Promoting mRNA Degradation Research Articles

MiR-589 promotes gastric cancer aggressiveness by a LIFR-PI3K/AKT-c-Jun regulatory feedback loop

BackgroundAs novel biomarkers for various cancers, microRNAs negatively regulate genes expression via promoting mRNA degradation and suppressing mRNA translation. miR-589 has been reported to be deregulated in several human cancer types. However, its biological role has not been functionally characterized in gastric cancer. Here, we aim to investigate the biological effect of miR-589 on gastric cancer and to reveal the possible mechanism.MethodsReal-time PCR was performed to evaluate the expression of miR-589 in 34 paired normal and stomach tumor specimens, as well as gastric cell lines. Functional assays, such as wound healing, transwell assays and in vivo assays, were used to detect the biological effect of miR-589 and LIFR. We determined the role of miR-589 in gastric cancer tumorigenesis in vivo using xenograft nude models. Dual-luciferase report assays and Chromatin immunoprecipitation (ChIP) assay were performed for target evaluation, and the relationships were confirmed by western blot assay.ResultMiR-589 expression was significantly higher in tumor tissues and gastric cancer cells than those in matched normal tissues and gastric epithelial cells, respectively. Clinically, overexpression of miR-589 is associated with tumor metastasis, invasion and poor prognosis of GC patients. Gain- and loss-of function experiments showed that miR-589 promoted cell migration, metastasis and invasion in vitro and lung metastasis in vivo. Mechanistically, we found that miR-589 directly targeted LIFR to activate PI3K/AKT/c-Jun signaling. Meanwhile, c-Jun bound to the promoter region of miR-589 and activated its transcription. Thus miR-589 regulated its expression in a feedback loop that promoted cell migration, metastasis and invasion.ConclusionOur study identified miR-589, as an oncogene, markedly induced cell metastasis and invasion via an atypical miR-589-LIFR-PI3K/AKT-c-Jun feedback loop, which suggested miR-589 as a potential biomarker and/or therapeutic target for the gastric cancer management.

Establishment of genetically encoded biosensors for cytosolic boric acid in plant cells.

Boron (B) is an essential micronutrient for plants. To maintain B concentration in tissues at appropriate levels, plants use boric acid channels belonging to the NIP subfamily of aquaporins and BOR borate exporters. To regulate B transport, these transporters exhibit different cell-type specific expression, polar localization, and B-dependent post-transcriptional regulation. Here, we describe the development of genetically encoded biosensors for cytosolic boric acid to visualize the spatial distribution and temporal dynamics of B in plant tissues. The biosensors were designed based on the function of the NIP5;1 5'-untranslated region (UTR), which promotes mRNA degradation in response to an elevated cytosolic boric acid concentration. The signal intensities of the biosensor coupled with Venus fluorescent protein and a nuclear localization signal (uNIP5;1-Venus) showed negative correlation with intracellular B concentrations in cultured tobacco BY-2 cells. When expressed in Arabidopsis thaliana, uNIP5;1-Venus enabled the quantification of B distribution in roots at single-cell resolution. In mature roots, cytosolic B levels in stele were maintained under low B supply, while those in epidermal, cortical, and endodermal cells were influenced by external B concentrations. Another biosensor coupled with a luciferase protein fused to a destabilization PEST sequence (uNIP5;1-Luc) was used to visualize changes in cytosolic boric acid concentrations. Thus, uNIP5;1-Venus/Luc enables visualization of B transport in various plant cells/tissues.

MiR-377-3p regulates adipogenic differentiation of human bone marrow mesenchymal stem cells by regulating LIFR.

MicroRNAs are members of the family of non-coding small RNAs that regulate gene expression either by inhibiting mRNA translation or by promoting mRNA degradation at the post-transcriptional level. They play an important role in the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into adipocytes. However, the role of microRNAs in this process remains to be poorly understood. Here, we observed that miR-377-3p expression was markedly decreased during adipogenic differentiation of hMSCs. Overexpression of miR-377-3p decreased adipocyte differentiation and downregulated the expression of adipogenic markers. Meanwhile, bioinformatics-based studies suggested that LIFR is a target of miR-377-3p. Further analysis confirmed that expression of LIFR present markedly increased during adipogenic differentiation of hMSCs. In addition, downregulation expression of LIFR significantly inhibited the process of adipocyte differentiation. To confirm the relation between miR-377-3p and LIFR, luciferase reporter assays were carried out. The results indicated that miR-377-3p bound directly to the 3'-untranslated region of LIFR. These data indicate that miR-377-3p suppressed adipogenesis of hMSCs by targeting LIFR, which provides novel insights into the molecular mechanism of miRNA-mediated cellular differentiation.

Ythdf2-mediated m6A mRNA clearance modulates neural development in mice

BackgroundN6-methyladenosine (m6A) modification in mRNAs was recently shown to be dynamically regulated, indicating a pivotal role in multiple developmental processes. Most recently, it was shown that the Mettl3-Mettl14 writer complex of this mark is required for the temporal control of cortical neurogenesis. The m6A reader protein Ythdf2 promotes mRNA degradation by recognizing m6A and recruiting the mRNA decay machinery.ResultsWe show that the conditional depletion of the m6A reader protein Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development. We demonstrate that neural stem/progenitor cell (NSPC) self-renewal and spatiotemporal generation of neurons and other cell types are severely impacted by the loss of Ythdf2 in embryonic neocortex. Combining in vivo and in vitro assays, we show that the proliferation and differentiation capabilities of NSPCs decrease significantly in Ythdf2−/− embryos. The Ythdf2−/− neurons are unable to produce normally functioning neurites, leading to failure in recovery upon reactive oxygen species stimulation. Consistently, expression of genes enriched in neural development pathways is significantly disturbed. Detailed analysis of the m6A-methylomes of Ythdf2−/− NSPCs identifies that the JAK-STAT cascade inhibitory genes contribute to neuroprotection and neurite outgrowths show increased expression and m6A enrichment. In agreement with the function of Ythdf2, delayed degradation of neuron differentiation-related m6A-containing mRNAs is seen in Ythdf2−/− NSPCs.ConclusionsWe show that the m6A reader protein Ythdf2 modulates neural development by promoting m6A-dependent degradation of neural development-related mRNA targets.

341 Structure Proteins of Hepatitis C Induce miR-494-3p and miR942-3p Expression

Hepatitis C virus (HCV) infection, estimated in 3% of the world’s population, remains a major risk factor for chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Based on the recognition of single-stranded viral RNA, TLR7 and TLR8 have been suggested to play important roles in antiviral immune responses to HCV. The miRNAs, endogenously expressed small noncoding RNAs, are known to promote mRNA degradation and block protein translation. In the present study, the specific candidate miRNAs that influence HCV protein-modulated TLR7/8 expression and subsequent signal modulation were investigated. miRNA microarray analysis was performed to screen for the miRNAs expression in HCV structure proteins (core, E1, and E2) stably transfected HepG 2 cells, and miRBase was used to predict the interaction between these miRNAs and TLR7/8 gene. miR-494-3p and miR942-3p were identified and validated. A total of 100 serum samples (30 samples from controls, 70 samples from HCV-infected patients) were collected. The levels of the two, miR-494-3p and miR942-3p, were then detected by probe-based stem-loop quantitative reverse-transcriptase PCR. We found that the expression of serum miR-494-3p and miR942-3p was distinctly increased in HCV patients compared with controls (mean ± SEM: 917.7 ± 192.2 vs 32.5 ± 14.2, P < .001 and 1615.0 ± 516.3 vs 8.0 ± 5.4, P < .001). In addition, interferon treatment significantly decreased miR942-3p expression in HCV patients (mean ± SEM: 67200 ± 55000 vs 263400 ± 195900, P = .03). The expression of miR-494-3p and miR942-3p in serum were significantly up-regulated in patients with HCV infection. However, the modulation mechanisms of miR494-3p and miR942-3p on TLR7/8 need further investigation.

Are miRNAs critical determinants in herpes simplex virus pathogenesis?

miRNAs are small noncoding RNA that play a crucial role in gene regulation by inhibiting translation or promoting mRNA degradation. Viruses themselves express miRNAs that can target either the host or viral mRNA transcriptome. Moreover, viral infection of cells causes a drastic change in host miRNAs. This complex interaction between the host and viruses often favors the virus to evade immune elimination and favors the establishment and maintenance of latency. In this review we discuss the function of both host and viral miRNAs in regulating herpes simplex virus pathogenesis and also discuss the prospect of using miRNAs as biomarkers and therapeutic tools.

MicroRNAs: a new avenue to understand, investigate and treat immunoglobulin A nephropathy?

IgA nephropathy (IgAN) is the most common cause of primary glomerulonephritis worldwide. Up to 30% of cases develop the progressive form of the disease, eventually requiring renal replacement therapy. Diagnosis and risk stratification relies on an invasive kidney biopsy and management options are limited, with recurrence following renal transplantation being common. Thus the quest to understand the pathophysiology of IgAN has been one of great importance. MicroRNAs (miRs) are short nucleotides that suppress gene expression by hybridizing to the 3′ untranslated region of messenger RNA (mRNAs), promoting mRNA degradation or disrupting translation. First discovered in 1993, miRs have since been implicated in a number of chronic conditions, including cancer, heart disease and kidney disease. The mounting interest in the field of miRs has led to fascinating developments in the field of nephrology, ranging from their roles as biomarkers for disease to the development of miR antagonists as avenues for treatment. The translational potential for miRs in IgAN is thus well grounded and may represent a paradigm shift in current approaches to the disease. This review aims to summarize the literature with regard to miRs and their roles in IgAN.

Altered Expression of MicroRNAs in Rheumatoid Arthritis.

Rheumatoid arthritis is one of the most common types of inflammatory joint diseases. Women, smokers, and people with positive family history are more susceptible to this disease. Diagnostic criteria include at least one swollen joint that has not been caused by other diseases. MicroRNAs are non-coding RNAs that are evolutionarily conserved and have a length of 18-25 nucleotides. MicroRNAs control gene expression at the post-transcriptional level via promoting mRNA degradation or translational repression. Recognition of alterations in microRNA status and their respective targets, may offer an opportunity to better identify the pathways that are involved in the etiopathogenesis of autoimmune diseases. It has been suggested that microRNAs may serve as potential biomarkers for both diagnosis and prognosis of autoimmune diseases. Here, we review the available evidence on the deregulations of microRNA expression in rheumatoid arthritis. More precisely, this review focuses on the microRNA involved in T cell regulation and gives perspectives on the use of this microRNA as biomarkers of diagnosis, prognosis, or intervention efficacy. J. Cell. Biochem. 119: 478-487, 2018. © 2017 Wiley Periodicals, Inc.

MicroRNA hsa-miR-370-3p suppresses the expression and induction of CYP2D6 by facilitating mRNA degradation

Cytochrome P450 2D6 (CYP2D6) participates in the metabolism of approximately 20–25% of prescribed drugs. Genetic polymorphisms influence the expression and/or activity of CYP2D6, and inter-individual differences in drug activation and elimination caused by CYP2D6 genetic variants were reported. However, little is known about the potential modulation of CYP2D6 expression by microRNAs (miRNAs). In the current study, by using in silico prediction of the stabilities of miRNA/mRNA complexes, we screened 38 miRNA candidates that may interact with the transcript of CYP2D6. An inverse correlation between the expression of miRNA hsa-miR-370-3p and the expression of CYP2D6 was observed in human liver tissue samples. Electrophoretic mobility shift assays confirmed that hsa-miR-370-3p was able to directly bind to its cognate target within the coding region of the CYP2D6 transcript. The transfection of hsa-miR-370-3p mimics into the HepG2CYP2D6 cell line, a genetically modified cell line that overexpresses exogenous CYP2D6, was able to suppress the expression of CYP2D6 significantly at both mRNA and protein levels. The transfection of hsa-miR-370-3p mimics was also able to inhibit endogenous mRNA expression and/or protein production of CYP2D6 in HepaRG cells. Furthermore, in HepaRG, HepG2, and Huh7 cells, dexamethasone-induced expression of CYP2D6 was inhibited by hsa-miR-370-3p mimics. To investigate whether the miRNA mediated suppression is caused by inhibiting protein translation or promoting mRNA degradation, an actinomycin D assay was used to measure the stability of CYP2D6 transcripts. The results indicated that hsa-miR-370-3p mimics facilitated significantly the degradation of CYP2D6 mRNA. In addition, proteomics analyses of proteins isolated from the miRNA/mRNA/protein complex suggested that a group of multifunctional proteins facilitated the interaction between hsa-miR-370-3p and CYP2D6, thereby promoting mRNA degradation.

Tis11 mediated mRNA decay promotes the reacquisition of Drosophila intestinal stem cell quiescence

Adult stem cell proliferation rates are precisely regulated to maintain long-term tissue homeostasis. Defects in the mechanisms controlling stem cell proliferation result in impaired regeneration and hyperproliferative diseases. Many stem cell populations increase proliferation in response to tissue damage and reacquire basal proliferation rates after tissue repair is completed. Although proliferative signals have been extensively studied, much less is known about the molecular mechanisms that restore stem cell quiescence. Here we show that Tis11, an Adenine-uridine Rich Element (ARE) binding protein that promotes mRNA degradation, is required to re-establish basal proliferation rates of adult Drosophila intestinal stem cells (ISC) after a regenerative episode. We find that Tis11 limits ISC proliferation specifically after proliferation has been stimulated in response to heat stress or infection, and show that Tis11 expression and activity are increased in ISCs during tissue repair. Based on stem cell transcriptome analysis and RNA immunoprecipitation, we propose that Tis11 activation represents an integral part of a negative feedback mechanism that limits the expression of key components of several signaling pathways that control ISC function and proliferation. Our results identify Tis11 mediated mRNA decay as an evolutionarily conserved mechanism of re-establishing basal proliferation rates of stem cells in regenerating tissues.

MicroRNAs, miR-23a-3p and miR-151-3p, Are Regulated in Dentate Gyrus Neuropil following Induction of Long-Term Potentiation In Vivo.

Translation of synaptic mRNA contributes to alterations in the proteome necessary to consolidate long-term potentiation (LTP), a model of memory processes. Yet, how this process is controlled is not fully resolved. MicroRNAs are non-coding RNAs that negatively regulate gene expression by suppressing translation or promoting mRNA degradation. As specific microRNAs are synaptically located, we hypothesized that they are ideally suited to couple synaptic activation, translational regulation, and LTP persistence. The aim of this study was to identify LTP-regulated microRNAs at or near synapses. Accordingly, LTP was induced unilaterally at perforant path-dentate gyrus synapses in awake adult Sprague-Dawley rats. Five hours later, dentate gyrus middle molecular layer neuropil, containing potentiated synapses, was laser-microdissected. MicroRNA expression profiling, using TaqMan Low Density MicroRNA Microarrays (n = 4), identified eight regulated microRNAs. Subsequent individual TaqMan assays confirmed upregulation of miR-23a-3p (1.30 ± 0.10; p = 0.015) and miR-151-3p (1.17 ± 0.19; p = 0.045) in a second cohort (n = 7). Interestingly, bioinformatic analysis indicated that miR-151-3p and miR-23a-3p regulate synaptic reorganisation and transcription, respectively. In summary, we have demonstrated for the first time that microRNAs are regulated in isolated neuropil following LTP induction in vivo, supporting the hypothesis that synaptic, LTP-responsive microRNAs contribute to LTP persistence via regulation of the synaptic proteome.

Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2

Osteoporosis is a serious health problem worldwide. MicroRNA is a post-transcriptional regulator of gene expression by either promoting mRNA degradation or interfering with mRNA translation of specific target genes. It plays a significant role in the pathogenesis of osteoporosis. Here, we first demonstrated that miR-106b (miR-106b-5p) negatively regulated osteogenic differentiation of mesenchymal stem cells in vitro. Then, we found that miR-106b expression increased in C57BL/6 mice with glucocorticoid-induced osteoporosis (GIOP), and that silencing of miR-106b signaling protected mice against GIOP through promoting bone formation and inhibiting bone resorption. At last, we showed that miR-106b inhibited osteoblastic differentiation and bone formation partly through directly targeting bone morphogenetic protein 2 (BMP2) both in vitro and in the GIOP model. Together, our findings have identified the role and mechanism of miR-106b in negatively regulating osteogenesis. Inhibition of miR-106b might be a potential new strategy for treating osteoporosis and bone defects.

Fatty acid synthase is a primary target of MiR-15a and MiR-16-1 in breast cancer.

Fatty acid synthase (FASN) is upregulated in breast cancer and correlates with poor prognosis. FASN contributes to mammary oncogenesis and serves as a bona fide target in cancer therapies. MicroRNAs inhibit gene expression through blocking mRNA translation or promoting mRNA degradation by targeting their 3′-UTRs. We identified four microRNAs in two microRNA clusters miR-15a-16-1 and miR-497-195 that share a common seed sequence to target the 3′-UTR of the FASN mRNA. In reporter assays, both of these microRNA clusters inhibited the expression of a reporter construct containing the FASN 3′-UTR. However, only ectopic miR-15a-16-1, but not miR-497-195, markedly reduced the levels of endogenous FASN in breast cancer cells. Both miR-15a and miR-16-1 contributes to inhibiting FASN expression and breast cancer cell proliferation. Consistently, a sponge construct consisting of eight repeats of the FASN 3′-UTR region targeted by these microRNAs could markedly increase endogenous FASN levels in mammary cells. When FASN expression was restored by ectopic expression in breast cancer cells, retarded cell proliferation caused by miR-15a-16-1 was partially rescued. In conclusion, we demonstrated that FASN expression is primarily downregulated by miR-15a and miR-16-1 in mammary cells and FASN is one of the major targets of these two tumor suppressive microRNAs.

Downregulated miR-486-5p acts as a tumor suppressor in esophageal squamous cell carcinoma.

microRNAs (miRNAs/miRs) are crucial regulators of gene expression at the post-translational level through promoting mRNA degradation or the repression of translation of target genes. miRs have been confirmed to serve a dominant role in tumor biology. miR-486-5p has been ascertained to be involved in non-small-cell lung cancer, breast cancer and hepatocellular carcinoma; however, the expression and function of miR-486-5p in esophageal squamous cell carcinoma (ESCC) has yet to be elucidated. The present study aimed to analyze the expression levels of miR-486-5p in ESCC tissues and paired normal adjacent tissues, and determine the effects of miR-486-5p on esophageal cancer cells using MTT, wound scratch and apoptosis assays. The current results showed that miR-486-5p was significantly downregulated in ESCC specimens. Ectopic expression of miR-486-5p by synthetic mimics reduced cell proliferation and migration and induced increased cell apoptosis. The results indicated miR-486-5p may function as a tumor suppressor in ESCC. The present study demonstrated that miR-486-5p was downregulated in ESCC and served a anti-oncogene role in ESCC via affecting cellular migration.

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-016-4043-6) contains supplementary material, which is available to authorized users.

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA cross-linking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.

Allogeneic skin transplantation triggers unique miRNA signature profiles in CD4+ T cells with potential therapeutic implications.

Abstract Solid organ transplantation is a life-saving last resort for many people undergoing end stage organ failure. Currently, immunosuppressive drugs are employed to combat rejection; however, it is at the expense of the host’s immune system, often exposing the patient to a host of maladies, with opportunistic infection and malignancy being the most dire. Thus, it is imperative to find novel ways to induce tolerance in transplant recipients to allografts while maintaining homeostatic immune responses. MicroRNAs (miRNAs) are a class of evolutionarily conserved small non-coding RNAs that regulate gene expression by blocking translation or promoting mRNA degradation. Using a murine model of skin transplantation, microarray analysis was used to evaluate miRNA expression profiles in draining CD4+ T cells from allogenic (allo) and syngenic (syn) grafts. We identified several distinct miRNAs (e.g. miR-27a-5p, 15a-5p and several members of the 466 family) that were increased in response to alloantigen. Using Ingenuity Pathway Analysis software, these miRNAs were found to target several genes in the SMAD and TGFβ pathway that control the differentiation into immuno-suppressive T regulatory (Treg) cells. The current study suggests that targeting miRNA may constitute a novel therapeutic modality to induce Treg generation and potentially program the host’s own cells to restore immunological tolerance, while safely prolonging the health of the transplanted organ.

Identification of the microRNA transcriptome during the early phases of mammalian fracture repair

Fracture repair is a complex process that involves multiple biological processes requiring spatiotemporal expression of thousands of genes. The molecular regulation of this process is not completely understood. MicroRNAs (miRNAs) regulate gene expression by promoting mRNA degradation or blocking translation. To identify miRNAs expressed during fracture repair, we generated murine bone fractures and isolated miRNA-enriched RNA from intact and post-fracture day (PFD) 1, 3, 5, 7, 11, and 14 femurs. RNA samples were individually hybridized to mouse miRNA microarrays. Results indicated that 959 (51%) miRNAs were absent while 922 (49%) displayed expression in at least one sample. Of the 922 miRNAs, 306 (33.2%) and 374 (40.6%) were up- and down-regulated, respectively, in the calluses in comparison to intact bone. Additionally, 20 (2.2%) miRNAs displayed combined up- and down-regulated expression within the time course and the remaining 222 (24%) miRNAs did not exhibit any changes between calluses and intact bone. Quantitative-PCR validated the expression of several miRNAs. Further, we identified 2048 and 4782 target genes that were unique to the up- and down-regulated miRNAs, respectively. Gene ontology and pathway enrichment analyses indicated relevant biological processes. These data provide the first complete analysis of the miRNA transcriptome during the early phases of fracture repair.

MiRComb: An R Package to Analyse miRNA-mRNA Interactions. Examples across Five Digestive Cancers.

MicroRNAs (miRNAs) are small RNAs that regulate the expression of target mRNAs by specific binding on the mRNA 3'UTR and promoting mRNA degradation in the majority of cases. It is often of interest to know the specific targets of a miRNA in order to study them in a particular disease context. In that sense, some databases have been designed to predict potential miRNA-mRNA interactions based on hybridization sequences. However, one of the main limitations is that these databases have too many false positives and do not take into account disease-specific interactions. We have developed an R package (miRComb) able to combine miRNA and mRNA expression data with hybridization information, in order to find potential miRNA-mRNA targets that are more reliable to occur in a specific physiological or disease context. This article summarizes the pipeline and the main outputs of this package by using as example TCGA data from five gastrointestinal cancers (colon cancer, rectal cancer, liver cancer, stomach cancer and esophageal cancer). The obtained results can be used to develop a huge number of testable hypotheses by other authors. Globally, we show that the miRComb package is a useful tool to deal with miRNA and mRNA expression data, that helps to filter the high amount of miRNA-mRNA interactions obtained from the pre-existing miRNA target prediction databases and it presents the results in a standardised way (pdf report). Moreover, an integrative analysis of the miRComb miRNA-mRNA interactions from the five digestive cancers is presented. Therefore, miRComb is a very useful tool to start understanding miRNA gene regulation in a specific context. The package can be downloaded in http://mircomb.sourceforge.net.

Uridylation and PABP Cooperate to Repair mRNA Deadenylated Ends in Arabidopsis.

Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis. Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis. Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A) tails. In vivo and in vitro data indicate that Poly(A) Binding Protein (PABP) binds to uridylated oligo(A) tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism.