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The majority of human protein-coding genes are predicted to be targets of miRNA-mediated post-transcriptional regulation. The widespread influence of miRNAs is illustrated by their essential roles in all biological processes. Regulated miRNA expression is essential for maintaining cellular differentiation; therefore alterations in miRNA expression patterns are associated with several diseases, including various cancers. High-throughput sequencing technologies revealed low level expressing miRNA isoforms, termed isomiRs. IsomiRs may differ in sequence, length, target preference and expression patterns from their parental miRNA and can arise from differences in miRNA biosynthesis, RNA editing, or SNPs inherent to the miRNA gene. The association between isomiR expression and disease progression is largely unknown. Misregulated miRNA expression is thought to contribute to the formation and/or progression of cancer. However, due to the diversity of targeted transcripts, miRNAs can function as both tumor-suppressor genes and oncogenes as defined by cellular context. Despite this, miRNA profiling studies concluded that the differential expression of particular miRNAs in diseased tissue could aid the diagnosis and treatment of some cancers.

Background miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, the so-called 5’isomiRs exhibit a shifted 5’ end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. Results Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5’isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5’isomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. miRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5’isomiR-140-3p were highly expressed in patients’ tumors compared to normal breast tissue. In the current work, we present the functional characterization of 5’isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A, MDA-MB-468 and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa-miR-140-3p, overexpression of the 5’isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5’isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5’ismoiR-140-3p overexpression was found to cause a decrease in cell migration in the three cell lines. We identified three novel direct target genes of the 5’isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5’isomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. Conclusions In summary, this work presents evidence that there is functional synergy between the canonical hsa-miR-140-3p and the newly identified 5’isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2869-x) contains supplementary material, which is available to authorized users.

Background Massive parallel sequencing of transcriptomes, revealed the presence of many miRNAs and miRNAs variants named isomiRs with a potential role in several cellular processes through their interaction with a target mRNA. Many methods and tools have been recently devised to detect and quantify miRNAs from sequencing data. However, all of them are implemented on top of general purpose alignment methods, thus providing poorly accurate results and no information concerning isomiRs and conserved miRNA-mRNA interaction sites. Results To overcome these limitations we present a novel algorithm named isomiR-SEA, that is able to provide users with very accurate miRNAs expression levels and both isomiRs and miRNA-mRNA interaction sites precise classifications. Tags are mapped on the known miRNAs sequences thanks to a specialized alignment algorithm developed on top of biological evidence concerning miRNAs structure. Specifically, isomiR-SEA checks for miRNA seed presence in the input tags and evaluates, during all the alignment phases, the positions of the encountered mismatches, thus allowing to distinguish among the different isomiRs and conserved miRNA-mRNA interaction sites. Conclusions isomiR-SEA performances have been assessed on two public RNA-Seq datasets proving that the implemented algorithm is able to account for more reliable and accurate miRNAs expression levels with respect to those provided by two compared state of the art tools. Moreover, differently from the few methods currently available to perform isomiRs detection, the proposed algorithm implements the evaluation of isomiRs and conserved miRNA-mRNA interaction sites already in the first alignment phases, thus avoiding any additional filtering stages potentially responsible for the loss of useful information. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-0958-0) contains supplementary material, which is available to authorized users.

The expression of microRNAs (miRNAs) is essential for the proper development of the mammalian embryo. A maternal exposure to endocrine disrupting chemicals during preimplantation bears the potential for transgenerational inheritance of disease through the epigenetic perturbation of the developing embryo. A comprehensive assembly of embryo-specific miRNAs and respective isoforms (isomiR) is lacking to date. We aimed at revealing the sex-specific miRNA expression profile of single porcine blastocysts developing in gilts orally exposed to exogenous estradiol-17 β (E2). Therefore we analyzed the miRNA profile specifically focusing on isomiRs and potentially embryo-specific miRNAs. Deep sequencing of small RNA (small RNA-seq) result in the detection of miRNA sequences mapping to known and predicted porcine miRNAs as well as novel miRNAs highly conserved in human and cattle. A set of highly abundant miRNAs and a large number of rarely expressed miRNAs were identified by using a small RNA analysis pipeline, which was integrated into a novel Galaxy workflow specifically benefits incompletely annotated species. In particular, orthologue species information increased the total number of annotated miRNAs, while mapping to other non-coding RNAs avoided falsely annotated miRNAs. Neither the low nor the high dose of E2 treatment (10 and 1000 µE2/kg body weight daily, respectively) affected the miRNA profile in blastocysts despite the distinct differential mRNA expression and DNA methylation found in previous studies. The high number of generated sequence reads enabled a comprehensive analysis of the isomiR repertoire showing various templated and non-templated modifications. Furthermore, potentially blastocyst-specific miRNAs were identified. In pre-implantation embryos, numerous distinct isomiRs were discovered indicating a high complexity of miRNA expression. Neither the sex of the embryo nor a maternal E2 exposure affected the miRNA expression profile of developing porcine blastocysts. The adaptation to the continuous duration of the E2 treatment might explain the lack of an effect.

Tese de mestrado em Bioinformática e Biologia Computacional, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2017 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2018-04-05T14:48:28Z No. of bitstreams: 1 ulfc124148_tm_Ines_Viegas.pdf: 1876598 bytes, checksum: 1d4ad77fdfc5c1d1ba00f7816c394cbc (MD5) Made available in DSpace on 2018-04-05T14:48:36Z (GMT). No. of bitstreams: 1 ulfc124148_tm_Ines_Viegas.pdf: 1876598 bytes, checksum: 1d4ad77fdfc5c1d1ba00f7816c394cbc (MD5) Previous issue date: 2017

miR-34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR-34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR-34b and miR-449c by one supplemental uridine at their 5′-end, leading to a one-base shift in their seed region. Overexpression of canonical miR-34/449 or 5′-isomiR-34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5′-isomiR-34/449 may represent additional mechanisms by which miR-34/449 family finely controls several pathways to drive multiciliogenesis.

Bovine Leukemia Virus (BLV), a delta-retrovirus related to humanT-cell leukemia virus-1 (HTLV-1), is associated with the development of B-cell leukemia in experimentally-infected sheep. Using this outbred animal model of B-cell transformation, oncogenic modifications reflected in altered microRNA expression can be identified and compared as the disease progresses. We have analyzed the miRNome of transformed B-cells isolated from leukemic sheep. Using Taqman Low Density Array (TLDA) assays and High-Throughput (HT) sequencing of small RNA libraries, we identified differentially-expressed microRNAs associated with B-cell transformation. For miRBase database-matched sheep orthologs there was a good overall quantitative correlation between data generated with both techniques. Furthermore, deep sequencing identified variants of mature microRNA transcripts, indicating that isomir distribution might be of biological significance. Finally, HT sequencing revealed unknown candidate microRNAs which were confirmed both in silico using miRDeep and experimentally using stem-loop RT-QPCR methods. Target prediction tools (miRanda, targetscan) suggest that these microRNAs might target both cellular and viral mRNAs. Down-regulation of viral mRNAs might contribute to tumor-associated virus silencing and play a role in immune escape mechanisms. Ongoing work aims at the validation of bioinformatics predictions of microRNA targets. Altogether, this work should lead to a better understanding of the microRNA-mRNA regulation network associated with leukemia progression.

Background: Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules. Results: To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs. Conclusions: Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.