Target repression induced by endogenous microRNAs: large differences, small effects
Ontology highlight
ABSTRACT: MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity M-bM-^@M-^S that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets. Two replicates of S2-DRSC cells under normal conditions
Project description:MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
Project description:Schmitz2014 - RNA triplex formation
The model is parameterized using the
parameters for gene CCDC3 from Supplementary Table S1. The two
miRNAs which form the triplex together with CCDC3 are miR-551b and
miR-138.
This model is described in the article:
Cooperative gene regulation
by microRNA pairs and their identification using a
computational workflow.
Schmitz U, Lai X, Winter F,
Wolkenhauer O, Vera J, Gupta SK.
Nucleic Acids Res. 2014 Jul; 42(12):
7539-7552
Abstract:
MicroRNAs (miRNAs) are an integral part of gene regulation
at the post-transcriptional level. Recently, it has been shown
that pairs of miRNAs can repress the translation of a target
mRNA in a cooperative manner, which leads to an enhanced
effectiveness and specificity in target repression. However, it
remains unclear which miRNA pairs can synergize and which genes
are target of cooperative miRNA regulation. In this paper, we
present a computational workflow for the prediction and
analysis of cooperating miRNAs and their mutual target genes,
which we refer to as RNA triplexes. The workflow integrates
methods of miRNA target prediction; triplex structure analysis;
molecular dynamics simulations and mathematical modeling for a
reliable prediction of functional RNA triplexes and target
repression efficiency. In a case study we analyzed the human
genome and identified several thousand targets of cooperative
gene regulation. Our results suggest that miRNA cooperativity
is a frequent mechanism for an enhanced target repression by
pairs of miRNAs facilitating distinctive and fine-tuned target
gene expression patterns. Human RNA triplexes predicted and
characterized in this study are organized in a web resource at
www.sbi.uni-rostock.de/triplexrna/.
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Project description:Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2–8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3′ untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered. 293T cells were transiently transfected with artificial miRNAs or non-targeting control (Allstars siRNA, Qiagen). Three replicate transfections were performed for each miRNA or control. Total RNA was extracted 48 hours after transfection.
Project description:Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2–8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3′ untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered.
Project description:microRNA dysregulation is a common feature of cancer cells, but the complex roles of microRNAs in cancer are not fully elucidated. Here we used functional genomics to identify oncogenic microRNAs in non-small cell lung cancer and to evaluate their impact on response to EGFR targeting therapy. Our data demonstrate that microRNAs with an AAGUGC-motif in their seed-sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors. Global transcriptomics, proteomics and target prediction resulted in the identification of several tumor suppressors involved in the G1/S transition as targets of AAGUGC-microRNAs. The clinical implications of our findings were evaluated by analysis of public domain data supporting the link between this microRNA seed-family, their tumor suppressor targets and cancer cell proliferation. In conclusion we propose that AAGUGC-microRNAs are an integral part of an oncogenic signaling network, and that these findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.
Project description:microRNA dysregulation is a common feature of cancer cells, but the complex roles of microRNAs in cancer are not fully elucidated. Here we used functional genomics to identify oncogenic microRNAs in non-small cell lung cancer and to evaluate their impact on response to EGFR targeting therapy. Our data demonstrate that microRNAs with an AAGUGC-motif in their seed-sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors. Global transcriptomics, proteomics and target prediction resulted in the identification of several tumor suppressors involved in the G1/S transition as targets of AAGUGC-microRNAs. The clinical implications of our findings were evaluated by analysis of public domain data supporting the link between this microRNA seed-family, their tumor suppressor targets and cancer cell proliferation. In conclusion we propose that AAGUGC-microRNAs are an integral part of an oncogenic signaling network, and that these findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.
Project description:Many mammalian microRNAs are embedded within introns of protein-coding genes, yet their functional relationship with the host gene is poorly understood. Here we identify Mir483, as having opposing developmental and physiological roles to those of its host gene, the paternally-expressed Igf2. Mechanistically, Mir483 expression is dependent on Igf2 transcription and the epigenetic regulation of the Igf2/H19 imprinting control region. Mir483 deletion in vivo resulted in normal growth, but induced distinct molecular signatures. Transgenic Mir483 overexpression in utero caused fetal, but not placental, growth restriction and cardiovascular defects leading to fetal demise. Overexpression of Mir483 postnatally resulted in growth stunting through IGF1 repression, increased hepatic lipid production, and excessive adiposity. IGF1 infusion rescued the post-natal growth restriction. Our data highlight a novel functional antagonism between a growth-suppressor microRNA and its growth-promoting host gene. We suggest that Mir483 was evolutionary co-opted to provide exquisite control of IGF signalling activity.
Project description:Mouse embryonic stem cells can spontaneously differentiate and assemble into a spherical embryoid body (EB) during suspension culture. The initial study aims to identify the up-regulated or down-regulated microRNAs during the differentiation process of pluripotent stem cells. From the microRNA profiling, we will focus on the microRNAs associated with mesodermal and endothelial differentiation of embryonic or induced pluripotent stem cells. In this study, the EBs were collected at 0 and 10-day differentiation of ESCs (ATCC: CRL-1934). The miRNA transcripts listed in Sanger miRBase Release 19.0 was detected by microarray.
Project description:MicroRNA expression profiling of human microvascular endothelial cells (HMVECs) treated with either vascular endothelial growth factor (VEGF) only or in combination with the natural angiogenesis inhibitor pigment epithelial-derived factor (PEDF). Originally we were interested in the microRNA-mediated regulation of angiogenesis by the endogenous anti-angiogenic PEDF. To identify the microRNAs involved in PEDF signaling in activated endothelial cells, we compared the levels of microRNAs in non-treated microvascular endothelial cells, cells treated with VEGF, and cells treated with a combination of VEGF and PEDF. After treatment, total RNA content was isolated and sent for analysis to LC Sciences, LLC. They performed expression profiling and completed statistical analysis, based on which we confirmed the regulation of one of the microRNAs, mir-27b. In the following experiments, we identified the targets of mir-27b relevant for angiogenesis and confirmed our findings in zebrafish and mouse models. The manuscript describes the key role of mir-27b in determination of the endothelial tip cell fate and venous differentiation by regulating Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). Three-condition experiment: untreated (control) HMVECs vs. VEGF-treated HMVECs vs. PEDF/VEGF-treated HMVECs.
Project description:Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high-throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5' or 3' ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5' end of mature tRNA using qRT-PCR. Examination of 2 different small RNA expression profilings in 2 developmental stages of meristems.