Christopher Nicchitta

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.

Aim/Purpose National and international survey studies have begun to identify heightened levels of depression, anxiety, and burnout among doctoral students. Nevertheless, little research has been done to evaluate which interventions may support doctoral student wellness. Background To guide future interventions research, this study evaluated perceptions of the acceptability and effectiveness of wellness approaches among biomedical doctoral students. Methodology In this study, 69 biomedical doctoral students were sampled from a research institution in the southeastern United States. Participants completed a structured psychiatric diagnostic interview and self-report questionnaires. Questionnaires assessed participants’ beliefs about the acceptability and effectiveness of 36 wellness approaches in reducing burnout symptoms and depression symptoms, and the participants’ attitudes towards psychological services. Contribution This study demonstrates that approaches to support biomedical doctoral student wellness should be tailored according to a student’s history of problems with mental health. Findings Among candidate approaches, those involving spending time socializing with friends and family were rated most favorably by the entire sample. However, participants with high burnout or depression symptoms negatively evaluated approaches involving social engagement. Participants with high burnout symptoms or a history of psychological diagnoses or treatment rated individual therapy more favorably. Recommendations Social engagement is highly valued by biomedical doctoral students, above for Practitioners and beyond institution-based wellness resources. University administrators should prioritize interventions favored by students struggling with symptoms of burnout and mental health problems, especially individual therapy. Recommendations Randomized trials should be conducted to assess the effectiveness in reducfor Researchers ing problems with mental health of the approaches rated favorably, particularly those involving social engagement. Studies should investigate facilitators and barriers to approaches rated highly likely to be effective, but not acceptable, including peer support groups and individual therapy. Impact on Society In the interest of preventing attrition from biomedical doctoral programs and promoting the wellness and success of future scientists, it is important to develop training programs sensitive to the mental health needs of their students. This study provides important insights guiding next steps in intervention testing and implementation to support biomedical doctoral students. Future Research Future studies should validate the findings in this study with large internationally representative samples of students across various fields of doctoral study. Future intervention studies should include rigorous evaluation of facilitators and barriers for approaches rated favorably in this study.

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

Although doctoral students in the biomedical sciences have been recognized as a population at particular risk for mental health problems such as burnout and depression, little research has been conducted to identify candidate targets for intervention. To this end, we used the stress process model to evaluate potential mediators of stress-burnout and stress-depression relationships in biomedical doctoral students. A cross-sectional sample (n = 69) completed validated self-report measures of stress; symptoms of burnout and depression; and perceptions of mastery, social support, and advisor support. In linear regression models, we found that academic stressors were most predictive of burnout, whereas family/monetary stressors were most predictive of depression. In mediation models, we found that the relationship between stress and burnout was partially mediated by mastery and advisor support, while the stress-depression relationship was partially mediated by mastery. These findings represent a first step in identifying interventional targets to improve mental health in this at-risk population. Whereas certain stressors are inherent to the doctoral training environment, psychosocial interventions to enhance one's sense of mastery and/or to improve advisor relationships may mitigate the influence of such stressors on burnout and depression.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5' RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.  Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.