Small RNA-seq Research Articles

Identification of microRNA and analysis of target genes in Panax ginseng

ObjectiveGinsenosides, polysaccharides and phenols, the main active ingredients in Panax ginseng, are not different significantly in content between 3 and 5 years old of ginsengs called Yuan ginseng and more than ten years old ones called Shizhu ginseng. The responsible chemical compounds cannot fully explain difference in efficacy between them. According to reports in Lonicerae Japonicae Flos (Jinyinhua in Chinese) and Glycyrrhizae Radix et Rhizoma (Gancao in Chinese), microRNA may play a role in efficacy, so we identified microRNAs in P. ginseng at the different growth years and analyzed their target genes. MethodsUsing high-throughput sequencing, the RNA-Seq, small RNA-Seq and degradome databases of P. ginseng were constructed. The differentially expressed microRNAs was identified by qRT-PCR. ResultsA total of 63,875 unigenes and 24,154,579 small RNA clean reads were obtained from the roots of P. ginseng. From these small RNAs, 71 miRNA families were identified by bioinformatics target prediction software, including 34 conserved miRNAs, 37 non-conserved miRNA families, as well as 179 target genes of 17 known miRNAs. Through degradome sequencing and computation, we finally verified 13 targets of eight miRNAs involved in transcription, energy metabolism, biological stress and disease resistance, suggesting the significance of miRNAs in the development of P. ginseng. Consistently, major miRNA targets exhibited tissue specificity and complexity in expression patterns. ConclusionDifferential expression microRNAs were found in different growth years of ginsengs (Shizhu ginseng and Yuan ginseng), and the regulatory roles and functional annotations of miRNA targets in P. ginseng need further investigation.

Small RNA sequencing and profiling of serum-derived exosomes from African swine fever virus-infected pigs.

African swine fever (ASF) virus (ASFV) is responsible for one of the most severe swine diseases worldwide, with a morbidity rate of up to 100%; no vaccines or antiviral medicines are available against the virus. Exosomal miRNAs from individual cells can regulate the immune response to infectious diseases. In this study, pigs were infected with an ASFV Pig/HN/07 strain that was classified as acute form, and exosomal miRNA expression in the serum of infected pigs was analyzed using small RNA sequencing (small RNA-seq). Twenty-seven differentially expressed (DE) miRNAs were identified in the ASFV-infected pigs compared to that in the uninfected controls. Of these, 10 were upregulated and 17 were downregulated in the infected pigs. All DE miRNAs were analyzed using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the DE miRNAs were found to be highly involved in T-cell receptor signaling, cGMP-PKG signaling, Toll-like receptor, MAPK signaling, and mTOR signaling pathways. Furthermore, the Cytoscape network analysis identified the network of interactions between DE miRNAs and target genes. Finally, the transcription levels of four miRNA genes (ssc-miR-24-3p, ssc-miR-130b-3p, ssc-let-7a, and ssc-let-7c) were examined using quantitative real-time PCR (qRT-PCR) and were found to be consistent with the small RNA-seq data. These DE miRNAs were associated with cellular genes involved in the pathways related to immune response, virus-host interactions, and several viral genes. Overall, our findings provide an important reference and improve our understanding of ASF pathogenesis and the immune or protective responses during an acute infection in the host.

Mouse primordial germ-cell-like cells lack piRNAs.

PIWI-interacting RNAs (piRNAs) are small RNAs bound by PIWI-clade Argonaute proteins that function to silence transposable elements (TEs). Following mouse primordial germ cell (mPGC) specification around E6.25, fetal piRNAs emerge in male gonocytes from E13.5 onward. The invitro differentiation of mPGC-like cells (mPGCLCs) has raised the possibility of studying the fetal piRNA pathway in greater depth. However, using single-cell RNA-seq and RT-qPCR along mPGCLC differentiation, we find that piRNA pathway factors are not fully expressed in Day 6 mPGCLCs. Moreover, we do not detect piRNAs across a panel of Day 6 mPGCLC lines using small RNA-seq. Our combined efforts highlight that invitro differentiated Day 6 mPGCLCs do not yet resemble E13.5 or later mouse gonocytes where the piRNA pathway is active. This Matters Arising paper is in response to von Meyenn etal. (2016), published in Developmental Cell. See also the correction by von Meyenn etal. published in this issue.

Abstract A030: Deciphering the micro-RNA landscape of AKT isoforms and its clinical impact in patients with lung adenocarcinoma

Abstract Over the last 25 years, AKT kinase has been thoroughly investigated and characterized as a crucial regulator of cellular homeostasis, affecting a plethora of essential molecular functions. The three identified isoforms of this kinase, namely AKT1, AKT2 and AKT3, often display opposing and antagonizing cellular functions. One of these important cellular functions is the expression and activation of micro-RNAs (miRNAs), an important RNA process that epigenetically regulates the human transcriptome. In the context of human cancer and based on several reports, AKT kinase has been associated with aberrant expression of oncogenic miRNAs, that in turn hijack oncogenic pathways, leading to tumor progression, metastasis and resistance to therapy, especially in lung adenocarcinoma (LUAD). Nevertheless, a comprehensive analysis of the impact of each AKT isoform on the regulation and enrichment of a specific miRNA signature in lung adenocarcinoma has yet to be performed. Here, we were willing to elucidate the miRNA epigenetic landscape and downstream targets, driven by each AKT isoform, and its clinical importance in patients with lung adenocarcinoma. To this extent, we utilized RNA-Seq and Small RNA-Seq data from The Cancer Genome Atlas (TCGA-LUAD) to identify the miRNA signatures related with the expression of each AKT isoform in LUAD. Gene Set Enrichment Analysis from RNA-Seq and miRNA levels analysis from Small RNA-Seq, identified a unique miRNA signature for each AKT isoform, demonstrating their largely opposing effect in miRNA profiling. In parallel analysis, in silico target analysis of the top 50 regulated miRNAs, demonstrated another set of unique targets for each isoform-dependent miRNA signature, which is involved in metabolism, DNA damage, RNA processing, cell survival, immune response and metastasis. More importantly, clinical and survival analysis revealed that the top 50 Differentially regulated miRNAs, ranked by q-value, for each isoform correlated with poor progression free survival in patients with LUAD. Collectively, our atlas of AKT isoform-driven miRNA landscapes implicates AKT as a global effector of miRNA landscape in lung adenocarcinoma. Due to the lack of reliable isoform-specific AKT inhibitors, the identification of the oncogenic miRNA networks regulated by each isoform, acts as an important transnational resource for the design and testing of inhibitors that bypass AKT inhibition toxicity, sparing its impactful physiological functions. Citation Format: George Laliotis, Pauline Lin. Deciphering the micro-RNA landscape of AKT isoforms and its clinical impact in patients with lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A030.

First Trimester Maternal Plasma Aberrant miRNA Expression Associated with Spontaneous Preterm Birth.

Spontaneous Preterm Delivery (sPTD) is one of the leading causes of perinatal mortality and morbidity worldwide. The present case−control study aims to detect miRNAs differentially expressed in the first trimester maternal plasma with the view to identify predictive biomarkers for sPTD, between 320/7 and 366/7 weeks, that will allow for timely interventions for this serious pregnancy complication. Small RNA sequencing (small RNA-seq) of five samples from women with a subsequent sPTD and their matched controls revealed significant down-regulation of miR-23b-5p and miR-125a-3p in sPTD cases compared to controls, whereas miR-4732-5p was significantly overexpressed. Results were confirmed by qRT-PCR in an independent cohort of 29 sPTD cases and 29 controls. Statistical analysis demonstrated that miR-125a is a promising early predictor for sPTL (AUC: 0.895; 95% CI: 0.814-0.972; p < 0.001), independent of the confounding factors tested, providing a useful basis for the development of a novel non-invasive predictive test to assist clinicians in estimating patient-specific risk.

Combined Noncoding RNA-mRNA Regulomics Signature in Reprogramming and Pluripotency in iPSCs

Somatic cells are reprogrammed with reprogramming factors to generate induced pluripotent stem cells (iPSCs), offering a promising future for disease modeling and treatment by overcoming the limitations of embryonic stem cells. However, this process remains inefficient since only a small percentage of transfected cells can undergo full reprogramming. Introducing miRNAs, such as miR-294 and miR302/3667, with reprogramming factors, has shown to increase iPSC colony formation. Previously, we identified five transcription factors, GBX2, NANOGP8, SP8, PEG3, and ZIC1, which may boost iPSC generation. In this study, we performed quantitative miRNAome and small RNA-seq sequencing and applied our previously identified transcriptome to identify the potential miRNA-mRNA regulomics and regulatory network of other ncRNAs. From each fibroblast (N = 4), three iPSC clones were examined (N = 12). iPSCs and original fibroblasts expressed miRNA clusters differently and miRNA clusters were compared to mRNA hits. Moreover, miRNA, piRNA, and snoRNAs expression profiles in iPSCs and original fibroblasts were assessed to identify the potential role of ncRNAs in enhancing iPSC generation, pluripotency, and differentiation. Decreased levels of let-7a-5p showed an increase of SP8 as described previously. Remarkably, the targets of identifier miRNAs were grouped into pluripotency canonical pathways, on stemness, cellular development, growth and proliferation, cellular assembly, and organization of iPSCs.

Meta-Analysis of RNA-Seq Datasets Identifies Novel Players in Glioblastoma

Glioblastoma is a devastating grade IV glioma with poor prognosis. Identification of predictive molecular biomarkers of disease progression would substantially contribute to better disease management. In the current study, we performed a meta-analysis of different RNA-seq datasets to identify differentially expressed protein-coding genes (PCGs) and long non-coding RNAs (lncRNAs). This meta-analysis aimed to improve power and reproducibility of the individual studies while identifying overlapping disease-relevant pathways. We supplemented the meta-analysis with small RNA-seq on glioblastoma tissue samples to provide an overall transcriptomic view of glioblastoma. Co-expression correlation of filtered differentially expressed PCGs and lncRNAs identified a functionally relevant sub-cluster containing DANCR and SNHG6, with two novel lncRNAs and two novel PCGs. Small RNA-seq of glioblastoma tissues identified five differentially expressed microRNAs of which three interacted with the functionally relevant sub-cluster. Pathway analysis of this sub-cluster identified several glioblastoma-linked pathways, which were also previously associated with the novel cell death pathway, ferroptosis. In conclusion, the current meta-analysis strengthens evidence of an overarching involvement of ferroptosis in glioblastoma pathogenesis and also suggests some candidates for further analyses.

Potential of Non-Coding RNA as Biomarkers for Progressive Supranuclear Palsy.

Objective markers for the neurodegenerative disorder progressive supranuclear palsy (PSP) are needed to provide a timely diagnosis with greater certainty. Non-coding RNA (ncRNA), including microRNA, piwi-interacting RNA, and transfer RNA, are good candidate markers in other neurodegenerative diseases, but have not been investigated in PSP. Therefore, as proof of principle, we sought to identify whether they were dysregulated in matched serum and cerebrospinal fluid (CSF) samples of patients with PSP. Small RNA-seq was undertaken on serum and CSF samples from healthy controls (n = 20) and patients with PSP (n = 31) in two cohorts, with reverse transcription-quantitative PCR (RT-qPCR) to confirm their dysregulation. Using RT-qPCR, we found in serum significant down-regulation in hsa-miR-92a-3p, hsa-miR-626, hsa-piR-31068, and tRNA-ValCAC. In CSF, both hsa-let-7a-5p and hsa-piR-31068 showed significant up-regulation, consistent with their changes observed in the RNA-seq results. Interestingly, we saw no correlation in the expression of hsa-piR-31068 within our matched serum and CSF samples, suggesting there is no common dysregulatory mechanism between the two biofluids. While these changes were in a small cohort of samples, we have provided novel evidence that ncRNA in biofluids could be possible diagnostic biomarkers for PSP and further work will help to expand this potential.

Cerebrospinal fluid and blood profiles of transfer RNA fragments show age, sex, and Parkinson's disease-related changes.

Transfer RNA fragments (tRFs) have recently been shown to be an important family of small regulatory RNAs with diverse functions. Recent reports have revealed modified tRF blood levels in a number of nervous system conditions including epilepsy, ischemic stroke, and neurodegenerative diseases, but little is known about tRF levels in the cerebrospinal fluid (CSF). To address this issue, we studied age, sex, and Parkinson's disease (PD) effects on the distributions of tRFs in the CSF and blood data of healthy controls and PD patients from the NIH and the Parkinson's Progression Markers Initiative (PPMI) small RNA-seq datasets. We discovered that long tRFs are expressed in higher levels in the CSF than in the blood. Furthermore, the CSF showed a pronounced age-associated decline in the level of tRFs cleaved from the 3'-end and anti-codon loop of the parental tRNA (3'-tRFs, i-tRFs), and more pronounced profile differences than the blood profiles between the sexes. In comparison, we observed moderate age-related elevation of blood 3'-tRF levels. In addition, distinct sets of tRFs in the CSF and in the blood segregated PD patients from controls. Finally, we found enrichment of tRFs predicted to target cholinergic mRNAs (Cholino-tRFs) among mitochondrial-originated tRFs, raising the possibility that the neurodegeneration-related mitochondrial impairment in PD patients may lead to deregulation of their cholinergic tone. Our findings demonstrate that the CSF and blood tRF profiles are distinct and that the CSF tRF profiles are modified in a sex-, age-, and disease-related manner, suggesting that they reflect the inter-individual cerebral differences and calling for incorporating this important subset of small RNA regulators into future studies.

VsRNAfinder: a novel method for identifying high-confidence viral small RNAs from small RNA-Seq data.

Virus-encoded small RNAs (vsRNA) have been reported to play an important role in viral infection. Unfortunately, there is still a lack of an effective method for vsRNA identification. Herein, we presented vsRNAfinder, a de novo method for identifying high-confidence vsRNAs from small RNA-Seq (sRNA-Seq) data based on peak calling and Poisson distribution and is publicly available at https://github.com/ZenaCai/vsRNAfinder. vsRNAfinder outperformed two widely used methods namely miRDeep2 and ShortStack in identifying viral miRNAs with a significantly improved sensitivity. It can also be used to identify sRNAs in animals and plants with similar performance to miRDeep2 and ShortStack. vsRNAfinder would greatly facilitate effective identification of vsRNAs from sRNA-Seq data.

Negative effects of heat stress on ovarian tissue in female rabbit.

Numerous studies have highlighted the role of miRNA in the deformation and necrosis of cells of ovarian tissue caused by heat stress (HS), which ultimately affects ovarian function. Although the role of small RNAs has been investigated in alterations in ovarian tissue functioning in response to HS, the expression profile of ovarian miRNA has been explored to a lesser extent. In this study, female rabbits were subject to HS treatment by using electrical heater. The current work demonstrated that HS could significantly change physiological performance of female rabbits including body weight, rectal temperature and relative ovary weight, and significantly reduce serum IL-2, IL-8, CAT, and GSH-Px concentrations by enzyme-linked immunosorbent assay (ELISA) technique. As a result, an increase in apoptosis in ovarian cells, as well as unhealthy follicles, were observed by Hematoxylin-eosin (HE) and TUNEL staining. Additionally, small RNA-seq revealed changes in the miRNA expression profile of rabbit ovaries under HS. Five hundred fourteen miRNAs were obtained including known miRNAs 442 and novel miRNAs 72. Among these miRNAs, 23 miRNAs were significantly expressed under HS. Eleven differentially expressed miRNAs (DE miRNAs) and 9 their predicted targets were confirmed by qPCR, which were expected miRNA-mRNA negative regulation pattern. Among the DE miRNAs and targets, miR-141-39 may target COQ6, miR-449a-5p and miR-34c-5p may control RFC5 and RTN2 together, miR-449a-5p may target ACADVL, miR-34c-5p potentially targets Bcl-2 and miR-196b-5p potentially regulates CASK and HOXB6. Thus, the current work suggested the negative effects of HS on the ovarian tissue of female rabbits, and in conclusion these changes could be caused by decreased serum IL-2, IL-8, CAT and GSH-Px levels, increased ovarian apoptosis, and changed the expression of miRNAs.

Abstract 12839: Integrated Multi-Omics and Single Nucleus Transcriptomics Highlight Enhanced Adaptive Immune Responses in Human Bicuspid Aortic Valve Stenosis

Introduction: Bicuspid aortic valve (BAV) is the most common congenital cardiac defect, occurring in 1.5% of humans. Due to unknown pathogenic mechanisms, over 70% of those with BAV develop calcific aortic valve disease (CAVD). These patients require valve replacement 25x more frequently and a decade earlier than those with a tricuspid aortic valve (TAV). Objective: Identify cellular and molecular drivers of accelerated disease in BAV-CAVD. Methods: Small RNA-seq, RNA-seq, and proteomics were performed on 70 human aortic valves (11 non-diseased TAVs; fibrotic and calcific stages of 32 TAVs and 27 BAVs excised for CAVD). Single nuclei RNA-seq (snRNA-seq) was completed on 54,608 nuclei from 13 additional human valves (4 non-diseased TAVs; 5 TAVs and 4 BAVs with CAVD). Results: 4,938 miRs, transcripts, and proteins were differentially enriched between stages of BAV- and TAV-CAVD (q&lt;0.05). Multi-omics data, CT-derived valve calcification, and 29 clinical parameters were integrated by latent factor analysis. Protein-protein interaction networks revealed significantly elevated adaptive immune responses in BAV-CAVD: T cell/B cell inflammatory activation was enhanced and SLIT-ROBO signaling disrupted in diseased BAVs. snRNA-seq identified 10 major cell types in human aortic valves: valvular interstitial cells (65% of cells; 3 states), endothelial cells (10%; 2), macrophages (13%), T cells (8%; 2), and B cells (3%; 2). CAVD drove differentiation of dual side-specific endothelial subpopulations, enrichment of CARMN/CACNA1C/ACTA2-high interstitial cells, and substantial immune cell accumulation. B cells were further enriched in BAV- vs. TAV-CAVD. Conclusions: Adaptive immunity underpins molecular differences in BAV- vs. TAV-CAVD and is a novel avenue for tailored pharmacotherapy. We potentiate targeting of key cellular subpopulations by defining the dissociation bias-free transcriptional and cellular diversity of human CAVD at single-cell resolution.

LBMON272 Chromatoid Bodies In The Regulation Of Spermatogenesis: Novel Role Of Grth

Abstract More than 5 million couples in US are affected by infertility. Male infertility is a major cause (&amp;gt;30%), and understanding spermatogenesis is important to address the problem. During early spermatogenesis, mRNAs are actively transcribed, transported and stored transiently in chromatoid bodies (CBs). CBs are membrane-less perinuclear organelles which serve as storehouses of mRNAs until later stages of spermatogenesis. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) plays critical role in spermatogenesis. In addition to its inherent helicase activity, GRTH transports mRNAs from nucleus to cytoplasm and from there to CBs for storage until translation. In Germ cells, there are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho (pGRTH) forms. Our early studies revealed a missense mutation (R242H) of GRTH in Japanese azoospermic men which resulted in the lack of pGRTH in in vitro studies. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H), show loss of the phospho-species from cytoplasm and CBs with preservation of the non-phospho form in the cytoplasm, Nucleus and CBs. GRTH-KI mice are sterile, lack elongated spermatids and spermatozoa with spermatogenic arrest at step 8 of round spermatids (RS). CBs isolated from GRTH KI mice are smaller, highly condensed, lack pGRTH and RNA-Seq analysis of isolated mRNA (from CBs) revealed differential abundance of mRNAs related to spermatid differentiation. Integrative analysis of small RNA and mRNA expression will provide cohesive outlook of molecular mechanisms of gene regulation during spermatogenesis. Hence, we proceeded with small RNAseq analysis which reveal differential expression (Padj&amp;lt;0. 05) of piRNAs and miRNAs. In total 226706 piRNAs upregulated and 166567 piRNAs downregulated in the round spermatids. Several miRNAs like miR32, miR7, miR184, miR335, miR140, miR141, miR1981, miR202, miR880, miR669c were downregulated. These are involved in the positive regulation of spermatid differentiation, sperm motility, mRNA processing and decay. The miRNAs, miR150, miR196a-2, miR7b, miR652, miR146, miR10b, miR379, miR122a, miR27a, miR127 and miR328 were upregulated, which negatively regulate the spermatid differentiation. Gene ontology analysis revealed that the majority of downregulated miRNAs belong to regulation of gene expression. Majority of upregulated miRNAs belong to miRNA mediated suppression of translation mostly associated with spermiogenesis and CBs. The identified miRNAs regulate essential genes like Tnp2, TSSK6, Pabpc1, Ppp1cc and UPF2 etc. These indicates that the CBs mediate highly specialized mRNA metabolism process and serve as a processing center of essential mRNAs. These results demonstrate the importance of GRTH and phospho-GRTH in the regulation of CB and their role in mRNA storage and stability of germ cell specific mRNAs during spermiogenesis and healthy sperm formation. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

SCRAP: a bioinformatic pipeline for the analysis of small chimeric RNA-seq data.

MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that function in post-transcriptional gene regulation through imperfect base pairing with mRNA targets which results in inhibition of translation and typically destabilization of bound transcripts. Sequence-based algorithms historically used to predict miRNA targets face inherent challenges in reliably reflecting in vivo interactions. Recent strategies have directly profiled miRNA-target interactions by crosslinking and ligation of sncRNAs to their targets within the RNA-induced silencing complex (RISC), followed by high throughput sequencing of the chimeric sncRNA:target RNAs. Despite the strength of these direct profiling approaches, standardized pipelines for effectively analyzing the resulting chimeric sncRNA:target RNA sequencing data are not readily available. Here we present SCRAP, a robust Small Chimeric RNA Analysis Pipeline for the bioinformatic processing of chimeric sncRNA:target RNA sequencing data. SCRAP consists of two parts, each of which are specifically optimized for the distinctive characteristics of chimeric small RNA sequencing reads: first, read processing and alignment and second, peak calling and annotation. We apply SCRAP to benchmark chimeric sncRNA:target RNA sequencing datasets generated by distinct molecular approaches, and compare SCRAP to existing chimeric RNA analysis pipelines. SCRAP has minimal hardware requirements, is cross-platform, and contains extensive annotation to broaden accessibility for processing small chimeric RNA sequencing data and enable insights about the targets of small non-coding RNAs in regulating diverse biological systems.

Identification of novel microRNAs associated with type 2 diabetes by an integrative bioinformatic analysis

To identify potential miRNAs involved in the development/progression of type 2 diabetes (T2D) we performed data screening and integration of microRNA (miRNA) and messenger RNA (mRNA) expression profiles available in public data repositories. We retrieved two independent sequencing datasets: GSE52314 (small RNA-Seq) and GSE50244 (RNA-Seq). Then, we identified Differentially Expressed Genes (DEGs) and Differentially Expressed miRNAs (DEMs) from non-diabetic (ND) vs. people with T2D. The integrative analysis of identified DEGs and DEMs revealed 303 interactions involving 33 miRNAs and 256 genes. Functional enrichment analysis showed these interactions could play a functional role in carbohydrate, lipid, and nucleotide impaired metabolism. Some interactions we identified could also be relevant at an early stage of T2D. We also performed a similar integration analysis using a single study from a rat model, including both RNA-Seq and small RNA-Seq in the same pancreatic tissue: CRA000791. Interestingly, in this analysis one identified miRNA was also identified in the integrative analysis in human transcriptome: hsa-miR-27a-5p. Moreover, functional enrichment analysis showed that the target genes are mainly enriched in biological processes and pathways related to lipid and nucleotide metabolism.The miRNAs and interactions we identified could be relevant to gene expression regulation in T2D. Although further validation is necessary for extensive use in clinics, they provide new and useful knowledge to develop potential novel strategies for early diagnosis, prevention, and treatment of T2D.

Integrated analysis of miRNA and mRNA expression profiles in testes of Landrace and Hezuo boars.

Precocious puberty is closely related to testicular development and spermatogenesis, and there is increasing evidence that miRNAs are involved in regulation of testicular development and spermatogenesis. However, little is known about the regulation of microRNAs (miRNAs) during precocious maturation in Hezuo (HZ) boars. In this study, serum Testosterone (T), Estradiol (E2), Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) levels were detected in HZ and Landrace (LC) boars in the postnatal period at 30, 90, 120, 180, and 240 days, and the testes of HZ and LC boars at 30 and 120 days were used for histological observation. In addition, we performed small RNA-Seq to identify miRNA at sexual immaturity (30-days-old) and maturity (120-days-old) of HZ boar testis (using LC boar as control) to reveal the key miRNA in regulation of precocious puberty. Hormone assay results showed that high levels of T, E2, FSH, and LH may be related to precocious sexual maturity of HZ boars, and that FSH may play an important function before sexual maturity. Histological observation showed that HZ boars developed earlier than LC boars and had reached sexual maturity at 120 days. Small RNA-Seq yielded a total of 359 exist miRNAs, 767 known miRNAs and 322 novel miRNAs in 12 samples; 549, 468, 133, and 247 differentially expressed (DE) miRNAs were identified between Ha vs. Hb, La vs. Lb, Ha vs. La, and Hb vs. Lb (log2 fold change >1 and p < 0.05). Enrichment analysis showed that target genes of these DE miRNAs were enriched in many gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways (such as PI3K-Akt, Hippo and Rap1 signaling pathways) were related to testicular development and spermatogenesis. Further screening, some miRNAs (such as ssc-miR-29b, ssc-miR-199b, ssc-miR-383, ssc-miR-149, ssc-miR-615, and ssc-miR-370) were possibly associated with precocious puberty. These results provide new light on miRNA regulatory mechanisms involved in precocious puberty.

Functional evaluation of LTR-derived lncRNAs in porcine oocytes and zygotes with RNA-seq and small RNA-seq.

Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of early embryonic development in mammals. However, they are seldom investigated in pigs. Here, to annotate full-length RNA transcripts, we performed annotation using a newly developed computational pipeline—an RNA-seq and small RNA-seq combined strategy—using our previously obtained RNA-seq and small RNA-seq data from porcine oocytes and zygotes. As evidenced by the length comparison, the frequency of the core promoter, and the polyadenylation signal motifs, the transcripts appear to be full-length. Furthermore, our strategy allowed the identification of a large number of endogenous retrovirus-associated lncRNAs (ERV-lncRNAs) and found that some of them were highly expressed in porcine zygotes, as compared to oocytes. Through the knockdown strategy, two ERV-lncRNAs (TCONS_00035465 and TCONS_00031520) were identified as playing potential roles in the early embryo development of pigs, laying a foundation for future research.

MicroRNA 1307 Is a Potential Target for SARS-CoV-2 Infection: An in Vitro Model.

microRNAs (miRs) are proposed as critical molecular targets in SARS-CoV-2 infection. Our recent in silico studies identified seven SARS-CoV-2 specific miR-like sequences, which are highly conserved with humans, including miR-1307-3p, with critical roles in COVID-19. In this current study, Vero cells were infected with SARS-CoV-2, and miR expression profiles were thereafter confirmed by qRT-PCR. miR-1307-3p was the most highly expressed miR in the infected cells; we, therefore, transiently inhibited its expression in both infected and uninfected cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay assessed cell viability following SARS-CoV-2 infection, identifying that miR-1307 expression is inversely correlated with cell viability. Lastly, changes in miR-1307-dependent pathways were analyzed through a detailed miRNOME and associated in silico analysis. In addition to our previously identified miRs, including miR-1307-3p, the upregulation of miR-193a-5p, miR-5100, and miR-23a-5p and downregulation of miR-130b-5p, miR34a-5p, miR-505-3p, miR181a-2-3p, miR-1271-5p, miR-598-3p, miR-34c-3p, and miR-129-5p were also established in Vero cells related to general lung disease-related genes following SARS-CoV-2 infection. Targeted anti-miR-1307-3p treatment rescued cell viability in infection when compared to SARS CoV-2 mediated cell cytotoxicity only. We furthermore identified by in silico analysis that miR-1307-3p is conserved in all SARS-CoV-2 sequences/strains, except in the BA.2 variant, possibly contributing to the lower disease severity of this variant, which warrants further investigation. Small RNA seq analysis was next used to evaluate alterations in the miRNOME, following miR-1307-3p manipulation, identifying critical pathobiological pathways linked to SARS-CoV-2 infection-mediated upregulation of this miR. On the basis of our findings, miRNAs like miR-1307-3p play a critical role in SARS-CoV-2 infection, including via effects on disease progression and severity.

DNA polymerase epsilon interacts with SUVH2/9 to repress the expression of genes associated with meiotic DSB hotspot in Arabidopsis

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)-triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

In-depth investigation of microRNA-mediated cross-kingdom regulation between Asian honey bee and microsporidian.

Asian honey bee Apis cerana is the original host for Nosema ceranae, a unicellular fungal parasite that causes bee nosemosis throughout the world. Currently, interaction between A. cerana and N. ceranae is largely unknown. Our group previously prepared A. c. cerana workers’ midguts at 7 days post inoculation (dpi) and 10 dpi with N. ceranae spores as well as corresponding un-inoculated workers’ midguts, followed by cDNA library construction and a combination of RNAs-seq and small RNA-seq. Meanwhile, we previously prepared clean spores of N. ceranae, which were then subjected to cDNA library construction and deep sequencing. Here, based on the gained high-quality transcriptome datasets, N. ceranae differentially expressed mRNAs (DEmiRNAs) targeted by host DEmiRNAs, and A. c. cerana DEmRNAs targeted by microsporidian DEmiRNAs were deeply investigated, with a focus on targets involved in N. ceranae glycolysis/glyconeogenesis as well as virulence factors, and A. c. cerana energy metabolism and immune response. In A. c. cerana worker’s midguts at 7 (10) dpi (days post inoculation), eight (seven) up-regulated and six (two) down-regulated miRNAs were observed to target 97 (44) down-regulated and 60 (15) up-regulated N. ceranae mRNAs, respectively. Additionally, two up-regulated miRNAs (miR-60-y and miR-676-y) in host midgut at 7 dpi could target genes engaged in N. ceranae spore wall protein and glycolysis/gluconeogenesis, indicating potential host miRNA-mediated regulation of microsporidian virulence factor and energy metabolism. Meanwhile, in N. ceranae at 7 (10) dpi, 121 (110) up-regulated and 112 (104) down-regulated miRNAs were found to, respectively, target 343 (247) down-regulated and 138 (110) down-regulated mRNAs in A. c. cerana workers’ midguts. These targets in host were relevant to several crucial cellular and humoral immune pathways, such as phagasome, endocytosis, lysosomes, regulation of autophagy, and Jak–STAT signaling pathway, indicative of the involvement of N. ceranae DEmiRNAs in regulating these cellular and humoral immune pathways. In addition, N. ceranae miR-21-x was up-regulated at 7 dpi and had a target relative to oxidative phosphorylation, suggesting that miR-21-x may be used as a weapon to modulate this pivotal energy metabolism pathway. Furthermore, potential targeting relationships between two pairs of host DEmiRNAs-microsporidian DEmRNAs and two pairs of microsporidian DEmiRNAs-host DEmRNAs were validated using RT-qPCR. Our findings not only lay a foundation for exploring the molecular mechanism underlying cross-kingdom regulation between A. c. cerana workers and N. ceranae, but also offer valuable insights into Asian honey bee-microsporidian interaction.