Gle1 is required for tRNA to stimulate Dbp5 ATPase activity in vitro and promote Dbp5-mediated tRNA export in vivo in Saccharomyces cerevisiae.

Cells must maintain a pool of processed and charged transfer RNAs (tRNA) to sustain translation capacity and efficiency. Numerous parallel pathways support the processing and directional movement of tRNA in and out of the nucleus to meet this cellular demand. Recently, several proteins known to control messenger RNA (mRNA) transport were implicated in tRNA export. The DEAD-box Protein 5, Dbp5, is one such example. In this study, genetic and molecular evidence demonstrates that Dbp5 functions parallel to the canonical tRNA export factor Los1. In vivo co-immunoprecipitation data further shows Dbp5 is recruited to tRNA independent of Los1, Msn5 (another tRNA export factor), or Mex67 (mRNA export adaptor), which contrasts with Dbp5 recruitment to mRNA that is abolished upon loss of Mex67 function. However, as with mRNA export, overexpression of Dbp5 dominant-negative mutants indicates a functional ATPase cycle and that binding of Dbp5 to Gle1 is required by Dbp5 to direct tRNA export. Biochemical characterization of the Dbp5 catalytic cycle demonstrates the direct interaction of Dbp5 with tRNA (or double-stranded RNA) does not activate Dbp5 ATPase activity, rather tRNA acts synergistically with Gle1 to fully activate Dbp5. These data suggest a model where Dbp5 directly binds tRNA to mediate export, which is spatially regulated via Dbp5 ATPase activation at nuclear pore complexes by Gle1.

Racial Differences in Patient-Reported Access to Telehealth: An Important and Unmeasured Social Determinant of Health.

PURPOSE: The COVID-19 pandemic expanded opportunities for remote oncology telehealth visits. However, reliable internet connectivity, digital literacy, and patient comfort with virtual medical visits may differ among patients, especially socially disadvantaged groups. The primary aim of this study was to identify barriers that might limit access to telehealth video services. METHODS: First, retrospective analysis was performed of composite administrative data of all patient visits to a large regional cancer center during the pandemic (March 2020 to April 2022). Second, a prospective, cross-sectional study was conducted of patients with known or suspected malignancy over a 6-month period (November 2021 to April 2022). A survey regarding video telehealth accessibility was verbally administered to patients at their clinic visit. RESULTS: Administrative data demonstrated that although Black patients comprised 43% (n = 9,021) of all patient visits (n = 20,953), the proportion of telehealth visits conducted among Black patients was significantly lower compared with White patients (29% v 71%; P < .0001). Of the prospective, cross-sectional study cohort (n = 148), 51.4% of patients (n = 76) were Black, 38.5% (n = 57) resided in a rural county, and 8.1% (n = 12) were Medicaid-insured. Black participants were more likely to self-report lack of internet access (73.7% v 90.4%; P < .01) and were less likely to report having access to or actively using a patient portal (29.0% v 47.2%; P < .001) compared with White patients. The independent association of race and internet access (P < .05) and patient portal use (P = .001) persisted after multivariable analysis. CONCLUSION: Black patients disproportionately underparticipated in telehealth visits, suggesting underlying structural disparities in access to digital care. A greater proportion of Black participants self-reported lack of internet access and access to a patient portal compared with White patients. Ensuring equal internet access and digital literacy will be critical to reduce disparities in cancer care among racial minorities.

Gle1 is required for tRNA to stimulate Dbp5 ATPase activity in vitro and to promote Dbp5 mediated tRNA export in vivo.

Cells must maintain a pool of processed and charged transfer RNAs (tRNA) to sustain translation capacity and efficiency. Numerous parallel pathways support the processing and directional movement of tRNA in and out of the nucleus to meet this cellular demand. Recently, several proteins known to control messenger RNA (mRNA) transport were implicated in tRNA export. The DEAD-box Protein 5, Dbp5, is one such example. In this study, genetic and molecular evidence demonstrates that Dbp5 functions parallel to the canonical tRNA export factor Los1. In vivo co-immunoprecipitation data further shows Dbp5 is recruited to tRNA independent of Los1, Msn5 (another tRNA export factor), or Mex67 (mRNA export adaptor), which contrasts with Dbp5 recruitment to mRNA that is abolished upon loss of Mex67 function. However, as with mRNA export, overexpression of Dbp5 dominant-negative mutants indicates a functional ATPase cycle and that binding of Dbp5 to Gle1 is required by Dbp5 to direct tRNA export. Biochemical characterization of the Dbp5 catalytic cycle demonstrates the direct interaction of Dbp5 with tRNA (or double stranded RNA) does not activate Dbp5 ATPase activity, rather tRNA acts synergistically with Gle1 to fully activate Dbp5. These data suggest a model where Dbp5 directly binds tRNA to mediate export, which is spatially regulated via Dbp5 ATPase activation at nuclear pore complexes by Gle1.

Emerging molecular functions and novel roles for the DEAD-box protein Dbp5/DDX19 in gene expression.

The DEAD-box protein (DBP) Dbp5, a member of the superfamily II (SFII) helicases, has multiple reported roles in gene expression. First identified as an essential regulator of mRNA export in Saccharomyces cerevisiae, the enzyme now has reported functions in non-coding RNA export, translation, transcription, and DNA metabolism. Localization of the protein to various cellular compartments (nucleoplasm, nuclear envelope, and cytoplasm) highlights the ability of Dbp5 to modulate different stages of the RNA lifecycle. While Dbp5 has been well studied for > 20 years, several critical questions remain regarding the mechanistic principles that govern Dbp5 localization, substrate selection, and functions in gene expression. This review aims to take a holistic view of the proposed functions of Dbp5 and evaluate models that accommodate current published data.

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2.

RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels in balance with PABPs and other RBPs with mutable substrate specificity across nucleoplasmic and nucleolar RNA processes.

Loss of Muscle Carnitine Palmitoyltransferase 2 Prevents Diet-Induced Obesity and Insulin Resistance despite Long-Chain Acylcarnitine Accumulation.

To assess the effects of acylcarnitine accumulation on muscle insulin sensitivity, a model of muscle acylcarnitine accumulation was generated by deleting carnitine palmitoyltransferase 2 (CPT2) specifically from skeletal muscle (Cpt2Sk-/- mice). CPT2 is an irreplaceable enzyme for mitochondrial long-chain fatty acid oxidation, converting matrix acylcarnitines to acyl-CoAs. Compared with controls, Cpt2Sk-/- muscles do not accumulate anabolic lipids but do accumulate ∼22-fold more long-chain acylcarnitines. High-fat-fed Cpt2Sk-/- mice resist weight gain, adiposity, glucose intolerance, insulin resistance, and impairments in insulin-induced Akt phosphorylation. Obesity resistance of Cpt2Sk-/- mice could be attributed to increases in lipid excretion via feces, GFD15 production, and energy expenditure. L-carnitine supplement intervention lowers acylcarnitines and improves insulin sensitivity independent of muscle mitochondrial fatty acid oxidative capacity. The loss of muscle CPT2 results in a high degree of long-chain acylcarnitine accumulation, simultaneously protecting against diet-induced obesity and insulin resistance.

A nuclear role for the DEAD-box protein Dbp5 in tRNA export.

Dbp5 is an essential DEAD-box protein that mediates nuclear mRNP export. Dbp5 also shuttles between nuclear and cytoplasmic compartments with reported roles in transcription, ribosomal subunit export, and translation; however, the mechanism(s) by which nucleocytoplasmic transport occurs and how Dbp5 specifically contributes to each of these processes remains unclear. Towards understanding the functions and transport of Dbp5 in Saccharomyces cerevisiae, alanine scanning mutagenesis was used to generate point mutants at all possible residues within a GFP-Dbp5 reporter. Characterization of the 456 viable mutants led to the identification of an N-terminal Xpo1-dependent nuclear export signal in Dbp5, in addition to other separation-of-function alleles, which together provide evidence that Dbp5 nuclear shuttling is not essential for mRNP export. Rather, disruptions in Dbp5 nucleocytoplasmic transport result in tRNA export defects, including changes in tRNA shuttling dynamics during recovery from nutrient stress.