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In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICI) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication., Experimental Design: We harnessed a variety of transcriptomic, spatial, and functional assays to characterize the differential impact of neoadjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to neoadjuvant chemotherapy (NACT)-naïve HGSOC samples from five independent patient cohorts., Results: We found NACT-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD1+ CD8+ T cells over their ICI-insensitive TIM-3+PD1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden., Conclusions: Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC. See related commentary by Bravo Melgar and Laoui, p. 10., (©2024 The Authors; Published by the American Association for Cancer Research.)

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in Dicer ΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. Dicer ΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo., (© 2024. The Author(s).)

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8 + effector T (T EFF ) cells and TIM3 + PD1 + , hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8 + T cells. Conversely, CD8 + T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1 + PD1 + T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8 + T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1 + PD1 + CD8 + T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC., (© 2024. The Author(s).)

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML., (© 2023. The Author(s).)

Background: Genes, principal units of genetic information, vary in complexity and evolutionary history. Less-complex genes (e.g., long non-coding RNA (lncRNA) expressing genes) readily emerge de novo from non-genic sequences and have high evolutionary turnover. Genesis of a gene may be facilitated by adoption of functional genic sequences from retrotransposon insertions. However, protein-coding sequences in extant genomes rarely lack any connection to an ancestral protein-coding sequence., Results: We describe remarkable evolution of the murine gene D6Ertd527e and its orthologs in the rodent Muroidea superfamily. The D6Ertd527e emerged in a common ancestor of mice and hamsters most likely as a lncRNA-expressing gene. A major contributing factor was a long terminal repeat (LTR) retrotransposon insertion carrying an oocyte-specific promoter and a 5' terminal exon of the gene. The gene survived as an oocyte-specific lncRNA in several extant rodents while in some others the gene or its expression were lost. In the ancestral lineage of Mus musculus, the gene acquired protein-coding capacity where the bulk of the coding sequence formed through CAG (AGC) trinucleotide repeat expansion and duplications. These events generated a cytoplasmic serine-rich maternal protein. Knock-out of D6Ertd527e in mice has a small but detectable effect on fertility and the maternal transcriptome., Conclusions: While this evolving gene is not showing a clear function in laboratory mice, its documented evolutionary history in Muroidea during the last ~ 40 million years provides a textbook example of how a several common mutation events can support de novo gene formation, evolution of protein-coding capacity, as well as gene's demise., (© 2022. The Author(s).)

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) have received considerable attention as potential targets for the development of novel cancer immunotherapies. However, the clinical efficacy of DC-based vaccines remains suboptimal, largely reflecting local and systemic immunosuppression at baseline. An autologous DC-based vaccine (DCVAC) has recently been shown to improve progression-free survival and overall survival in randomized clinical trials enrolling patients with lung cancer (SLU01, NCT02470468) or ovarian carcinoma (SOV01, NCT02107937), but not metastatic castration-resistant prostate cancer (SP005, NCT02111577), despite a good safety profile across all cohorts. We performed biomolecular and cytofluorometric analyses on peripheral blood samples collected prior to immunotherapy from 1000 patients enrolled in these trials, with the objective of identifying immunological biomarkers that may improve the clinical management of DCVAC-treated patients. Gene signatures reflecting adaptive immunity and T cell activation were associated with favorable disease outcomes and responses to DCVAC in patients with prostate and lung cancer, but not ovarian carcinoma. By contrast, the clinical benefits of DCVAC were more pronounced among patients with ovarian carcinoma exhibiting reduced expression of T cell-associated genes, especially those linked to T H2 -like signature and immunosuppressive regulatory T (T REG ) cells. Clinical responses to DCVAC were accompanied by signs of antitumor immunity in the peripheral blood. Our findings suggest that circulating signatures of antitumor immunity may provide a useful tool for monitoring the potency of autologous DC-based immunotherapy., Competing Interests: IV declares consulting for AstraZeneca, Clovis Oncology Inc., Carrick Therapeutics, Deciphera Pharmaceuticals, Elevar Therapeutics, F. Hoffmann-La Roche Ltd, Genmab, GSK, Immunogen Inc., Jazzpharma, Mersana, Millennium Pharmaceuticals, MSD, Novocure, Octimet Oncology NV, Oncoinvent AS, Sotio a.s., Verastem Oncology, Zentalis; contracted research for: Oncoinvent AS, Genmab; and research funding from Amgen and Roche. RS and JB are minority shareholders of Sotio. ADG received fees for consultancy, lectures or services from Boehringer Ingelheim (Germany), Miltenyi Biotec (Germany), Isoplexis (USA) and Novigenix (Switzerland). AR declares advisory services and invited lectures for Amgen, AstraZeneca, BMS, Eli-Lilly, Janssen-Cilag, MSD, and Roche. AC is a contracted researcher for Oncoinvent AS and Novocure and a consultant for Sotio Biotech a.s. MH, JR, LK, TL, JF, PH, MH, TH, PK, KS, DR, LS, JB, RS, and JF are employees of Sotio a.s. The other authors declare no conflicts of interest., (© 2022 Sotio Biotech. Published with license by Taylor & Francis Group, LLC.)

Purpose: The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937)., Patients and Methods: We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC., Results: Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood., Conclusions: Our findings suggest that while patients with highly infiltrated, "hot" EOCs benefit from chemotherapy, women with "cold" EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses., (©2022 American Association for Cancer Research.)

PIWI-interacting RNAs (piRNAs) support the germline by suppressing retrotransposons. Studies of the pathway in mice have strongly shaped the view that mammalian piRNAs are essential for male but not for female fertility. Here, we report that the role of the piRNA pathway substantially differs in golden hamsters (Mesocricetus auratus), the piRNA pathway setup of which more closely resembles that of other mammals, including humans. The loss of the Mov10l1 RNA helicase-an essential piRNA biogenesis factor-leads to striking phenotypes in both sexes. In contrast to mice, female Mov10l1 -/- hamsters are sterile because their oocytes do not sustain zygotic development. Furthermore, Mov10l1 -/- male hamsters have impaired establishment of spermatogonia accompanied by transcriptome dysregulation and an expression surge of a young retrotransposon subfamily. Our results show that the mammalian piRNA pathway has essential roles in both sexes and its adaptive nature allows it to manage emerging genomic threats and acquire new critical roles in the germline., (© 2021. The Author(s).)

Accumulating evidence indicates that immune checkpoint inhibitors (ICIs) can restore CD8 + cytotoxic T lymphocyte (CTL) functions in preclinical models of acute myeloid leukemia (AML). However, ICIs targeting programmed cell death 1 (PDCD1, best known as PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) have limited clinical efficacy in patients with AML. Natural killer (NK) cells are central players in AML-targeting immune responses. However, little is known on the relationship between co-inhibitory receptors expressed by NK cells and the ability of the latter to control AML. Here, we show that hepatitis A virus cellular receptor 2 (HAVCR2, best known as TIM-3) is highly expressed by NK cells from AML patients, correlating with improved functional licensing and superior effector functions. Altogether, our data indicate that NK cell frequency as well as TIM-3 expression levels constitute prognostically relevant biomarkers of active immunity against AML., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: There are no clinical longitudinal studies exploring the associations between sport specialization and intense training patterns and injuries in young athletes., Purpose: To prospectively determine the relationship between young athletes' degree of sport specialization and their risk of injuries and reinjuries., Study Design: Case-control study; Level of evidence, 2., Methods: Young athletes aged between 7 and 18 years presenting for sports-related injuries or sports physical examinations were recruited from either sports medicine clinics or pediatric/family medicine offices. Each participant completed a baseline survey at enrollment and an identical follow-up survey every 6 months for 3 years. Surveys assessed training patterns and injuries. Injury type (acute, overuse, or serious overuse) and clinical diagnosis were also recorded from electronic medical records., Results: Of the 1208 participants who provided consent, 579 (48%) completed the baseline survey and first follow-up survey at 6 months (mean age, 14.1 ± 2.3 years; 53% female). Of this sample, 27% (158/579) of participants were uninjured, and 73% (421/579) were injured, with 29% (121/421) of injuries classified as reinjuries. Consistent with previous studies, over the 3-year study period, the degree of sport specialization had an effect such that more specialized athletes were significantly more likely to be injured ( P = .03) or have an overuse injury ( P = .02) compared with less specialized athletes after adjusting for potential confounders. Additionally, female athletes were more at risk for all injuries ( P = .01) and overuse injuries ( P = .02) after adjusting for covariates. Finally, young athletes who trained in weekly hours in excess of their age or who trained twice as many hours as their free play were significantly more likely to be injured on univariate analysis (both P < .001)., Conclusion: Our study confirms that over time, young athletes, and in particular young female athletes, were more likely to be injured and sustain an overuse injury if they had a higher degree of sport specialization. Similarly, those athletes whose training hours exceeded thair age or whose sports hours exceeded their free play by a factor of greater than 2 were also more likely to develop injuries and overuse injuries., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: Research funding for this study was provided by consecutive grants from the American Medical Society for Sports Medicine (AMSSM). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2020.)

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Germline genome defense evolves to recognize and suppress retrotransposons. One of defensive mechanisms is the PIWI-associated RNA (piRNA) pathway, which employs small RNAs for sequence-specific repression. The loss of the piRNA pathway in mice causes male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes posses also RNA interference (RNAi), another small RNA pathway capable of retrotransposon suppression. To examine whether RNAi compensates the loss of the piRNA pathway, we produced a new RNAi pathway mutant DicerSOM and crossed it with a catalytically-dead mutant of Mili, an essential piRNA gene. Normal follicular and oocyte development in double mutants showed that RNAi does not suppress a strong ovarian piRNA knock-out phenotype. However, we observed redundant and non-redundant targeting of specific retrotransposon families illustrating stochasticity of recognition and targeting of invading retrotransposons. Intracisternal A Particle retrotransposon was mainly targeted by the piRNA pathway, MaLR and RLTR10 retrotransposons were targeted mainly by RNAi. Double mutants showed accumulations of LINE-1 retrotransposon transcripts. However, we did not find strong evidence for transcriptional activation and mobilization of retrotransposition competent LINE-1 elements suggesting that while both defense pathways are simultaneously expendable for ovarian oocyte development, yet another transcriptional silencing mechanism prevents mobilization of LINE-1 elements., Competing Interests: The authors have declared that no competing interests exist.

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy., (© 2019 Demeter et al.)

Background: Published training recommendations exist for youth athletes aimed at reducing injury risk. No studies have assessed the impact of counselling interventions using training recommendations on risk of injury in young athletes., Objectives: To determine if online training counselling regarding safe sport participation can reduce injury risk in youth athletes and to assess recommendation compliance, including barriers to compliance., Methods: A multicentre randomised intervention trial was performed at two Midwestern academic institutions with expertise in treating young athletes. Enrolled subjects ages 8-17 completed a baseline risk assessment survey and were randomised to a control or intervention group. Both groups completed exposure surveys every 3 months for 1 year. The intervention group also received online training counselling on safe sport participation every 3 months. Training characteristics including training volume, degree of specialisation and adherence to recommendations were captured. Differences in self-reported injury between groups, compliance to recommendations and barriers to compliance were evaluated., Results: At baseline, n=357 subjects were enrolled (n=172 control and n=185 intervention). Controls were nearly twice as likely to be injured during the intervention period after controlling for age, sex, baseline injury and level of specialisation. No improvement in recommendation compliance was detected among intervention subjects. Primary barriers to compliance were no prior knowledge of recommendations, personal choice and following coaches' recommendations., Conclusions: In this convenience sample of youth athletes, electronic training counselling surrounding safe sports participation was not determined to affect injury risk. Lack of knowledge and adherence to appropriate training recommendations is evident and barriers to compliance exist., Competing Interests: Competing interests: NJ reports grants from Loyola University Medical Center Intramural Research Grant, personal fees from University of Hawaii, personal fees from Young Athlete Foundation (YAF) and personal fees from United States Tennis Association (USTA), outside the submitted work., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Current trends among young athletes towards earlier specialization age and year-round training on multiple teams has raised concern for increased injury risk. Our previous analyses showed higher risk for injury in highly specialized young athletes. The goal of this research was to determine whether sports specialization and injury patterns vary by sports type., Methods: In this clinical case-control study, injured athletes (aged 7-18 years) were recruited from sports medicine clinics and compared to similarly aged uninjured athletes recruited from primary care clinics. Participants completed a survey reporting age, gender, sport type, specialization patterns, and details regarding sports-related injuries in the previous 6 months. Clinical diagnoses were collected from patients' medical records. Injuries were classified as acute, overuse, or serious overuse., Results: Of 1,190 athletes enrolled, 26% (313) were single-sport specialized (reported participation in one sport and trained >8 months/year). Sports with the highest proportion of single-sport specialized athletes were tennis (46.7%), gymnastics (30.1%), and dance (26.3%). Single-sport specialized athletes in individual sports started specializing at a younger age (11.2 ± 2.4 vs. 12.0 ± 2.7, p = 0.05) and reported higher training volumes (11.8 vs. 10.3 h/week, p = 0.04) than those in team sports. Sports with the youngest specialization age were gymnastics (8.9 ± 1.7), dance (10.8 ± 3.0), and soccer (10.9 ± 2.4). Single-sport specialized athletes in individual sports accounted for a higher proportion of overuse injuries (44.3% vs 32.2%, OR = 1.67, p = 0.037) and serious overuse injuries (23.4% vs 11.6%, OR = 2.38, p = 0.011), but a lower proportion of acute injuries (28.8% vs 13.8%, OR = 0.37, p = 0.001) compared to single-sport specialized athletes involved in team sports., Conclusions: Athletes in individual sports may be more likely to specialize in a single sport than team sport athletes. Single-sport specialized athletes in individual sports also reported higher training volumes and greater rates of overuse injuries than single-sport specialized athletes in team sports.

Retrotransposons are "copy-and-paste" insertional mutagens that substantially contribute to mammalian genome content. Retrotransposons often carry long terminal repeats (LTRs) for retrovirus-like reverse transcription and integration into the genome. We report an extraordinary impact of a group of LTRs from the mammalian endogenous retrovirus-related ERVL retrotransposon class on gene expression in the germline and beyond. In mouse, we identified more than 800 LTRs from ORR1, MT, MT2, and MLT families, which resemble mobile gene-remodeling platforms that supply promoters and first exons. The LTR-mediated gene remodeling also extends to hamster, human, and bovine oocytes. The LTRs function in a stage-specific manner during the oocyte-to-embryo transition by activating transcription, altering protein-coding sequences, producing noncoding RNAs, and even supporting evolution of new protein-coding genes. These functions result, for example, in recycling processed pseudogenes into mRNAs or lncRNAs with regulatory roles. The functional potential of the studied LTRs is even higher, because we show that dormant LTR promoter activity can rescue loss of an essential upstream promoter. We also report a novel protein-coding gene evolution- D6Ertd527e -in which an MT LTR provided a promoter and the 5' exon with a functional start codon while the bulk of the protein-coding sequence evolved through a CAG repeat expansion. Altogether, ERVL LTRs provide molecular mechanisms for stochastically scanning, rewiring, and recycling genetic information on an extraordinary scale. ERVL LTRs thus offer means for a comprehensive survey of the genome's expression potential, tightly intertwining with gene expression and evolution in the germline., (© 2017 Franke et al.; Published by Cold Spring Harbor Laboratory Press.)

The care for adults with spina bifida is an important area to study. As increasing numbers of patients with spina bifida survive into adulthood, they expect to thrive and receive the best possible care into adulthood to maintain their health. Understanding the health needs in this emerging and changing population will help clinicians provide the best anticipatory care for adults with spina bifida and continue to improve outcomes. This will also impact pediatric care by improving the ability to determine preventive methods from early on and understand the impacts of pediatric care and decisions over the lifespan., Competing Interests: The authors report no conflicts of interest.

Uridylation of various cellular RNA species at the 3' end has been generally linked to RNA degradation. In mammals, uridylated pre-let-7 miRNAs and mRNAs are targeted by the 3' to 5' exoribonuclease DIS3L2. Mutations in DIS3L2 have been associated with Perlman syndrome and with Wilms tumor susceptibility. Using in vivo cross-linking and immunoprecipitation (CLIP) method, we discovered the DIS3L2-dependent cytoplasmic uridylome of human cells. We found a broad spectrum of uridylated RNAs including rRNAs, snRNAs, snoRNAs, tRNAs, vault, 7SL, Y RNAs, mRNAs, lncRNAs, and transcripts from pseudogenes. The unifying features of most of these identified RNAs are aberrant processing and the presence of stable secondary structures. Most importantly, we demonstrate that uridylation mediates DIS3L2 degradation of short RNA polymerase II-derived RNAs. Our findings establish the role of DIS3L2 and oligouridylation as the cytoplasmic quality control for highly structured ncRNAs., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Data are lacking regarding the independent risk of injury related to intense single-sport training or growth rate in young athletes., Purpose: To determine whether sports specialization, weekly training volumes, and growth rates are associated with increased risk for injury and serious overuse injury in young athletes., Study Design: Case-control study; Level of evidence, 3., Methods: Injured athletes aged 7 to 18 years were recruited from 2 hospital-based sports medicine clinics and compared with healthy controls from affiliated primary care clinics undergoing sports physicals (2010-2013). Participants completed surveys reporting hours per week spent in organized sports, physical education class, and free play, as well as degree of sports specialization and Tanner stage. Heights and weights were measured. Injury details were obtained from athlete surveys and electronic medical records., Results: Of 1214 athletes enrolled, 1190 (50.7% male) had data satisfactory for analysis. There were 822 injured participants (49.5% male; unique injuries, n = 846) and 368 uninjured participants (55% male). Injured athletes were older than uninjured athletes (14.1 ± 2.1 vs. 12.9 ± 2.6 years; P < .001) and reported more total hours of physical activity (19.6 ± 9.2 vs. 17.6 ± 8.9 h/wk; P < .001) and organized sports activity (11.2 ± 2.6 vs. 9.1 ± 6.3 h/wk; P < .01). After accounting for age and hours in sports activity spent per week, sports-specialized training was an independent risk for injury (odds ratio [OR], 1.27; 95% CI, 1.07-1.52; P < .01) and serious overuse injury (OR, 1.36; 95% CI, 1.08-1.72; P < .01). Young athletes participating in more hours of sports per week than number of age in years (OR, 2.07; 95% CI, 1.40-3.05; P < .001) or whose ratio of organized sports to free play time was >2:1 hours/week had increased odds of having a serious overuse injury (OR, 1.87; 95% CI, 1.26-2.76; P < .01). Growth rates were similar between injured and uninjured athletes (4.8 cm/y for both groups; P = .96)., Conclusion: Injured young athletes were older and spent more hours per week in organized sports. There is an independent risk of injury and serious overuse injury in young athletes who specialize in a single sport. Growth rate was not related to injury risk. The study data provide guidance for clinicians counseling young athletes and their parents regarding injury risks associated with sports specialization., (© 2015 The Author(s).)

In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Asymmetric dimethylarginine (aDMA) marks are placed on histones and the C-terminal domain (CTD) of RNA Polymerase II (RNAP II) and serve as a signal for recruitment of appropriate transcription and processing factors in coordination with transcription cycle. In contrast to other Tudor domain-containing proteins, Tudor domain-containing protein 3 (TDRD3) associates selectively with the aDMA marks but not with other methylarginine motifs. Here, we report the solution structure of the Tudor domain of TDRD3 bound to the asymmetrically dimethylated CTD. The structure and mutational analysis provide a molecular basis for how TDRD3 recognizes the aDMA mark. The unique aromatic cavity of the TDRD3 Tudor domain with a tyrosine in position 566 creates a selectivity filter for the aDMA residue. Our work contributes to the understanding of substrate selectivity rules of the Tudor aromatic cavity, which is an important structural motif for reading of methylation marks.

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.

In this article, we report the resonance assignment of CTD-interacting domain (CID) of pre-mRNA down-regulation (Nrd)1 bound to Ser5-phosphorylated CTD (pSer5) of RNA Polymerase II. The presented assignment of backbone and side-chain resonances of the Nrd1 CID proton, carbon and nitrogen nuclei will allow studies of the structure and interaction of CID with carboxy-terminal domain (CTD) of the RNA polymerase II.

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.

Nuclear polyadenylated RNA-binding (Nab)3 protein is an RNA-binding protein that is involved in the poly(A) independent termination pathway. Here, we report the NMR spectral assignments of RNA-recognition motif (RRM) of Nab3. The assignment will allow performing NMR structural and RNA-binding studies of Nab3 with the aim to investigate its role in the poly(A) independent termination pathway.