level. This methodology could be replicated in other countries.