Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs

miRDeepFinder is a software package developed to identify and functionally analyze plant microRNAs (miRNAs) and their targets from small RNA datasets obtained from deep sequencing. The functions available in miRDeepFinder include pre-processing of raw data, identifying conserved miRNAs, mining and classifying novel miRNAs, miRNA expression profiling, predicting miRNA targets, and gene pathway and gene network analysis involving miRNAs. The fundamental design of miRDeepFinder is based on miRNA biogenesis, miRNA-mediated gene regulation and target recognition, such as perfect or near perfect hairpin structures, different read abundances of miRNA and miRNA*, and targeting patterns of plant miRNAs. To test the accuracy and robustness of miRDeepFinder, we analyzed a small RNA deep sequencing dataset of Arabidopsis thaliana published in the GEO database of NCBI. Our test retrieved 128 of 131 (97.7%) known miRNAs that have a more than 3 read count in Arabidopsis. Because many known miRNAs are not associated with miRNA*s in small RNA datasets, miRDeepFinder was also designed to recover miRNA candidates without the presence of miRNA*. To mine as many miRNAs as possible, miRDeepFinder allows users to compare mature miRNAs and their miRNA*s with other small RNA datasets from the same species. Cleaveland software package was also incorporated into miRDeepFinder for miRNA target identification using degradome sequencing analysis. Using this new computational tool, we identified 13 novel miRNA candidates with miRNA*s from Arabidopsis and validated 12 of them experimentally. Interestingly, of the 12 verified novel miRNAs, a miRNA named AC1 spans the exons of two genes (UTG71C4 and UGT71C3). Both the mature AC1 miRNA and its miRNA* were also found in four other small RNA datasets. We also developed a tool, ??miRNA primer designer?? to design primers for any type of miRNAs. miRDeepFinder provides a powerful tool for analyzing small RNA datasets from all species, with or without the availability of genome information. miRDeepFinder and miRNA primer designer are freely available at http://www.leonxie.com/DeepFinder.php and at http://www.leonxie.com/miRNAprimerDesigner.php, respectively. A program (called RefFinder: http://www.leonxie.com/referencegene.php) was also developed for assessing the reliable reference genes for gene expression analysis, including miRNAs.     

Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis

HEN1-dependent methylation of the 3'-terminal nucleotide is a crucial step in plant microRNA (miRNA) biogenesis. Here we report that several viral RNA silencing suppressors (P1/HC-Pro, p21 and p19) inhibit miRNA methylation. These suppressors have distinct effects on different miRNAs. We also show that miRNA* is methylated in vivo in a suppressor-sensitive manner, suggesting that the viral proteins interfere with miRNA/miRNA* duplexes. p19 and p21 bind both methylated and unmethylated miRNA/miRNA* duplexes in vivo. These findings suggest miRNA/miRNA* as the in vivo substrates for the HEN1 miRNA methyltransferase and raise intriguing possibilities regarding the cellular location of miRNA methylation.     

Implementation of a de novo genome-wide computational approach for updating Brachypodium miRNAs

Plant microRNAs (miRNAs) are single-stranded 20-22 nt small RNAs (sRNA) that are produced from their own genes. We have developed a de novo genome-wide approach for the computational identification of novel plant miRNAs based on the integration of the complete genome sequence with sRNA libraries. It comprises three modules - the clustering module identifies genomic regions that have two closely-located unidirectional sRNA clusters, the mirplan module explores the secondary structure of the genomic regions, and the duplex module predicts miRNA/miRNA* duplexes. We applied our approach to the Brachypodium genome and publicly available sRNA libraries and predicted 102 miRNAs. Our results extend the list of known miRNAs with 58 novel miRNAs and define the genomic loci of all predicted miRNAs. Because this approach considers specific features of plant miRNAs, it can be employed for the analysis of the genome and sRNA libraries generated for plant species to achieve systematic miRNA discovery.     

Characterization of microRNAs from sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.