Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis.
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ABSTRACT: The epididymis in male reproductive system plays a crucial role in regulating the development of sperm motility and fertilizing capacity. Small non-coding RNAs (sncRNAs), especially microRNAs (miRNAs), can participate in the regulation of various physiological pathways. However, their abundance and whether they are involved in the regulation of gene expression in the human epididymis are unknown. By adopting the Solexa deep sequencing approach, we systematically investigated the sncRNAs in the adult human epididymis. A total of 4,903 unique reads representing 527 known miRNA were discovered. Eighteen novel miRNA genes encoding 23 mature miRNAs were also identified, suggesting a complicated miRNA regulatory network. The expression of some of the novel miRNA was confirmed by qRT-PCR. The presence of Piwi-interacting RNAs (piRNAs) in the library also adds to the diversity of sncRNA population in the human epididymis. This research lays the foundation for the functional study of sncRNAs in huamn reproductive system.
Project description:Paternal exposure to a range of environmental and lifestyle factors elicits distinct changes to the sperm sncRNA profile; modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures effect the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the potent reproductive toxicant, acrylamide. Further, we traced the differential accumulation of acrylamide responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure altered the expression of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identified extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa, the implications of which manifest in the form of dysregulated gene expression during early embryonic development. These data provide a causative mechanistic link to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos
Project description:Paternal exposure to environmental stressors elicits distinct changes to the sperm sncRNA profile; modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures modify the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the reproductive toxicant, acrylamide. Further, we traced the differential accumulation of acrylamide-responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure altered the abundance of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identified extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa, and dysregulated gene expression during early embryonic development following fertilisation by acrylamide-exposed spermatozoa. These data provide mechanistic links to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.
Project description:Background: Aberrant expression of small non-coding RNAs (sncRNAs), in particular microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) define several pathological processes. Asthma is characterized by airway hyper-reactivity, chronic inflammation and airway wall remodeling. Asthma-specific miRNA profiles were reported for bronchial epithelial cells, but no information on sncRNA expression in asthmatic bronchial smooth muscle (BSM) cells is available. Objective: To determine whether primary BSM sncRNA expression profile is altered in asthma and identify targets of differentially expressed sncRNAs. Methods: SmallRNA sequencing was used for sncRNA profiling in BSM cells (8 asthma, 6 non-asthma). sncRNA identification and differential expression analysis was performed with iMir, . experimentally validated miRNA targets were identified with Ingenuity Pathway Analysis and putative piRNA targets with miRanda. Results: Asthmatic BSM cells showed abnormal expression of 32 sncRNAs (26 miRNAs, 5 piRNAs, and 1 snoRNA). Target prediction for deregulated miRNAs and piRNAs revealed experimentally validated and predicted mRNA targets expressed in the BSM cells. 38 of these mRNAs represent major targets for deregulated miRNAs and may play important roles in the pathophysiology of asthma. Interestingly, 6 such miRNAs were previously associated with asthma and/or considered as novel therapeutic targets for treatment of this disease. Signaling pathway analysis revealed involvement of these sncRNAs in increased cell proliferation via PTEN and PI3K/Akt signaling pathways. Conclusions: BSM cells from asthma patients are characterized by aberrant sncRNA expression that recapitulates multiple pathological phenotypes of these cells. Implications: sncRNA expression profiling performed in this study further improve our understanding of the molecular mechanisms underlying asthma-associated processes in lungs.
Project description:This study annotates the NGS reads in a Sertoli cell sncRNA library. In addition to known sncRNAs, we also identified numerous novel sncRNAs expressed by Sertoli cells. Our data suggest that the Sertoli cell sncRNA transcriptome predominantly consists of miRNAs, piRNA-like RNAs, tRNAs and snoRNAs. This study reports the first comprehensive annotation of the Sertoli cell sncRNA transcriptome.
Project description:Proper functioning of the placenta, the temporary organ crucial in fetal development during pregnancy, is critical for maternal and fetal health. While microRNAs (miRNAs) are known to impact placental gene expression, the effects of other small non-coding RNAs (sncRNAs) on the placental transcriptome are not well-established. Here, we performed small RNA sequencing of 30 chorionic villi samples of varying gestational ages (M = 23.5 ± 11.4 weeks). We observed expression of 1543 sncRNAs, including miRNAs, piRNAs, snRNAs, snoRNAs, and tRNAs, as well as 18,003 miRNA variants (isomiRs), and identified 48 previously-unannotated miRNAs. This novel characterization of the placental sncRNA transcriptome may aid future investigations of sncRNA regulatory functions within the human placenta.
Project description:The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract. Examination of the microRNA expression profile in the whole mouse epididymis and mouse epididymal epithelial cells using next generation sequencing in duplicate.
Project description:This study annotates the NGS reads in a Sertoli cell sncRNA library. In addition to known sncRNAs, we also identified numerous novel sncRNAs expressed by Sertoli cells. Our data suggest that the Sertoli cell sncRNA transcriptome predominantly consists of miRNAs, piRNA-like RNAs, tRNAs and snoRNAs. This study reports the first comprehensive annotation of the Sertoli cell sncRNA transcriptome. Sertoli cells were purified from post-natal day 6 C57Bl/6 mice using the STAPUT unit gravity sedimentation apparatus. Small RNA was isolated from cells and a cDNA library was generated for 454 sequencing. Sequences from FastA files were initially mapped to known sequences and the genome and subsequently annotated upon novel small RNA categorization.
Project description:The epididymis is an important component of the male reproductive system and a crucial site for sperm maturation. During the process of sperm maturation through the epididymis, the effective payload of SncRNA significantly changes, which may be related to epigenetic modifications. Unfortunately, the N6 Methyladenosine (m6A) modification profile of the epididymis has not yet been established. In this study, the MeRIP seq combined with RNA seq method was used to analyze the m6A modification levels in the head, body, and tail of yak epididymis. It was found that the level of m6A in the epididymis significantly increased, and differentially methylated RNAs (DMRs) in the proximal end of the epididymis were significantly enriched in Gap junction, ErbB signaling pathway, and mTOR signaling pathway, participating in cell communication and sperm maturation processes. In the distal epididymis, DMRs are enriched in Apoptosis, FoxO signaling pathway, PI3K Akt signaling pathway, and TNF signaling pathway, which are involved in sperm autophagy, oxidative stress, and sperm maturation. In addition, we identified key genes with significant changes in m6A levels but no differences in RNA levels, including YY1 associated factor 2 (YAF2), Fork head box J2 (FOXJ2), and Fork head box O1 (FOXO1), suggesting that these genes are modified by m6A to affect translation processes and participate in sperm maturation. In summary, our study established an m6A map of the epididymis, which is beneficial for further understanding the maturation process of sperm and revealing more information related to male infertility.
Project description:Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs. In order to understand sncRNAsM-bM-^@M-^Y roles in plant cold response and stress acclimation, we studied miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate cold stresses, that underwent further severe cold stress after cold acclimation at moderate stress, and that grew under the normal condition. We also included Castor bean (Ricinus communis) to understand conservation of sncRNAs. In addition to known miRNAs, we identified dozens of novel miRNAs as well as ta-siRNA-yielding and nat-siRNA-yielding loci in Cassava and Castor bean, respectively. Among the expressed sncRNAs, many sncRNAs were differentially expressed under cold stresses. Our study provided the results on gene regulation by sncRNAs in cold acclimation of Euphorbiaceous plants and the role of sncRNA-mediated pathways affected by cold stress and stress acclimation in Cassava. Examination of small RNA populations in Cassava cultivar SC124 under the normal condition (NC), gradual cold acclimation (CA), cold shock (CS) and stress acclimation Cold stress after cold acclimation (CCA).
Project description:The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract.