ABSTRACT
Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.
Subject(s)
MicroRNAs , Semen , Male , Animals , Mice , Semen/metabolism , Spermatogenesis/genetics , Spermatozoa/metabolism , Testis/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Mammals/geneticsABSTRACT
As the genome is organized into a three-dimensional structure in intracellular space, epigenomic information also has a complex spatial arrangement. However, most epigenetic studies describe locations of methylation marks, chromatin accessibility regions, and histone modifications in the horizontal dimension. Proper spatial epigenomic information has rarely been obtained. In this study, we designed spatial chromatin accessibility sequencing (SCA-seq) to resolve the genome conformation by capturing the epigenetic information in single-molecular resolution while simultaneously resolving the genome conformation. Using SCA-seq, we are able to examine the spatial interaction of chromatin accessibility (e.g. enhancer-promoter contacts), CpG island methylation, and spatial insulating functions of the CCCTC-binding factor. We demonstrate that SCA-seq paves the way to explore the mechanism of epigenetic interactions and extends our knowledge in 3D packaging of DNA in the nucleus.
Subject(s)
Chromatin , Epigenomics , Chromatin/genetics , Chromosomes , DNA , Regulatory Sequences, Nucleic Acid , DNA MethylationABSTRACT
Despite rapid evolution across eutherian mammals, the X-linked miR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (Slitrk2 and Fmr1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked miR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernable defects, but simultaneous ablation of five clusters containing nineteen members of the miR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked miR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the miR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.
ABSTRACT
We presented an experimental method called FLOUR-seq, which combines BD Rhapsody and nanopore sequencing to detect the RNA lifecycle (including nascent, mature, and degrading RNAs) in cells. Additionally, we updated our HIT-scISOseq V2 to discover a more accurate RNA lifecycle using 10x Chromium and Pacbio sequencing. Most importantly, to explore how single-cell full-length RNA sequencing technologies could help improve the RNA velocity approach, we introduced a new algorithm called 'Region Velocity' to more accurately configure cellular RNA velocity. We applied this algorithm to study spermiogenesis and compared the performance of FLOUR-seq with Pacbio-based HIT-scISOseq V2. Our findings demonstrated that 'Region Velocity' is more suitable for analyzing single-cell full-length RNA data than traditional RNA velocity approaches. These novel methods could be useful for researchers looking to discover full-length RNAs in single cells and comprehensively monitor RNA lifecycle in cells.
Subject(s)
Nanopore Sequencing , Sequence Analysis, RNA , Single-Cell Analysis , Algorithms , High-Throughput Nucleotide Sequencing/methods , Nanopore Sequencing/methods , Sequence Analysis, RNA/methodsABSTRACT
Epigenetic modifications of histones are associated with development and pathogenesis of disease. Existing approaches cannot provide insights into long-range interactions and represent the average chromatin state. Here we describe BIND&MODIFY, a method using long-read sequencing for profiling histone modifications and transcription factors on individual DNA fibers. We use recombinant fused protein A-M.EcoGII to tether methyltransferase M.EcoGII to protein binding sites to label neighboring regions by methylation. Aggregated BIND&MODIFY signal matches bulk ChIP-seq and CUT&TAG. BIND&MODIFY can simultaneously measure histone modification status, transcription factor binding, and CpG 5mC methylation at single-molecule resolution and also quantifies correlation between local and distal elements.
Subject(s)
Eukaryota , Histones , Eukaryota/genetics , Histones/metabolism , Chromatin , Methylation , DNA/metabolism , DNA MethylationABSTRACT
BACKGROUND: Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In most conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. METHODS: Here, we developed a method of sequencing enzyme-accessible chromatin in circular DNA (CCDA-seq) based on the use of methylase to label open chromatin without fragmentation and exonuclease to enrich ecDNA sequencing depth, followed by long-read nanopore sequencing. RESULTS: Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding to ecDNA at a single-molecule resolution. CONCLUSIONS: These results deepen the understanding of ecDNA regulatory mechanisms.
Subject(s)
Chromatin , High-Throughput Nucleotide Sequencing , Chromatin/genetics , DNA/genetics , Epigenomics , MethyltransferasesABSTRACT
Coordinated gastric smooth muscle contraction is critical for proper digestion and is adversely affected by a number of gastric motility disorders. In this study we report that the secreted protein Mfge8 (milk fat globule-EGF factor 8) inhibits the contractile responses of human gastric antrum muscles to cholinergic stimuli by reducing the inhibitory phosphorylation of the MYPT1 (myosin phosphatase-targeting subunit (1) subunit of MLCP (myosin light chain phosphatase), resulting in reduced LC20 (smooth muscle myosin regulatory light chain (2) phosphorylation. Mfge8 reduced the agonist-induced increase in the F-actin/G-actin ratios of ß-actin and γ-actin1. We show that endogenous Mfge8 is bound to its receptor, α8ß1 integrin, in human gastric antrum muscles, suggesting that human gastric antrum muscle mechanical responses are regulated by Mfge8. The regulation of gastric antrum smooth muscles by Mfge8 and α8 integrin functions as a brake on gastric antrum mechanical activities. Further studies of the role of Mfge8 and α8 integrin in regulating gastric antrum function will likely reveal additional novel aspects of gastric smooth muscle motility mechanisms.
Subject(s)
Muscle Contraction , Pyloric Antrum , Antigens, Surface/metabolism , Humans , Milk Proteins/metabolism , Muscle, Smooth , Myosin Light Chains/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Phosphorylation , Pyloric Antrum/metabolismABSTRACT
Previous studies have shown that Dnmt2-null sperm block the paternal transmission (through sperm) of certain acquired traits, e.g., high-fat diet-induced metabolic disorders or white tails due to a Kit paramutation. Here, we report that DNMT2 is also required for the transmission of a Kit paramutant phenotype (white tail tip) through the female germline (i.e., oocytes). Specifically, ablation of Dnmt2 led to aberrant profiles of tRNA-derived small RNAs (tsRNAs) and other small noncoding RNAs (sncRNAs) in sperm, which correlate with altered mRNA transcriptomes in pronuclear zygotes derived from wild-type oocytes carrying the Kit paramutation and a complete blockage of transmission of the paramutant phenotype through oocytes. Together, the present study suggests that both paternal and maternal transmissions of epigenetic phenotypes require intact DNMT2 functions in the male germline.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/deficiency , Epigenesis, Genetic , Mice/genetics , Mutation , Pigmentation/genetics , Proto-Oncogene Proteins c-kit/genetics , Tail/physiology , Animals , Color , DNA (Cytosine-5-)-Methyltransferases/metabolism , Female , Male , PhenotypeABSTRACT
Small noncoding RNAs deep sequencing (sncRNA-Seq) has become a routine for sncRNA detection and quantification. However, the software packages currently available for sncRNA annotation can neither recognize sncRNA variants in the sequencing reads, nor annotate all known sncRNA simultaneously. Here, we report a novel anchor alignment-based small RNA annotation (AASRA) software package (https://github.com/biogramming/AASRA). AASRA represents an all-in-one sncRNA annotation pipeline, which allows for high-speed, simultaneous annotation of all known sncRNA species with the capability to distinguish mature from precursor microRNAs, and to identify novel sncRNA variants in the sncRNA-Seq sequencing reads.
Subject(s)
Mice/genetics , MicroRNAs/analysis , Molecular Sequence Annotation/methods , Software , AnimalsABSTRACT
The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.
Subject(s)
Focal Adhesions/metabolism , Immunohistochemistry/methods , Integrin alpha Chains/metabolism , Integrin beta1/metabolism , Multiprotein Complexes/metabolism , Oligonucleotides/metabolism , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Antibodies/chemistry , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Focal Adhesions/ultrastructure , Gastric Mucosa/metabolism , Gastric Mucosa/ultrastructure , Humans , Image Processing, Computer-Assisted , Integrin alpha Chains/chemistry , Integrin beta1/chemistry , Microscopy, Fluorescence , Molecular Probes/chemistry , Molecular Probes/metabolism , Multiprotein Complexes/chemistry , Muscle, Smooth/metabolism , Muscle, Smooth/ultrastructure , Oligonucleotides/chemical synthesis , Protein BindingABSTRACT
[This corrects the article DOI: 10.3389/fphys.2020.00230.].
ABSTRACT
Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca2+-activated Cl- channels (CaCC) encoded by Ano1. Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca2+ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na+/Ca2+ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca2+ exit mode, helps to regulate basal [Ca2+] i in these cells. Shifting NCX into Ca2+ entry mode by replacing extracellular Na+ with Li+ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from -80 to -40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca2+ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca2+ during the inter-slow wave interval and providing Ca2+ for sustained activation of ANO1 during the slow wave plateau phase.
ABSTRACT
The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.
Subject(s)
Adenosine/analogs & derivatives , Open Reading Frames , RNA, Circular/metabolism , Spermatocytes/metabolism , Spermatogenesis , Adenosine/metabolism , Adult , Animals , Humans , Male , Mice , Mice, Inbred C57BL , Spermatocytes/cytology , Young AdultABSTRACT
Comment on "A microRNA cluster in the Fragile-X region expressed during spermatogenesis targets FMR1" by Ramaiah et al.
Subject(s)
MicroRNAs , Animals , Fragile X Mental Retardation Protein , Gene Expression Regulation , Male , Mice , Spermatogenesis , SpermatogoniaABSTRACT
Male reproductive health has been in decline for decades with dropping sperm counts and increasing infertility, which has created a significant societal and economic burden. Between the 1970s and now, a general decline of over 50% in sperm concentration has been observed in the population. Environmental toxicant-induced epigenetic transgenerational inheritance has been shown to affect testis pathology and sperm count. Sertoli cells have an essential role in spermatogenesis by providing physical and nutritional support for developing germ cells. The current study was designed to further investigate the transgenerational epigenetic changes in the rat Sertoli cell epigenome and transcriptome that are associated with the onset of testis disease. Gestating female F0 generation rats were transiently exposed during the period of fetal gonadal sex determination to the environmental toxicants, such as dichlorodiphenyltrichloroethane (DDT) or vinclozolin. The F1 generation offspring were bred (i.e. intercross within the lineage) to produce the F2 generation grand-offspring that were then bred to produce the transgenerational F3 generation (i.e. great-grand-offspring) with no sibling or cousin breeding used. The focus of the current study was to investigate the transgenerational testis disease etiology, so F3 generation rats were utilized. The DNA and RNA were obtained from purified Sertoli cells isolated from postnatal 20-day-old male testis of F3 generation rats. Transgenerational alterations in DNA methylation, noncoding RNA, and gene expression were observed in the Sertoli cells from vinclozolin and DDT lineages when compared to the control (vehicle exposed) lineage. Genes associated with abnormal Sertoli cell function and testis pathology were identified, and the transgenerational impacts of vinclozolin and DDT were determined. Alterations in critical gene pathways, such as the pyruvate metabolism pathway, were identified. Observations suggest that ancestral exposures to environmental toxicants promote the epigenetic transgenerational inheritance of Sertoli cell epigenetic and transcriptome alterations that associate with testis abnormalities. These epigenetic alterations appear to be critical factors in the developmental and generational origins of testis pathologies and male infertility.
ABSTRACT
Antibody-based in situ proximity ligation assays (isPLA) have the potential to study protein phosphorylation and protein interactions with spatial resolution in intact tissues. However, the application of isPLA at the tissue level is limited by a lack of appropriate positive and negative controls and the difficulty in accounting for changes in tissue shape. Here we demonstrate a set of experimental and computational approaches using gastric fundus smooth muscles to improve the validity of quantitative isPLA. Appropriate positive and negative biological controls and PLA technical controls were selected to ensure experimental rigor. To account for changes in morphology between relaxed and contracted smooth muscles, target PLA spots were normalized to smooth muscle myosin light chain 20 PLA spots or the cellular cross-sectional areas. We describe the computational steps necessary to filter out false-positive improperly sized spots and set the thresholds for counting true positive PLA spots to quantify the PLA signals. We tested our approach by examining protein phosphorylation and protein interactions in smooth muscle myofilament Ca2+ sensitization pathways from resting and contracted gastric fundus smooth muscles. In conclusion, our tissue-level isPLA method enables unbiased quantitation of protein phosphorylation and protein-protein interactions in intact smooth muscle tissues, suggesting the potential for quantitative isPLA applications in other types of intact tissues.
Subject(s)
Fluorescent Antibody Technique/methods , Intracellular Signaling Peptides and Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Smooth/metabolism , Phosphoproteins/metabolism , Animals , Male , Mice , Mice, Inbred C57BL , Muscle Contraction , PhosphorylationABSTRACT
Prostate diseases include prostate cancer, which is the second most common male neoplasia, and benign prostatic hyperplasia (BPH), which affects approximately 50% of men. The incidence of prostate disease is increasing, and some of this increase may be attributable to ancestral exposure to environmental toxicants and epigenetic transgenerational inheritance mechanisms. The goal of the current study was to determine the effects that exposure of gestating female rats to vinclozolin has on the epigenetic transgenerational inheritance of prostate disease, and to characterize by what molecular epigenetic mechanisms this has occurred. Gestating female rats (F0 generation) were exposed to vinclozolin during E8-E14 of gestation. F1 generation offspring were bred to produce the F2 generation, which were bred to produce the transgenerational F3 generation. The transgenerational F3 generation vinclozolin lineage males at 12 months of age had an increased incidence of prostate histopathology and abnormalities compared to the control lineage. Ventral prostate epithelial and stromal cells were isolated from F3 generation 20-day old rats, prior to the onset of pathology, and used to obtain DNA and RNA for analysis. Results indicate that there were transgenerational changes in gene expression, noncoding RNA expression, and DNA methylation in both cell types. Our results suggest that ancestral exposure to vinclozolin at a critical period of gestation induces the epigenetic transgenerational inheritance of prostate stromal and epithelial cell changes in both the epigenome and transcriptome that ultimately lead to prostate disease susceptibility and may serve as a source of the increased incidence of prostate pathology observed in recent years.
Subject(s)
Epigenesis, Genetic , Epithelial Cells/metabolism , Gene Expression Regulation , Hazardous Substances/adverse effects , Prostatic Diseases/etiology , Prostatic Diseases/pathology , Stromal Cells/metabolism , DNA Methylation , Disease Susceptibility , Epigenome , Epithelial Cells/pathology , Humans , Inheritance Patterns , Male , RNA, Untranslated , Stromal Cells/pathology , TranscriptomeABSTRACT
Two of the most prevalent ovarian diseases affecting women's fertility and health are Primary Ovarian Insufficiency (POI) and Polycystic Ovarian Syndrome (PCOS). Previous studies have shown that exposure to a number of environmental toxicants can promote the epigenetic transgenerational inheritance of ovarian disease. In the current study, transgenerational changes to the transcriptome and epigenome of ovarian granulosa cells are characterized in F3 generation rats after ancestral vinclozolin or DDT exposures. In purified granulosa cells from 20-day-old F3 generation females, 164 differentially methylated regions (DMRs) (P < 1 x 10-6) were found in the F3 generation vinclozolin lineage and 293 DMRs (P < 1 x 10-6) in the DDT lineage, compared to controls. Long noncoding RNAs (lncRNAs) and small noncoding RNAs (sncRNAs) were found to be differentially expressed in both the vinclozolin and DDT lineage granulosa cells. There were 492 sncRNAs (P < 1 x 10-4) in the vinclozolin lineage and 1,085 sncRNAs (P < 1 x 10-4) in the DDT lineage. There were 123 lncRNAs and 51 lncRNAs in the vinclozolin and DDT lineages, respectively (P < 1 x 10-4). Differentially expressed mRNAs were also found in the vinclozolin lineage (174 mRNAs at P < 1 x 10-4) and the DDT lineage (212 mRNAs at P < 1 x 10-4) granulosa cells. Comparisons with known ovarian disease associated genes were made. These transgenerational epigenetic changes appear to contribute to the dysregulation of the ovary and disease susceptibility that can occur in later life. Observations suggest that ancestral exposure to toxicants is a risk factor that must be considered in the molecular etiology of ovarian disease.
Subject(s)
Environmental Pollutants/toxicity , Epigenesis, Genetic , Granulosa Cells/drug effects , Polycystic Ovary Syndrome/genetics , Primary Ovarian Insufficiency/genetics , Transcriptome , Animals , DDT/toxicity , DNA Methylation , Female , Granulosa Cells/metabolism , Male , Oxazoles/toxicity , RNA, Small Nuclear/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Epigenetic transgenerational inheritance of disease and phenotypic variation can be induced by several toxicants, such as vinclozolin. This phenomenon can involve DNA methylation, non-coding RNA (ncRNA) and histone retention, and/or modification in the germline (e.g. sperm). These different epigenetic marks are called epimutations and can transmit in part the transgenerational phenotypes. This study was designed to investigate the vinclozolin-induced concurrent alterations of a number of different epigenetic factors, including DNA methylation, ncRNA, and histone retention in rat sperm. Gestating females (F0 generation) were exposed transiently to vinclozolin during fetal gonadal development. The directly exposed F1 generation fetus, the directly exposed germline within the fetus that will generate the F2 generation, and the transgenerational F3 generation sperm were studied. DNA methylation and ncRNA were altered in each generation rat sperm with the direct exposure F1 and F2 generations being distinct from the F3 generation epimutations. Interestingly, an increased number of differential histone retention sites were found in the F3 generation vinclozolin sperm, but not in the F1 or F2 generations. All three different epimutation types were affected in the vinclozolin lineage transgenerational sperm (F3 generation). The direct exposure generations (F1 and F2) epigenetic alterations were distinct from the transgenerational sperm epimutations. The genomic features and gene pathways associated with the epimutations were investigated to help elucidate the integration of these different epigenetic processes. Our results show that the three different types of epimutations are involved and integrated in the mediation of the epigenetic transgenerational inheritance phenomenon.
ABSTRACT
OBJECTIVE: To determine the expression profile of small noncoding RNAs (sncRNAs) in leiomyoma, which has not been investigated to date. DESIGN: Laboratory-based investigation. SETTING: Academic center. PATIENT(S): Women undergoing hysterectomy for benign indications. INTERVENTION(S): Next-generation sequencing and screening of an sncRNA database with confirmatory analysis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). MAIN OUTCOME MEASURE(S): Expression profile of sncRNAs in leiomyoma and matched myometrium. RESULT(S): Screening our previously determined RNA sequencing data with the sncRNA database resulted in identification of 15 small nuclear (sn) RNAs, 284 small nucleolar (sno) RNAs, 98 Piwi-interacting (pi) RNAs, 152 transfer (t) RNAs, and 45 ribosomal (r) RNAs, of which 15 snoRNAs, 24 piRNAs, 7 tRNAs, and 6 rRNAs were differentially expressed at a 1.5-fold change cutoff in leiomyoma compared with myometrium. We selected 5 snoRNAs, 4 piRNAs, 1 tRNA, and 1 rRNA that were differentially expressed and confirmed their expression in paired tissues (n = 20) from both phases of the menstrual cycle with the use of qRT-PCR. The results indicated up-regulation of the snoRNAs (SNORD30, SNORD27, SNORA16A, SNORD46, and SNORD56) and down-regulation of the piRNAs (piR-1311, piR-16677, piR-20365, piR-4153), tRNA (TRG-GCC5-1), and rRNA (RNA5SP202) expression in leiomyoma compared with myometrium (P<.05). The pattern of expression of these sncRNAs was similar to RNA sequencing analysis, with no menstrual cycle-dependent differences detected except for SNORD30. Because Argonaute 2 (AGO2) is required for sncRNA-mediated gene silencing, we determined its expression and found greater abundance in leiomyoma. CONCLUSION(S): Our results provide the first evidence for the differential expression of additional classes of sncRNAs and AGO2 in leiomyoma, implicating their roles as a gene regulatory mechanism.