Transposable elements (TEs) are of interest as immunomodulators for cancer therapies. TEs can fold into dsRNAs that trigger the interferon response. Here, we investigated the effect of different HDAC inhibitors (HDACIs) on the expression of TEs in leiomyosarcoma cells. Our data show that endogenous retroviruses (ERVs), especially ERV1 elements, are upregulated after treatment with HDAC1/2/3-specific inhibitors. Surprisingly, the interferon response was not activated. We observed an increase in A-to-I editing of upregulated ERV1. This could have an impact on the stability of dsRNAs and the activation of the interferon response. We also found that H3K27ac levels are increased in the LTR12 subfamilies, which could be regulatory elements controlling the expression of proapoptotic genes such as TNFRSF10B. In summary, we provide a detailed characterization of TEs modulation in response to HDACIs and suggest the use of HDACIs in combination with ADAR inhibitors to induce cell death and support immunotherapy in cancer.

The National Institute of Health R25 Research Education Program was evaluated in the second year of implementation. Twelve mentors and 20 underrepresented minority students (URMs) scholars from partnerships and collaborations among five colleges and universities were added to the program to provide a more diverse research experience. Findings reveal that 100% of research mentors agree that the approachableness and accessibility of the program coordinator were beneficial in achieving mentorship goals and objectives. In addition, 85% of the students strongly agreed that the presentation of their research findings and the weekly reflection on goals, identification of accomplishments, and obstacles through the individual development plan were very effective. Of the 23 successfully tracked students for 2 years, six URMs (26.09%) obtained a bachelor\'s degree and were admitted into a graduate program; two were directly admitted to a PhD program in biomedical sciences.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2\'s preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes\' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Cancer is a complex and heterogeneous disease, in which a number of genetic and epigenetic changes occur in tumor onset and progression. Recent studies indicate that changes at the RNA level are also involved in tumorigenesis, such as adenosine-to-inosine (A-to-I) RNA editing. Here, we systematically investigate transcriptome-wide A-to-I editing events in a large number of samples from Non-Hodgkin lymphomas (NHLs). Using a computational pipeline that determines significant differences in editing level between NHL and normal samples at known A-to-I editing sites, we identify a number of differentially edited editing sites between NHL subtypes and normal samples. Most of the differentially edited sites are located in non-coding regions, and many such sites show a strong correlation between gene expression level and editing efficiency, indicating that RNA editing might have direct consequences for the cancer cell\'s aberrant gene regulation status in these cases. Moreover, we establish a strong link between RNA editing and NHL by demonstrating that NHL and normal samples and even NHL subtypes can be distinguished based on genome-wide RNA editing profiles alone. Our study establishes a strong link between RNA editing, cancer and aberrant gene regulation in NHL.

We examined the relationship between subjective and objective sleep outcomes and loneliness in older women at risk for Alzheimer\'s disease (AD). Our sample consisted of 39 participants (aged 65+) with mild cognitive deficits who completed the UCLA Loneliness Scale, the Pittsburgh Sleep Quality Index (PSQI), and an at home sleep test, to determine presence of obstructive sleep apnea. Based on sleep quality scores, individuals categorized as \"poor sleepers\" had significantly higher loneliness scores than \"good sleepers.\" However, total loneliness scores did not significantly differ between groups with or without sleep apnea. We found that higher loneliness was significantly associated to lower habitual sleep efficiency and sleep duration and was also influenced by use of sleep medication. Our findings suggest that increased loneliness relates to worse subjective sleep quality, but not to sleep apnea. These findings suggest that combined interventions targeting loneliness and sleep quality may be important for older women.

Measures of life outlook in older adults have been investigated in connection to pain, as both pain management and outlook are important factors of successful aging. We hypothesized that higher pain is associated with lower optimism among community-dwelling older adults. We utilized data from the UC San Diego Successful Aging Evaluation (SAGE), a prospective longitudinal cohort study initiated in 2010, to evaluate the relationship between pain and optimism in 378 community-dwelling adults aged ≥50 years. We used the revised Life Orientation Test (LOT-R) to measure optimism and three pain subscales-PROMIS Pain Interference, PROMIS Pain Intensity, and MOS 36-Item Short-Form Health Survey (SF-36)-as pain measures. Regression analyses reveal negative relationships between pain and optimism for all three pain scales, with regression coefficients of -0.277 (p < .0001), -0.246 (p < .0001), and 0.269 (p < .0001) respectively. This indicates value in considering physical and psychological elements in future intervention research to promote healthy aging.

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community\'s ability to interrogate both forms of RNA variation simultaneously.
We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.
Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

The dynamic process of Drosophila spermatogenesis involves asymmetric division, mitosis, and meiosis, which ultimately results in the production of mature spermatozoa. Disorders of spermatogenesis can lead to infertility in males. ADAR (adenosine deaminase acting on RNA) mutations in Drosophila cause male infertility, yet the causative factors remain unclear. In this study, immunofluorescence staining was employed to visualize endogenous ADAR proteins and assess protein levels via fluorescence-intensity analysis. In addition, the early differentiation disorders and homeostatic alterations during early spermatogenesis in the testes were examined through quantification of transit-amplifying region length, counting the number of GSCs (germline stem cells), and fertility experiments. Our findings suggest that deletion of ADAR causes testicular tip transit-amplifying cells to accumulate and become infertile in older male Drosophila. By overexpressing ADAR in early germline cells, male infertility can be partially rescued. Transcriptome analysis showed that ADAR maintained early spermatogenesis homeostasis through the bone-morphogenetic-protein (BMP) signaling pathway. Taken together, these findings have the potential to help explore the role of ADAR in early spermatogenesis.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.