Brief guide to RNA sequencing analysis for nonexperts in bioinformatics.

Transcriptome analysis is widely used for current biological research but remains challenging for many experimental scientists. Here, we present a brief but broad guideline for transcriptome analysis, focusing on RNA sequencing, by providing the list of publicly available datasets, tools, and R packages for practical transcriptome analysis. This work will be useful for biologists to perform key transcriptomic analysis with minimum expertise in bioinformatics.

Combinatorial transcriptomic and genetic dissection of insulin/IGF-1 signaling-regulated longevity in Caenorhabditis elegans.

Classical genetic analysis is invaluable for understanding the genetic interactions underlying specific phenotypes, but requires laborious and subjective experiments to characterize polygenic and quantitative traits. Contrarily, transcriptomic analysis enables the simultaneous and objective identification of multiple genes whose expression changes are associated with specific phenotypes. Here, we conducted transcriptomic analysis of genes crucial for longevity using datasets with daf-2/insulin/IGF-1 receptor mutant Caenorhabditis elegans. Our analysis unraveled multiple epistatic relationships at the transcriptomic level, in addition to verifying genetically established interactions. Our combinatorial analysis also revealed transcriptomic changes associated with longevity conferred by daf-2 mutations. In particular, we demonstrated that the extent of lifespan changes caused by various mutant alleles of the longevity transcription factor daf-16/FOXO matched their effects on transcriptomic changes in daf-2 mutants. We identified specific aging-regulating signaling pathways and subsets of structural and functional RNA elements altered by different genes in daf-2 mutants. Lastly, we elucidated the functional cooperation between several longevity regulators, based on the combination of transcriptomic and molecular genetic analysis. These data suggest that different biological processes coordinately exert their effects on longevity in biological networks. Together our work demonstrates the utility of transcriptomic dissection analysis for identifying important genetic interactions for physiological processes, including aging and longevity.

Meta-analysis of the transcriptome identifies aberrant RNA processing as common feature of aging in multiple species.

Aging is accompanied by the gradual deregulation of the transcriptome. However, whether age-dependent changes in the transcriptome are evolutionarily conserved or diverged remains largely unexplored. Here, we performed a meta-analysis examining the age-dependent changes in the transcriptome using publicly available datasets of 11 representative metazoans, ranging from Caenorhabditis elegans to humans. To identify the transcriptomic changes associated with aging, we analyzed various aspects of the transcriptome, including genome composition, RNA processing, and functional consequences. The use of introns and novel splice sites tended to increase with age, particularly in the brain. In addition, our analysis suggests that the age-dependent accumulation of premature termination codon-containing transcripts is a common feature of aging across multiple animal species. Using C. elegans as a test model, we showed that several splicing factors that are evolutionarily conserved and age-dependently downregulated were required to maintain a normal lifespan. Thus, aberrant RNA processing appears to be associated with aging and a short lifespan in various species.

OASIS portable: User-friendly offline suite for secure survival analysis.

Online application for survival analysis (OASIS) and its update, OASIS 2, have been widely used for survival analysis in biological and medical sciences. Here, we provide a portable version of OASIS, an all-in-one offline suite, to facilitate secure survival analysis without uploading the data to online servers. OASIS portable provides a virtualized and isolated instance of the OASIS 2 webserver, operating on the users' personal computers, and enables user-friendly survival analysis without internet connection and security issues.

The Role of mRNA Quality Control in the Aging of Caenorhabditis elegans.

The proper maintenance of mRNA quality that is regulated by diverse surveillance pathways is essential for cellular homeostasis and is highly conserved among eukaryotes. Here, we review findings regarding the role of mRNA quality control in the aging and longevity of Caenorhabditis elegans, an outstanding model for aging research. We discuss the recently discovered functions of the proper regulation of nonsense-mediated mRNA decay, ribosome-associated quality control, and mRNA splicing in the aging of C. elegans. We describe how mRNA quality control contributes to longevity conferred by various regimens, including inhibition of insulin/insulin-like growth factor 1 (IGF-1) signaling, dietary restriction, and reduced mechanistic target of rapamycin signaling. This review provides valuable information regarding the relationship between the mRNA quality control and aging in C. elegans, which may lead to insights into healthy longevity in complex organisms, including humans.

Mitochondrial aconitase suppresses immunity by modulating oxaloacetate and the mitochondrial unfolded protein response.

Accumulating evidence indicates that mitochondria play crucial roles in immunity. However, the role of the mitochondrial Krebs cycle in immunity remains largely unknown, in particular at the organism level. Here we show that mitochondrial aconitase, ACO-2, a Krebs cycle enzyme that catalyzes the conversion of citrate to isocitrate, inhibits immunity against pathogenic bacteria in C. elegans. We find that the genetic inhibition of aco-2 decreases the level of oxaloacetate. This increases the mitochondrial unfolded protein response, subsequently upregulating the transcription factor ATFS-1, which contributes to enhanced immunity against pathogenic bacteria. We show that the genetic inhibition of mammalian ACO2 increases immunity against pathogenic bacteria by modulating the mitochondrial unfolded protein response and oxaloacetate levels in cultured cells. Because mitochondrial aconitase is highly conserved across phyla, a therapeutic strategy targeting ACO2 may eventually help properly control immunity in humans.

The role of tRNA-derived small RNAs in aging.

Aging is characterized by a gradual decline in biological functions, leading to the increased probability of diseases and deaths in organisms. Previous studies have identified biological factors that modulate aging and lifespan, including non-coding RNAs (ncRNAs). Here, we review the relationship between aging and tRNA-derived small RNAs (tsRNAs), ncRNAs that are generated from the cleavage of tRNAs. We describe age-dependent changes in tsRNA levels and their functions in age-related diseases, such as cancer and neurodegenerative diseases. We also discuss the association of tsRNAs with aging-regulating processes, including mitochondrial respiration and reduced mRNA translation. We cover recent findings regarding the potential roles of tsRNAs in cellular senescence, a major cause of organismal aging. Overall, our review will provide useful information for understanding the roles of tsRNAs in aging and age-associated diseases. [BMB Reports 2023; 56(2): 49-55].

Systematic transcriptome analysis associated with physiological and chronological aging in Caenorhabditis elegans.

Aging is associated with changes in a variety of biological processes at the transcriptomic level, including gene expression. Two types of aging occur during a lifetime: chronological and physiological aging. However, dissecting the difference between chronological and physiological ages at the transcriptomic level has been a challenge because of its complexity. We analyzed the transcriptomic features associated with physiological and chronological aging using Caenorhabditis elegans as a model. Many structural and functional transcript elements, such as noncoding RNAs and intron-derived transcripts, were up-regulated with chronological aging. In contrast, mRNAs with many biological functions, including RNA processing, were down-regulated with physiological aging. We also identified an age-dependent increase in the usage of distal 3' splice sites in mRNA transcripts as a biomarker of physiological aging. Our study provides crucial information for dissecting chronological and physiological aging at the transcriptomic level.

Immunosenescence in Caenorhabditis elegans.

Immunosenescence is an age-dependent decline in immune functions and hallmark of aging in diverse species, ranging from invertebrates to mammals. However, identifying the factors responsible for immunosenescence is challenging because of the complexity of immune systems and aging in mammals. The roundworm Caenorhabditis elegans is suitable for understanding immunosenescence because of its simple immune system and rapid aging process. In this review, we discuss the advances in our understanding of immunosenescence in C. elegans. PMK-1/p38 mitogen-activated protein kinase (MAPK), SKN-1/NRF, and ZIP-10/bZIP transcription factor regulate immunosenescence through p38 MAPK and insulin/IGF-1 signaling pathways. Because these factors and pathways are evolutionarily conserved, the findings discussed in this review may help understand the mechanisms underlying immunosenescence and develop new treatment therapy for immunosenescence in humans.

Recent Progress in Regulation of Aging by Insulin/IGF-1 Signaling in Caenorhabditis elegans.

Caenorhabditis elegans has been used as a major model organism to identify genetic factors that regulate organismal aging and longevity. Insulin/insulin-like growth factor 1 (IGF- 1) signaling (IIS) regulates aging in many species, ranging from nematodes to humans. C. elegans is a nonpathogenic genetic nematode model, which has been extensively utilized to identify molecular and cellular components that function in organismal aging and longevity. Here, we review the recent progress in the role of IIS in aging and longevity, which involves direct regulation of protein and RNA homeostasis, stress resistance, metabolism and the activities of the endocrine system. We also discuss recently identified genetic factors that interact with canonical IIS components to regulate aging and health span in C. elegans. We expect this review to provide valuable insights into understanding animal aging, which could eventually help develop anti-aging drugs for humans.

Double-stranded RNA induction asa potential dynamic biomarkerfor DNA-demethylating agents.

Hypomethylating agents (HMAs), such as azacitidine and decitabine, induce cancer cell death by demethylating DNAs to promote the expression of tumor-suppressor genes. HMAs also reactivate the transcription of endogenous double-stranded RNAs (dsRNAs) that trigger the innate immune response and subsequent apoptosis via viral mimicry. However, the expression patterns of endogenous dsRNAs and their relevance in the efficacy of HMAs remain largely uninvestigated. Here, we employ amidine-conjugated spiropyran (Am-SP) to examine the dynamic expression pattern of total dsRNAs regulated by HMAs. By analyzing the bone-marrow aspirates of myelodysplastic syndrome or acute myeloid leukemia patients who received the HMAs, we find a dramatic increase in total dsRNA levels upon treatment only in patients who later benefited from the therapy. We further apply our approach in solid tumor cell lines and show that the degree of dsRNA induction correlates with the effectiveness of decitabine in most cases. Notably, when dsRNA induction is accompanied by increased expression of nc886 RNA, decitabine becomes ineffective. Collectively, our study establishes the potential application of monitoring the total dsRNA levels by a small molecule as an analytical method and a dynamic marker to predict the clinical outcome of the HMA therapy.

MON-2, a Golgi protein, promotes longevity by upregulating autophagy through mediating inter-organelle communications.

The Golgi apparatus regulates the process of modification and subcellular localization of macromolecules, including proteins and lipids. Aberrant protein sorting caused by defects in the Golgi leads to various diseases in mammals. However, the role of the Golgi apparatus in organismal longevity remained largely unknown. By employing a quantitative proteomic approach, we demonstrated that MON-2, an evolutionarily conserved Arf-GEF protein implicated in Golgi-to-endosome trafficking, promotes longevity via upregulating macroautophagy/autophagy in C. elegans. Our data using cultured mammalian cells indicate that MON2 translocates from the Golgi to the endosome under starvation conditions, subsequently increasing autophagic flux by binding LGG-1/GABARAPL2. Thus, Golgi-to-endosome trafficking appears to be an evolutionarily conserved process for the upregulation of autophagy, which contributes to organismal longevity.

MON-2, a Golgi protein, mediates autophagy-dependent longevity in Caenorhabditis elegans.

The Golgi apparatus plays a central role in trafficking cargoes such as proteins and lipids. Defects in the Golgi apparatus lead to various diseases, but its role in organismal longevity is largely unknown. Using a quantitative proteomic approach, we found that a Golgi protein, MON-2, was up-regulated in long-lived Caenorhabditis elegans mutants with mitochondrial respiration defects and was required for their longevity. Similarly, we showed that DOP1/PAD-1, which acts with MON-2 to traffic macromolecules between the Golgi and endosome, contributed to the longevity of respiration mutants. Furthermore, we demonstrated that MON-2 was required for up-regulation of autophagy, a longevity-associated recycling process, by activating the Atg8 ortholog GABARAP/LGG-1 in C. elegans. Consistently, we showed that mammalian MON2 activated GABARAPL2 through physical interaction, which increased autophagic flux in mammalian cells. Thus, the evolutionarily conserved role of MON2 in trafficking between the Golgi and endosome is an integral part of autophagy-mediated longevity.

Age-dependent upregulation of Y RNAs in Caenorhabditis elegans.

Y RNA is a conserved small non-coding RNA whose functions in aging remain unknown. Here, we sought to determine the role of C. elegans Y RNA homologs, CeY RNA (CeY) and stem-bulge RNAs (sbRNAs), in aging. We found that the levels of CeY and sbRNAs generally increased during aging. We showed that CeY was downregulated by oxidative and thermal stresses, whereas several sbRNAs were upregulated by oxidative stress. We did not observe lifespan phenotypes by mutations in CeY-coding yrn-1. Future research under various genetic and environmental conditions is required to further evaluate the role of Y RNA in C. elegans aging.

A PTEN variant uncouples longevity from impaired fitness in Caenorhabditis elegans with reduced insulin/IGF-1 signaling.

Insulin/IGF-1 signaling (IIS) regulates various physiological aspects in numerous species. In Caenorhabditis elegans, mutations in the daf-2/insulin/IGF-1 receptor dramatically increase lifespan and immunity, but generally impair motility, growth, and reproduction. Whether these pleiotropic effects can be dissociated at a specific step in insulin/IGF-1 signaling pathway remains unknown. Through performing a mutagenesis screen, we identified a missense mutation daf-18(yh1) that alters a cysteine to tyrosine in DAF-18/PTEN phosphatase, which maintained the long lifespan and enhanced immunity, while improving the reduced motility in adult daf-2 mutants. We showed that the daf-18(yh1) mutation decreased the lipid phosphatase activity of DAF-18/PTEN, while retaining a partial protein tyrosine phosphatase activity. We found that daf-18(yh1) maintained the partial activity of DAF-16/FOXO but restricted the detrimental upregulation of SKN-1/NRF2, contributing to beneficial physiological traits in daf-2 mutants. Our work provides important insights into how one evolutionarily conserved component, PTEN, can coordinate animal health and longevity.

Combinatorial Approach Using Caenorhabditis elegans and Mammalian Systems for Aging Research.

Aging is associated with functional and structural declines in organisms over time. Organisms as diverse as the nematode Caenorhabditis elegans and mammals share signaling pathways that regulate aging and lifespan. In this review, we discuss recent combinatorial approach to aging research employing C. elegans and mammalian systems that have contributed to our understanding of evolutionarily conserved aging-regulating pathways. The topics covered here include insulin/IGF-1, mechanistic target of rapamycin (mTOR), and sirtuin signaling pathways; dietary restriction; autophagy; mitochondria; and the nervous system. A combinatorial approach employing high-throughput, rapid C. elegans systems, and human model mammalian systems is likely to continue providing mechanistic insights into aging biology and will help develop therapeutics against age-associated disorders.

Transfer RNA-derived fragments in aging Caenorhabditis elegans originate from abundant homologous gene copies.

Small RNAs that originate from transfer RNA (tRNA) species, tRNA-derived fragments (tRFs), play diverse biological functions but little is known for their association with aging. Moreover, biochemical aspects of tRNAs limit discovery of functional tRFs by high throughput sequencing. In particular, genes encoding tRNAs exist as multiple copies throughout genome, and mature tRNAs have various modified bases, contributing to ambiguities for RNA sequencing-based analysis of tRFs. Here, we report age-dependent changes of tRFs in Caenorhabditis elegans. We first analyzed published RNA sequencing data by using a new strategy for tRNA-associated sequencing reads. Our current method used unique mature tRNAs as a reference for the sequence alignment, and properly filtered out false positive enrichment for tRFs. Our analysis successfully distinguished de novo mutation sites from differences among homologous copies, and identified potential RNA modification sites. Overall, the majority of tRFs were upregulated during aging and originated from 5'-ends, which we validated by using Northern blot analysis. Importantly, we revealed that the major source of tRFs upregulated during aging was the tRNAs with abundant gene copy numbers. Our analysis suggests that tRFs are useful biomarkers of aging particularly when they originate from abundant homologous gene copies.

Emerging functions of circular RNA in aging.

Circular RNA (circRNA) is a closed, single-stranded transcript widely detected in eukaryotes. Recent studies indicate that the levels of circRNAs change with age in various tissues in multiple species, ranging from nematodes to mammals. Here we discuss the functional roles of circRNAs in animal aging and longevity. We review studies regarding the differential expression of circRNAs that contributes to cellular senescence and the pathogenesis of aging-associated diseases. We explore the features of aging-associated circRNAs by discussing their potential as biomarkers of aging, tissue specificity, physiological roles, action mechanisms, and evolutionarily conserved characteristics. Our review provides insights into current progress in circRNA research and their significant functions in the aging process.