Small RNA-seq and clinical evaluation of tRNA-derived fragments in multiple myeloma: Loss of mitochondrial i-tRFHisGTG results in patients' poor treatment outcome.

Despite the substantial progress in multiple myeloma (MM) therapy nowadays, treatment resistance and disease relapse remain major clinical hindrances. Herein, we have investigated tRNA-derived fragment (tRF) profiles in MM and precursor stages (smoldering MM/sMM; monoclonal gammopathy of undetermined significance/MGUS), aiming to unveil potential MM-related tRFs in ameliorating MM prognosis and risk stratification. Small RNA-seq was performed to profile tRFs in bone marrow CD138+ plasma cells, revealing the significant deregulation of the mitochondrial internal tRFHisGTG (mt-i-tRFHisGTG) in MM versus sMM/MGUS. The screening cohort of the study consisted of 147 MM patients, and mt-i-tRFHisGTG levels were quantified by RT-qPCR. Disease progression was assessed as clinical end-point for survival analysis, while internal validation was performed by bootstrap and decision curve analyses. Screening cohort analysis highlighted the potent association of reduced mt-i-tRFHisGTG levels with patients' bone disease (p = 0.010), osteolysis (p = 0.023) and with significantly higher risk for short-term disease progression following first-line chemotherapy, independently of patients' clinical data (HR = 1.954; p = 0.036). Additionally, mt-i-tRFHisGTG-fitted multivariate models led to superior risk stratification of MM patients' treatment outcome and prognosis compared to disease-established markers. Notably, our study highlighted mt-i-tRFHisGTG loss as a powerful independent indicator of post-treatment progression of MM patients, leading to superior risk stratification of patients' treatment outcome.

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.

miRNA-seq identification and clinical validation of CD138+ and circulating miR-25 in treatment response of multiple myeloma.

BACKGROUND: Despite significant advancements in multiple myeloma (MM) therapy, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics. Herein, we have studied the clinical utility of miRNAs in ameliorating patients' management. METHODS: miRNA-seq was performed in bone marrow CD138+ plasma cells (PCs) of 24 MM and smoldering MM (sMM) patients to analyze miRNAs profile. CD138+ and circulating miR-25 levels were quantified using in house RT-qPCR assays in our screening MM/sMM cohort (CD138+ plasma cells n = 167; subcohort of MM peripheral plasma samples n = 69). Two external datasets (Kryukov et al. cohort n = 149; MMRF CoMMpass study n = 760) served as institutional-independent validation cohorts. Patients' mortality and disease progression were assessed as clinical endpoints. Internal validation was performed by bootstrap analysis. Clinical benefit was estimated by decision curve analysis. RESULTS: miRNA-seq highlighted miR-25 of CD138+ plasma cells to be upregulated in MM vs. sMM, R-ISS II/III vs. R-ISS I, and in progressed compared to progression-free patients. The analysis of our screening cohort highlighted that CD138+ miR-25 levels were correlated with short-term progression (HR = 2.729; p = 0.009) and poor survival (HR = 4.581; p = 0.004) of the patients; which was confirmed by Kryukov et al. cohort (HR = 1.878; p = 0.005) and MMRF CoMMpass study (HR = 1.414; p = 0.039) validation cohorts. Moreover, multivariate miR-25-fitted models contributed to superior risk-stratification and clinical benefit in MM prognostication. Finally, elevated miR-25 circulating levels were correlated with poor survival of MM patients (HR = 5.435; p = 0.021), serving as a potent non-invasive molecular prognostic tool. CONCLUSIONS: Our study identified miR-25 overexpression as a powerful independent predictor of poor treatment outcome and post-treatment progression, aiding towards modern non-invasive disease prognosis and personalized treatment decisions.

Characteristics of Human and Microbiome RNA Profiles in Saliva.

Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. By comparing the expression profiles of total RNA from these fractions, we found that MV was most enriched in microbiome RNA (76.2% of total reads on average), whereas EV-D was notably enriched in human RNA (70.3% of total reads on average). As for human RNA composition, CFS and EV-D were both enriched in snoRNA and tRNA compared with the two EV fractions (EXO and MV, P < 0.05). Interestingly, EXO and MV had highly correlated expression profiles for various noncoding RNAs such as miRNA, tRNA, and yRNA. Our study revealed unique characteristics of circulating RNAs in various saliva fractions, which provides a guideline on preparing saliva samples to study specific RNA biomarkers of interest.

Circadian Rhythm Does Not Affect the miRNA Cargo of Bovine Raw Milk Extracellular Vesicles.

Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.

3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation.

Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs-the canonical and a 3'isomiR variant (3' G addition)-which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests.

Comparative Analysis of microRNAs that Stratify in vitro Mammary stem and Progenitor Activity Reveals Functionality of Human miR-92b-3p.

Mammary stem/progenitor cells are fundamental for mammary gland development and function. However, much remains to be elucidated regarding their function in mammals beyond the traditionally studied rodents, human, and to a lesser extent, ruminants. Due to the growing appreciation for microRNAs (miRNAs) as regulators of stem cells and their progenitors, we compared miRNA expression in mammary stem/progenitor cells from mammals with varying mammary stem/progenitor activity in vitro, in order to identify miRNA candidates that regulate stem/progenitor self-renewal and function. Mammosphere-derived epithelial cells (MDECs), which are primary cell lines enriched in mammary stem and progenitor cells, were generated from six mammalian species (i.e., cow, human, pig, horse, dog, and rat) and small RNA sequencing was performed. We identified 9 miRNAs that were significantly differentially expressed in MDEC cultures with a low versus high mammary stem/progenitor activity. miR-92b-3p was selected for functional follow-up studies, as this miRNA is understudied in primary mammary cells but has well-described gene targets that are known to regulate mammary stem/progenitor activity. Altering the expression of miR-92b-3p in MDECs from species with low stem/progenitor activity (human and cow) and those with high stem/progenitor activity (dog and rat) via inhibition and overexpression, respectively, resulted in significantly decreased mammosphere formation of human MDECs, but showed no significant effects in cow, dog, or rat MDECs. This study is the first to perform small RNA sequencing in MDECs from various mammals and highlights that conserved miRNAs can have different functions in mammary stem/progenitor cells across species.

Determining extracellular vesicles properties and miRNA cargo variability in bovine milk from healthy cows and cows undergoing subclinical mastitis.

BACKGROUND: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. RESULTS: We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. CONCLUSIONS: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Myelination defects in the medial prefrontal cortex of Fkbp5 knockout mice.

The hypothalamic-pituitary-adrenal (HPA) axis plays a principal role in stress response regulation and has been implicated in the etiology of stress-related disorders. The HPA axis regulates the normal synthesis and release of glucocorticoids; dysregulation of the HPA axis causes abnormal responses to stress. FK506-binding protein 5 (FKBP5), a co-chaperone of heat shock protein 90 in the glucocorticoid receptor (GR) molecular complex, is a key GR sensitivity regulator. FKBP5 single nucleotide polymorphisms are associated with dysregulated HPA axis and increased risk of stress-related disorders, including posttraumatic stress disorder (PTSD) and depression. In this study, we profiled the microRNAs (miRNAs) in the medial prefrontal cortex of Fkbp5 knockout (Fkbp5-/- ) mice and identified the target genes of differentially expressed miRNAs using sequence-based miRNA target prediction. Gene ontology analysis revealed that the differentially expressed miRNAs were involved in nervous system development, regulation of cell migration, and intracellular signal transduction. The validation of the expression of predicted target genes using quantitative polymerase chain reaction revealed that the expression of axon development-related genes, specifically actin-binding LIM protein 1 (Ablim1), lemur tyrosine kinase 2 (Lmtk2), kinesin family member 5c (Kif5c), neurofascin (Nfasc), and ephrin type-A receptor 4 (Epha4), was significantly decreased, while that of brain-derived neurotrophic factor (Bdnf) was significantly increased in the brain of Fkbp5-/- mice. These results suggest that axonal development-related genes can serve as potential targets in future studies focused on understanding the pathophysiology of PTSD.

miRNA repertoires of cystic fibrosis ex vivo models highlight miR-181a and miR-101 that regulate WISP1 expression.

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic lung disease. Small non-coding RNAs are key regulators of gene expression and participate in various processes, which are dysregulated in CF; however, they remain poorly studied. Here, we determined the complete microRNAs (miRNAs) expression pattern in three CF ex vivo models. The miRNA profiles of air-liquid interface cultures of airway epithelia (bronchi, nasal cells, and nasal polyps) samples from patients with CF and non-CF controls were obtained by deep sequencing. Compared with non-CF controls, several miRNAs were deregulated in CF samples; for instance, miR-181a-5p and the miR-449 family were upregulated. Moreover, mature miRNAs often showed variations (i.e. isomiRs) relative to their reference sequence, such as miR-101, suggesting that miRNAs consist of heterogeneous repertoires of multiple isoforms with different effects on gene expression. Analysis of miR-181a-5p and miR-101-3p roles indicated that they regulate the expression of WISP1, a key component of cell proliferation/migration programs. We showed that miR-101 and miR-181a-5p participated in aberrant recapitulation of wound healing programs by controlling WISP1 mRNA and protein level. Our miRNA expression data bring new insights into CF physiopathology and define new potential therapeutic targets in CF. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

HIV-infection and cocaine use regulate semen extracellular vesicles proteome and miRNAome in a manner that mediates strategic monocyte haptotaxis governed by miR-128 network.

BACKGROUND: Extracellular vesicles (EVs) are regulators of cell-cell interactions and mediators of horizontal transfer of bioactive molecules between cells. EV-mediated cell-cell interactions play roles in physiological and pathophysiological processes, which maybe modulated by exposure to pathogens and cocaine use. However, the effect of pathogens and cocaine use on EV composition and function are not fully understood. RESULTS: Here, we used systems biology and multi-omics analysis to show that HIV infection (HIV +) and cocaine (COC) use (COC +) promote the release of semen-derived EVs (SEV) with dysregulated extracellular proteome (exProtein), miRNAome (exmiR), and exmiR networks. Integrating SEV proteome and miRNAome revealed a significant decrease in the enrichment of disease-associated, brain-enriched, and HIV-associated miR-128-3p (miR-128) in HIV + COC + SEV with a concomitant increase in miR-128 targets-PEAK1 and RND3/RhoE. Using two-dimensional-substrate single cell haptotaxis, we observed that in the presence of HIV + COC + SEV, contact guidance provided by the extracellular matrix (ECM, collagen type 1) network facilitated far-ranging haptotactic cues that guided monocytes over longer distances. Functionalizing SEV with a miR-128 mimic revealed that the strategic changes in monocyte haptotaxis are in large part the result of SEV-associated miR-128. CONCLUSIONS: We propose that compositionally and functionally distinct HIV + COC + and HIV-COC- SEVs and their exmiR networks may provide cells relevant but divergent haptotactic guidance in the absence of chemotactic cues, under both physiological and pathophysiological conditions.

miRNome Profiling and Functional Analysis Reveal Involvement of hsa-miR-1246 in Colon Adenoma-Carcinoma Transition by Targeting AXIN2 and CFTR.

Regulatory changes occurring early in colorectal cancer development remain poorly investigated. Since the majority of cases develop from polyps in the adenoma-carcinoma transition, a search of early molecular features, such as aberrations in miRNA expression occurring prior to cancer development, would enable identification of potentially causal, rather than consequential, candidates in the progression of polyp to cancer. In the current study, by employing small RNA-seq profiling of colon biopsy samples, we described differentially expressed miRNAs and their isoforms in the adenoma-carcinoma transition. Analysis of healthy-adenoma-carcinoma sequence in an independent validation group enabled us to identify early deregulated miRNAs including hsa-miR-1246 and hsa-miR-215-5p, the expressions of which are, respectively, gradually increasing and decreasing. Loss-of-function experiments revealed that inhibition of hsa-miR-1246 lead to reduced cell viability, colony formation, and migration rate, thereby indicating an oncogenic effect of this miRNA in vitro. Subsequent western blot and luciferase reporter assay provided evidence of hsa-miR-1246 being involved in the regulation of target AXIN2 and CFTR genes' expression. To conclude, the present study revealed possible involvement of hsa-miR-1246 in early colorectal cancer development and regulation of tumor suppressors AXIN2 and CFTR.

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.

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting.

BACKGROUND: The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRRconv and NRRss, respectively) was recorded for each bull. RESULTS: In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21-5p were significantly correlated to NRRss but not to NRRconv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRRss. CONCLUSIONS: A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire's suitability for the production of sex-sorted sperm.

Identification and molecular characterization of miRNAs and their target genes associated with seed development through small RNA sequencing in chickpea.

Multiple studies have attempted to dissect the molecular mechanism underlying seed development in chickpea (Cicer arietinum L.). These studies highlight the need to focus on the role of miRNAs in regulating storage protein accumulation in seeds. Therefore, a total of 8,856,691 short-read sequences were generated from a small RNA library of developing chickpea seeds and were analyzed using miRDeep-P to identify 74 known and 26 novel miRNA sequences. Known miRNAs were classified into 22 miRNA families with miRNA156 family being most abundant. Of the 26 putative novel miRNAs identified, only 22 could be experimentally validated using stem loop end point PCR. Differential expression analyses led to the identification of known as well as novel miRNAs that could regulate various stages of chickpea seed development. In silico target prediction revealed several important target genes and transcription factors like SPL, mediator of RNA Polymerase II transcription subunit 12, aspartic proteinase and NACs, which were further validated by real-time PCR analysis. A comparative expression analysis in chickpea genotypes with contrasting seed protein content revealed one known (Car-miR156h) and two novel miRNA (Car-novmiR7 and Car-novmiR23) candidates to be highly expressed in the LPC (low protein content) chickpea genotypes, targets of which are known to regulate seed storage protein accumulation. Therefore, this study provides a useful resource in the form of miRNA and their targets which can be further utilized to understand and manipulate various regulatory mechanisms involved in seed development with the overall aim of improving yield and nutrition attributes in chickpea.

Analysis of microRNA expression profiles in human bronchial epithelial cells infected by Chlamydia psittaci.

BACKGROUND: Chlamydia psittaci is a pathogen of birds that can cause zoonotic disease in mammals including pneumonia in humans. MicroRNAs (miRNAs) are a class of small non-coding RNA fragments with a length of about 22 nt, which play an important role in regulating gene expression after transcription. Chlamydia infection can cause changes in host cell miRNA expression, but the potential biological function of miRNAs in C. psittaci infection and pathogenesis is not well understood. METHODS: Small RNA sequencing (sRNA-Seq) technology was used to characterise miRNA expression in human bronchial epithelial (HBE) cells after C. psittaci infection, and differentially expressed miRNAs were identified. Candidate target genes for these miRNAs were then functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The sRNA-Seq results were partially validated by quantitative real time polymerase chain reaction (qRT-PCR) and miRNA-target networks were constructed using visualization software. RESULTS: We identified 151 differentially expressed miRNAs (46 known miRNAs and 105 novel miRNAs) in C. psittaci-infected HBE cells, of which 140 were upregulated and 11 were downregulated. Of these, 17 known miRNAs were significantly upregulated and two were downregulated using P < 0.05 and |log2FoldChange|>1.5 as threshold criteria. GO enrichment results showed that the predicted targets of these differentially expressed miRNAs were mainly involved in transcriptional regulation and ATP binding. KEGG pathway analysis suggested that the candidate target genes were involved in several important signaling pathways such as MAPK, ErbB, cGMP-PKG, cAMP, mTOR, GNRH, oxytocin, PI3K-Akt and AMPK, which are primarily related to biological processes such as transcription and signal transduction. The qRT-PCR results for miR-2116-3p, miR-3195, miR-663a, miR-10401-5p, miR-124-3p, miR-184, miR-744-5p and hsa-miR-514b-5p were consistent with the sRNA-Seq data. CONCLUSIONS: A large amount of miRNA expression profile data relating to C. psittaci infection was obtained, which provides a useful experimental and theoretical basis for further understanding the pathogenic mechanisms of C. psittaci infection.

Discovery and annotation of novel microRNAs in the porcine genome by using a semi-supervised transductive learning approach.

Despite the broad variety of available microRNA (miRNA) prediction tools, their application to the discovery and annotation of novel miRNA genes in domestic species is still limited. In this study we designed a comprehensive pipeline (eMIRNA) for miRNA identification in the yet poorly annotated porcine genome and demonstrated the usefulness of implementing a motif search positional refinement strategy for the accurate determination of precursor miRNA boundaries. The small RNA fraction from gluteus medius skeletal muscle of 48 Duroc gilts was sequenced and used for the prediction of novel miRNA loci. Additionally, we selected the human miRNA annotation for a homology-based search of porcine miRNAs with orthologous genes in the human genome. A total of 20 novel expressed miRNAs were identified in the porcine muscle transcriptome and 27 additional novel porcine miRNAs were also detected by homology-based search using the human miRNA annotation. The existence of three selected novel miRNAs (ssc-miR-483, ssc-miR484 and ssc-miR-200a) was further confirmed by reverse transcription quantitative real-time PCR analyses in the muscle and liver tissues of Göttingen minipigs. In summary, the eMIRNA pipeline presented in the current work allowed us to expand the catalogue of porcine miRNAs and showed better performance than other commonly used miRNA prediction approaches. More importantly, the flexibility of our pipeline makes possible its application in other yet poorly annotated non-model species.

Microprocessor-dependent processing of splice site overlapping microRNA exons does not result in changes in alternative splicing.

MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These splice site overlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in noncoding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons. Two SO-miRNAs, located in genes that code for basal membrane proteins, are known to inhibit proliferation in primary keratinocytes. These SO-miRNAs were up-regulated during differentiation and the host mRNAs were down-regulated, but again there was no change in inclusion ratio of the SO-miRNA exons. Interestingly, Drosha silencing increased nascent RNA density, on chromatin, downstream from SO-miRNA exons. Overall our data suggest a novel mechanism for regulating gene expression in which MPC-dependent cleavage of SO-miRNA exons could cause premature transcriptional termination of coding genes rather than affecting alternative splicing.

An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits.

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits.

Systematic assessment of commercially available low-input miRNA library preparation kits.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.