Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors ...that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.
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•An unbiased genetic screen identifies effectors of AKT signaling as regulators of NMD•AKT phosphorylation of UPF1 overcomes autoinhibition of UPF1 helicase activity•AKT signaling promotes formation of EJCs containing AKT at the expense of UPF2•AKT and UPF2 constitute alternative EJCs with distinct mechanisms of UPF1 activation
Cho, Abshire, and coworkers demonstrate that the serine/threonine kinase AKT functionally replaces UPF2 in alternative EJCs that contain CASC3 and promotes NMD by phosphorylating UPF1. AKT-mediated NMD is stimulated by insulin. FMR1-KO NSCs treated with the AKT inhibitor Afuresertib manifest suppression of hyperactivated NMD, which typifies Fragile X syndrome.
Abstract
The circadian protein Nocturnin (NOCT) belongs to the exonuclease, endonuclease and phosphatase superfamily and is most similar to the CCR4-class of deadenylases that degrade the ...poly-adenosine tails of mRNAs. NOCT-deficient mice are resistant to high-fat diet induced weight gain, and exhibit dysregulation of bone formation. However, the mechanisms by which NOCT regulates these processes remain to be determined. Here, we describe a pair of high-resolution crystal structures of the human NOCT catalytic domain. The active site of NOCT is highly conserved with other exoribonucleases, and when directed to a transcript in cells, NOCT can reduce translation and abundance of that mRNA in a manner dependent on key active site residues. In contrast to the related deadenylase CNOT6L, purified recombinant NOCT lacks in vitro ribonuclease activity, suggesting that unidentified factors are necessary for enzymatic activity. We also find the ability of NOCT to repress reporter mRNAs in cells depends upon the 3′ end of the mRNA, as reporters terminating with a 3′ MALAT1 structure cannot be repressed by NOCT. Together, these data demonstrate that NOCT is an exoribonuclease that can degrade mRNAs to inhibit protein expression, suggesting a molecular mechanism for its regulatory role in lipid metabolism and bone development.
Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and ...cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type–specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates NADP(H) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells.
Post-transcriptional control of messenger RNA (mRNA) is an important layer of gene regulation that modulates mRNA decay, translation, and localization. Eukaryotic mRNA decay begins with the catalytic ...removal of the 3′ poly-adenosine tail by deadenylase enzymes. Multiple deadenylases have been identified in vertebrates and are known to have distinct biological roles; among these proteins is Nocturnin, which has been linked to circadian biology, adipogenesis, osteogenesis, and obesity. Multiple studies have investigated Nocturnin's involvement in these processes; however, a full understanding of its molecular function remains elusive. Recent studies have provided new insights by identifying putative Nocturnin-regulated mRNAs in mice and by determining the structure and regulatory activities of human Nocturnin. This review seeks to integrate these new discoveries into our understanding of Nocturnin's regulatory functions and highlight the important remaining unanswered questions surrounding its regulation, biochemical activities, protein partners, and target mRNAs.
The circadian protein Nocturnin (NOCT) belongs to the exonuclease, endonuclease and phosphatase (EEP) superfamily and is most similar to the CCR4-class of deadenylases that degrade the poly-adenosine ...poly(A) tails of mRNAs. NOCT-deficient mice are resistant to diet-induced obesity and exhibit increased bone density; however, the mechanisms by which NOCT regulates these processes remain unknown.Based on homology, we predicted that NOCT adopts the α/β hydrolase fold that is characteristic of EEP deadenylases, has activity against poly(A) RNAs in vitro and regulates mRNA expression and half-lives in vivo. Since the NOCT null animals display alterations in lipid metabolism, we expected that we would identify target mRNAs of NOCT involved in lipid metabolism and adipogenesis.To determine the structure of the NOCT catalytic domain, we solved a pair of crystal structures of NOCT, demonstrating that the structure is highly conserved with other EEP deadenylases. Purified recombinant NOCT lacked ribonuclease activity in vitro. However, in cell-based assays, we found that NOCT reduces translation and abundance of reporter mRNAs. Incomplete reversal of repression when the NOCT active site is mutated suggests a role for translational repression in addition to ribonuclease activity. NOCT-mediated repression of reporter mRNAs is additionally dependent upon the 3′ end, as reporters terminating with a 3′ MALAT1 structure cannot be repressed by NOCT. Together, these data suggest a model by which NOCT represses expression from its target mRNAs through RNA decay and translational repression.We then examined the NOCT protein sequence for motifs indicating subcellular localization. Sequence features of the NOCT mRNA suggests that NOCT may be localized to the cytoplasm and the mitochondria. Additionally, the NOCT protein sequence has predicted motifs for mitochondrial localization. Consistent with these observations, we observed that the full-length NOCT is a preprotein that can be processed. Surprisingly, processing of NOCT appears to occur in a tissue-type specific manner, suggesting that NOCT is differentially located to the cytoplasm or mitochondria in different cell types.To identify potential targets of NOCT we generated HEK293 cells with cytoplasmic overexpression of NOCT and performed RNA-Seq. A dataset of transcripts that change in abundance with NOCT overexpression was identified, demonstrating that NOCT is able to affect steady state levels of mRNAs. In addition to these potential NOCT targets, we further confirmed a report that NOCT has phosphatase activity against NADPH and NADP+, indicating that NOCT can hydrolyze non-mRNA substrates. These data suggest that NOCT may regulate metabolism through modulating the steady state levels of mRNAs as well as NADP(H).
1. Become aware of TIMS audio files and their use by clinicians
2. Understand the utility of TIMS files in benefiting the clinical team, patients and loved ones.
TIMS (This is My Story) is 2-minute ...patient recorded audio file placed in the medical record that improves communication between patients, loved ones and the medical team and increases the team's feelings of empathy for the patient.
TIMS helps medical teams view the patient more holistically and begins to address rampant moral distress and burnout experienced by many clinicians even before the Covid-19 pandemic, by allowing them to resonate with their own humanity in their care delivery.
Our objective was to assess whether a streamlined TIMS file distilled from our previous work could achieve the same outcomes we observed with our initial open-ended interview.
Both quantitative and qualitative methods were used to assess the impact of TIMS recordings on medical staff. Pre and post listening questionnaires quantified attitudes and knowledge of the patient and loved ones, while qualitative interviews explored these in greater depth.
Fifty-three participants responded to our TIMS evaluation survey. Almost all respondents reported the TIMS file contained useful information (50/51 reported yes or some useful info.) There was limited self-reported effect of the TIMS file on distress and empathy; 24% of respondents reported listening to the file decreased their distress. The majority response was that the respondent either did not have distress or the TIMS file did not change their distress. Of concern, 3 people reported that listening to the file increased their distress. Importantly, most respondents reported feeling greater empathy for the patient after listening (53%) and of those who communicated with family members, most reported listening improved their communication with family members (63%, n=9/13). Qualitative analysis revealed most participants had positive impressions about their use of TIMS.
TIMS files help improve communications between clinicians caring for a patient and the patient and their loved ones. TIMS files increase empathy on the part of the medical team for the patient.
The TIMS intervention is one tool that can be easily employed and deployed to help medical teams communicate more easily and feel more empathy for patients. Future studies will assess whether this reduces burnout and moral distress for medical staff. Two other avenues of inquiry include a current study on the patient or loved one's perception of recording the TIMS file, and a longitudinal look at use of TIMS in patients undergoing head and neck surgery, where the file is played in the OR during the time out, is available for the subsequent hospitalization and in outpatient follow up care. An app to facilitate TIMS recordings is also under development.
INTRODUCTIONChildhood obesity has been associated with numerous poor health conditions, with geographic disparities demonstrated. Limited research has examined the association between rurality and ...food security, physical activity, and overweight or obesity among children. We examined rates of food security, physical inactivity, and overweight or obesity among rural and urban children and adolescents, and associations between rurality and these 3 outcomes.METHODSWe used cross-sectional data from a nationally representative sample of children and adolescents aged 10 to 17 years from the 2019-2020 National Survey of Children's Health (N = 23,199). We calculated frequencies, proportions, and unadjusted associations for each variable by using descriptive statistics and bivariate analyses. We used multivariable logistic regression models to examine the association between rurality and food security, physical activity, and overweight or obesity.RESULTSAfter adjusting for sociodemographic factors, rural children and adolescents had higher odds than urban children and adolescents of being overweight or obese (adjusted odds ratio = 1.30; 95% CI, 1.11-1.52); associations between rurality and physical inactivity and food insecurity were not significant.CONCLUSIONThe information from this study is timely for policy makers and community partners to make informed decisions on the allocation of healthy weight and obesity prevention programs for children and adolescents in rural settings. Our study provides information for public health programming and the designing of appropriate dietary and physical activity interventions needed to reduce disparities in obesity prevention among children and adolescents.
The purpose of this study is to examine how a novel intervention known as TIMS, “This is My Story,” impacted clinicians caring for patients during the COVID-19 pandemic in the medical intensive care ...unit (MICU) at the Johns Hopkins Hospital. An eight-question survey was administered to MICU staff on their experience with TIMS files for pre- and post-listening reflections. Qualitative interviews were conducted with 17 staff members who prospectively agreed to participate. A total of 97 pre-listening and 88 post-listening questionnaires were completed. Responses indicated that the audio recording was appropriate to discover more about the patient beyond the immediately observable and useful (98%), “considerably” increased staff empathy for the patient (74%), and thought it would “some” or “considerably” improve subsequent interactions with the patient’s loved ones (99%). The qualitative analysis revealed that medical staff found the audio format easy to use and helpful in humanizing patients in their clinical practice. The study demonstrates that TIMS audio files are an important addition to the electronic medical record, enabling clinicians to practice with greater awareness of the patient’s context and increased empathy for patients and families.
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