Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-ß (MENß) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENß triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENß stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5-13 °C more than the Watson-Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENß stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENß. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENß.

Global RNA modifications to the MALAT1 triple helix differentially affect thermostability and weaken binding to METTL16.

Therapeutic mRNAs are generated using modified nucleotides, namely N1-methylpseudouridine (m1Ψ) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple-stranded RNA, globally modified with N6-methyladenosine (m6A), pseudouridine (Ψ), or m1Ψ. UV thermal denaturation assays showed that m6A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by ∼20 °C, Ψ stabilizes the Hoogsteen and Watson-Crick faces of the RNA by ∼12 °C, and m1Ψ has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by ∼9 °C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m6A, Ψ, or m1Ψ. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means.

Ghost authors revealed: The structure and function of human N6 -methyladenosine RNA methyltransferases.

Despite the discovery of modified nucleic acids nearly 75 years ago, their biological functions are still being elucidated. N6 -methyladenosine (m6 A) is the most abundant modification in eukaryotic messenger RNA (mRNA) and has also been detected in non-coding RNAs, including long non-coding RNA, ribosomal RNA, and small nuclear RNA. In general, m6 A marks can alter RNA secondary structure and initiate unique RNA-protein interactions that can alter splicing, mRNA turnover, and translation, just to name a few. Although m6 A marks in human RNAs have been known to exist since 1974, the structures and functions of methyltransferases responsible for writing m6 A marks have been established only recently. Thus far, there are four confirmed human methyltransferases that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the N6 position of adenosine, producing m6 A: methyltransferase-like protein (METTL) 3/METTL14 complex, METTL16, METTL5, and zinc-finger CCHC-domain-containing protein 4. Though the methyltransferases have unique RNA targets, all human m6 A RNA methyltransferases contain a Rossmann fold with a conserved SAM-binding pocket, suggesting that they utilize a similar catalytic mechanism for methyl transfer. For each of the human m6 A RNA methyltransferases, we present the biological functions and links to human disease, RNA targets, catalytic and kinetic mechanisms, and macromolecular structures. We also discuss m6 A marks in human viruses and parasites, assigning m6 A marks in the transcriptome to specific methyltransferases, small molecules targeting m6 A methyltransferases, and the enzymes responsible for hypermodified m6 A marks and their biological functions in humans. Understanding m6 A methyltransferases is a critical steppingstone toward establishing the m6 A epitranscriptome and more broadly the RNome. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

A Mixed-Methods Exploration of the Experience of People With Aphasia Using Text-to-Speech Technology to Support Virtual Book Club Participation.

PURPOSE: This mixed-methods research sought to examine the experience of people with aphasia who used text-to-speech (TTS) support to read a novel for virtual book club participation. METHOD: Six people with chronic aphasia used a TTS system to review portions of a novel about which they conversed during eight virtual book club meetings occurring over 5 weeks. During one-on-one interactions prior to each meeting, participants answered comprehension questions and provided feedback about reading experiences. Then, during group meetings, they reviewed and discussed relevant book content and predicted upcoming content. During a structured individual interview, participants reflected on their supported reading and book club experience. RESULTS: Participants reported a range of reading confidence prior to study participation, mostly influenced by decreased comprehension or reading speed. After book club participation, four participants expressed increased confidence. Some reported searching for key words and skipping difficult words as strategies additional to TTS support. All reviewed at least some book sections more than once either with or without TTS support. Highly motivated participants expressed low frustration and high reading ease and enjoyment. Perceived comprehension was roughly consistent with actual comprehension across participants. Most believed TTS support promoted faster reading than otherwise possible. Participants liked adjustable features affecting speech output rate, word or sentence highlighting, and font size. Psychosocial benefits included decreased isolation and increased friendship. CONCLUSIONS: The findings extend previous evidence about perceived and actual benefits associated with TTS support. People with aphasia express positive experiences when given TTS support during book club participation.

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 µM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 µM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 µM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

Eye Fixation Behaviors and Processing Time of People With Aphasia and Neurotypical Adults When Reading Narratives With and Without Text-to-Speech Support.

BACKGROUND: Researchers have used eye-tracking technology to investigate eye movements in neurotypical adults (NAs) when reading. The technology can provide comparable information about people with aphasia (PWA). Eye fixations occurring when PWA do and do not have access to text-to-speech (TTS) technology are of interest because the support improves reading comprehension and decreases processing time for at least some PWA. AIMS: This study's purpose was to examine forward, regressive, and off-track eye fixations when PWA and NAs read narratives in read-only (RO) and TTS conditions. A secondary aim was to examine the influence of eye fixations on processing time. METHOD AND PROCEDURE: A Tobii Dynavox Pro Spectrum eye tracker recorded eye movements of nine PWA and nine NAs while reading narratives in two conditions. Movements of interest were forward fixations; within-word, within-sentence, and previous-sentence regressive fixations; and off-track fixations. OUTCOMES AND RESULTS: PWA exhibited significantly more forward and regressive fixations in the RO than TTS condition, whereas NAs showed opposite behaviors. NAs had significantly more off-track fixations in the TTS than RO condition, whereas PWA exhibited no difference across conditions. PWA took significantly longer to process content in the RO condition, whereas NAs took longer in the TTS condition. CONCLUSIONS: PWA and NAs differ in important ways when processing texts with and without TTS support. Examining eye-tracking data provides a means of gaining insight into the decoding and reading comprehension challenges of PWA and helps elucidate how assistive technology can mediate these challenges.

Comprehension, Processing Time, and Modality Preferences When People With Aphasia and Neurotypical Healthy Adults Read Books: A Pilot Study.

BACKGROUND: Many people with aphasia (PWA) want to read books. Text-to-speech (TTS) technology sometimes provides comprehension and processing time benefits when PWA read short, multisentence passages. Currently, no research examines the effect of TTS support when PWA read books. AIMS: This study's primary purpose was to examine comprehension accuracy and total processing time of PWA and neurotypical healthy adults (NHAs) when reading book sections in read-only versus TTS-supported conditions. A secondary aim was to examine condition preference and perceived degree of understanding by people in both participant groups. METHOD AND PROCEDURE: Ten PWA and 10 NHAs alternated between read-only and TTS-supported conditions to read a book. Participants answered comprehension questions and provided feedback about their reading experience, condition preference, and desire to use TTS technology for future book reading. Outcomes and Result: Overall, PWA exhibited less accurate comprehension and slower processing times compared to NHAs in both conditions. No significant comprehension accuracy difference occurred between conditions for either group. However, four PWA exhibited a 10% or greater increase in comprehension accuracy when receiving TTS support. A significant processing time difference occurred with PWA processing text faster with TTS support, whereas NHAs did not demonstrate processing time differences. Most PWA preferred the TTS condition and expressed a desire to use TTS technology in the future. Most NHAs expressed the opposite preference. CONCLUSIONS: TTS support during book reading promotes faster processing without compromising comprehension for PWA. Clinicians should discuss with PWA the relative importance of comprehension accuracy, processing time, and comfort with technology when determining whether using TTS support during book reading is desirable.

A single natural RNA modification can destabilize a U•A-T-rich RNA•DNA-DNA triple helix.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming RNA•DNA-DNA triple helices, as a mechanism to regulate transcription. One way cells could regulate the formation of these triple helices is through RNA modifications. With over 140 naturally occurring RNA modifications, we hypothesize that some modifications stabilize RNA•DNA-DNA triple helices while others destabilize them. Here, we focus on a pyrimidine-motif triple helix composed of canonical U•A-T and C•G-C base triples. We employed electrophoretic mobility shift assays and microscale thermophoresis to examine how 11 different RNA modifications at a single position in an RNA•DNA-DNA triple helix affect stability: 5-methylcytidine (m5C), 5-methyluridine (m5U or rT), 3-methyluridine (m3U), pseudouridine (Ψ), 4-thiouridine (s4U), N 6-methyladenosine (m6A), inosine (I), and each nucleobase with 2'-O-methylation (Nm). Compared to the unmodified U•A-T base triple, some modifications have no significant change in stability (Um•A-T), some have ∼2.5-fold decreases in stability (m5U•A-T, Ψ•A-T, and s4U•A-T), and some completely disrupt triple helix formation (m3U•A-T). To identify potential biological examples of RNA•DNA-DNA triple helices controlled by an RNA modification, we searched RMVar, a database for RNA modifications mapped at single-nucleotide resolution, for lncRNAs containing an RNA modification within a pyrimidine-rich sequence. Using electrophoretic mobility shift assays, the binding of DNA-DNA to a 22-mer segment of human lncRNA Al157886.1 was destabilized by ∼1.7-fold with the substitution of m5C at known m5C sites. Therefore, the formation and stability of cellular RNA•DNA-DNA triple helices could be influenced by RNA modifications.

Practical opportunities for microbiome analyses and bioinformatics in poultry processing.

Poultry processing is undergoing changes both in operations as well as microbial methodologies. Traditionally, microbial data has been gathered through a series of culturing methods using liquid media and plating for isolation and enumeration. Both foodborne pathogens and nonpathogenic bacterial populations are estimated to assess food safety risks as well as the potential for spoilage. Bacterial loads from carcasses are important for estimating processing control and the effectiveness of antimicrobial applications. However, these culture-based approaches may only provide part of the microbial ecology landscape associated with chicken carcasses and the subsequent changes that occur in these populations during processing. Newer molecular-based approaches, such as 16S sequencing of the microbiota, offer a means to retrieve a more comprehensive microbial compositional profile. However, such approaches also result in large data sets which must be analyzed and interpreted. As more data is generated, this will require not only bioinformatic programs to process the data but appropriate educational forums to present the processed data to a broad audience.

Reading behaviors and text-to-speech technology perceptions of people with aphasia.

People with aphasia often have reading impairments that affect participation in daily activities. Text-to-speech (TTS) devices are technology-based supports that can facilitate processing of written materials. The purpose of this study was to gather information about the reading behaviors and TTS technology perceptions of people with aphasia who had first learned about system features and options. Sixteen people with chronic aphasia participated in single, one-on-one instructional and guided practice sessions using TTS systems. They answered close-ended questions about current reading behaviors and materials and ways they believed these would change given TTS system access. Participants reported reading at home and community locations. Most read calendars, newspapers, magazines, and mail. Participants who did not read lengthy materials - such as newspapers, magazines, and novels - indicated their interest in these materials would likely increase given TTS support. Although participants did not predict substantial comprehension changes given TTS support, most expressed interest in the technology after learning about it. Thus, people with aphasia perceive TTS systems as helpful for comprehending lengthy materials. Given modest predictions about comprehension benefits, presenting TTS as one of several support strategies is an appropriate recommendation.

Reading Comprehension and Processing Time When People With Aphasia Use Text-to-Speech Technology With Personalized Supports and Features.

BACKGROUND: Person-centered approaches promote consistent use of supportive technology and feelings of empowerment for people with disabilities. Feature personalization is an aspect of person-centered approaches that can affect the benefit people with aphasia (PWA) derive from using text-to-speech (TTS) technology as a reading support. AIMS: This study's primary purpose was to compare the comprehension and processing time of PWA when performing TTS-supported reading with preferred settings for voice, speech output rate, highlighting type, and highlighting color versus unsupported reading. A secondary aim was to examine initial support and feature preference selections, preference changes following TTS exposure, and anticipated functional reading activities for utilizing TTS technology. METHOD AND PROCEDURE: Twenty PWA read passages either via written text or text combined with TTS output using personally selected supports and features. Participants answered comprehension questions, reevaluated their preference selections, and provided feedback both about feature selections and possible future TTS technology uses. OUTCOMES AND RESULTS: Comprehension accuracy did not vary significantly between reading conditions; however, processing time was significantly less in the TTS-supported condition, thus suggesting TTS support promoted greater reading speed without compromising comprehension. Most participants preferred the TTS condition and several anticipated benefits when reading lengthy and difficult materials. Alterations to initial settings were relatively rare. CONCLUSIONS: Personalizing TTS systems is relevant to person-centered interventions. Reading with desired TTS system supports and features promotes improved reading efficiency by PWA compared with reading without TTS support. Attending to client preferences is important when customizing and implementing TTS technology as a reading support.

First-Line Health Care Providers' Reported Knowledge of and Referrals to Speech-Language Pathologists for Clients With Mild Traumatic Brain Injury.

Purpose People with mild traumatic brain injury (mTBI) may experience deficits in cognition or communication that go unnoticed by first-line health care providers (FHPs). Speech-language pathologists (SLPs) assess and treat these domains yet are often underrepresented on mTBI multidisciplinary teams. This study's aim was to evaluate FHPs' reported knowledge of and referral practices to SLPs for individuals across the life span with mTBI. Method Physicians, physician assistants, nurse practitioners, nurses, and athletic trainers (n = 126) completed an online survey, including two Likert scale questions and one free response question relating to SLPs' role in mTBI. Results More than half of FHPs rate their knowledge of the SLP's role in mTBI management as low (somewhat knowledgeable, 29%; not very knowledgeable, 23%). Similarly, nearly two thirds of FHPs indicated rarely (19%) or never (44%), referring to SLPs for management of patients with mTBI. The majority of FHPs' open responses on the role of the SLP in mTBI management were incomplete, with many including domains that were not relevant to an SLP's role in the management of mTBI (e.g., dysphagia). Within the article, we provide results overall and according to individual profession. Conclusions Results suggest a majority of FHPs lack knowledge in the role of the SLP in the management of mTBI, which may underpin the low referral patterns reported by FHPs for SLP services. Future educational efforts for FHPs regarding the role of SLPs in mTBI care are necessary.

Effect of Digital Highlighting on Reading Comprehension Given Text-to-Speech Technology for People with Aphasia.

BACKGROUND: Many people with aphasia have a strong desire to participate in reading activities despite persistent reading challenges. Digital reading devices and text-to-speech (TTS) technology are increasing in popularity and have the potential to help people with aphasia. Systematic investigation of modifiable TTS features provides a means of exploring this potential. AIMS: This study's aim was to evaluate the effect of digital highlighting synchronised with TTS auditory and written output on reading comprehension by people with aphasia and to determine their highlighting preferences. METHODS & PROCEDURES: This work was registered with clinicaltrials.gov and assigned the clinical trial registry number 01446r prior to initiation of data collection. Twenty-five adults with aphasia read and listened to passages presented in three synchronised highlighting conditions: sentence highlighting, single word highlighting, and no highlighting. Participants answered comprehension questions, selected most and least preferred conditions, and provided feedback explaining highlighting preferences. OUTCOME & RESULTS: Comprehension accuracy did not vary significantly across presentation conditions, but participants preferred either single word or sentence highlighting over no highlighting. CONCLUSIONS: Neither word nor sentence highlighting benefitted or hindered comprehension by people with aphasia as a group, but individual differences may occur. Clinicians should attend to personal preferences when implementing digital highlighting as a reading support strategy.

Perceptions of people with aphasia about supporting reading with text-to-speech technology: A convergent mixed methods study.

INTRODUCTION: Text-to-speech (TTS) technology is a possible reading support for people with aphasia; however, adoption for functional reading remains rare, and people with aphasia may have limited knowledge about TTS. Given this situation coupled with inherent communication challenges associated with aphasia, the purpose of this convergent mixed methods study was to explore the perceptions of participants about desired features, benefits, and drawbacks of TTS technology after having engaged in one-on-one education and guided practice activities. METHODS: Nineteen adults with chronic aphasia completed a single education and guided practice session followed by close-ended questions and participation in a semi-structured interview to explain preferences, concerns, beliefs, and opinions about potential TTS technology benefits and drawbacks. Three participants had previously used TTS technology for functional reading; all others had some prior exposure but did not use a system for functional purposes. RESULTS: Seventeen of 19 participants expressed TTS technology interest after education and guided practice activities. Participants endorsed selection of a preferred voice, control of speech output rate, and highlighting as priority features. Frequently endorsed benefits were improved comprehension and increased reading independence; some participants believed they would succeed in reading a greater variety of materials, communicate more with others, participate in more reading activities, and/or read faster. The greatest concern was mastering device operation; other concerns related to understanding the voice output, needing another person's help for system use, and matching the speech output rate to a preferred reading rate. CONCLUSIONS: Overall, most participants had positive perceptions about possible benefits afforded by TTS technology. Practitioners need to provide opportunities for people with aphasia to learn about and explore TTS systems to determine whether adoption is desired.

Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer.

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

An automated method for defining anatomic coordinate systems in the hindfoot.

The absence of a standardized method for defining hindfoot bone coordinate systems makes it difficult to compare kinematics results from different research studies. The purpose of this study was to develop a reliable and robust procedure for defining anatomical coordinate systems for the talus and calcaneus. Four methods were evaluated based upon their anatomic consistency across subjects, repeatability, and their correspondence to functional axes of rotation. The four systems consisted of: 1) interactively identified bony landmarks, 2) a principal component analysis, 3) automatically identified bony landmarks, and 4) translating the tibial coordinate system to the hindfoot bones. The four systems were evaluated on 40 tali and 40 calcanei. The functional axes of rotation were determined using dynamic biplane radiography to image the hindfoot during gait. Systems 2 and 3 were the most repeatable and consistent due to the lack of operator intervention when defining coordinate systems. None of the coordinate systems corresponded well to functional axes of rotation during gait. System 3 is recommended over System 2 because it more closely mimics established bone angles measured clinically, especially for the calcaneus. This study presents an automated method for defining anatomic coordinate systems in the talus and calcaneus that does not rely on manual placement of markers or fitting of spheres to the bone surfaces which are less reliable due to operator-dependent measurements. Using this automated method will make it easier to compare hindfoot kinematics results across research studies.

Unraveling the structure and biological functions of RNA triple helices.

It has been nearly 63 years since the first characterization of an RNA triple helix in vitro by Gary Felsenfeld, David Davies, and Alexander Rich. An RNA triple helix consists of three strands: A Watson-Crick RNA double helix whose major-groove establishes hydrogen bonds with the so-called "third strand". In the past 15 years, it has been recognized that these major-groove RNA triple helices, like single-stranded and double-stranded RNA, also mediate prominent biological roles inside cells. Thus far, these triple helices are known to mediate catalysis during telomere synthesis and RNA splicing, bind to ligands and ions so that metabolite-sensing riboswitches can regulate gene expression, and provide a clever strategy to protect the 3' end of RNA from degradation. Because RNA triple helices play important roles in biology, there is a renewed interest in better understanding the fundamental properties of RNA triple helices and developing methods for their high-throughput discovery. This review provides an overview of the fundamental biochemical and structural properties of major-groove RNA triple helices, summarizes the structure and function of naturally occurring RNA triple helices, and describes prospective strategies to isolate RNA triple helices as a means to establish the "triplexome". This article is categorized under: RNA Structure and Dynamics > RNA Structure and Dynamics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Naturally occurring modified ribonucleosides.

The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as the "fifth" ribonucleotide in 1951. Since then, the ever-increasing number and complexity of modified ribonucleosides have been found in viruses and throughout all three domains of life. Such modifications can be as simple as methylations, hydroxylations, or thiolations, complex as ring closures, glycosylations, acylations, or aminoacylations, or unusual as the incorporation of selenium. While initially found in transfer and ribosomal RNAs, modifications also exist in messenger RNAs and noncoding RNAs. Modifications have profound cellular outcomes at various levels, such as altering RNA structure or being essential for cell survival or organism viability. The aberrant presence or absence of RNA modifications can lead to human disease, ranging from cancer to various metabolic and developmental illnesses such as Hoyeraal-Hreidarsson syndrome, Bowen-Conradi syndrome, or Williams-Beuren syndrome. In this review article, we summarize the characterization of all 143 currently known modified ribonucleosides by describing their taxonomic distributions, the enzymes that generate the modifications, and any implications in cellular processes, RNA structure, and disease. We also highlight areas of active research, such as specific RNAs that contain a particular type of modification as well as methodologies used to identify novel RNA modifications. This article is categorized under: RNA Processing > RNA Editing and Modification.