miRmap: Comprehensive prediction of microRNA target repression strength

MicroRNAs, or miRNAs, post-transcriptionally repress the expression of protein-coding genes. The human genome encodes over 1000 miRNA genes that collectively target the majority of messenger RNAs (mRNAs). Base pairing of the so-called miRNA ‘seed' region with mRNAs identifies many thousands of putative targets. Evaluating the strength of the resulting mRNA repression remains challenging, but is essential for a biologically informative ranking of potential miRNA targets. To address these challenges, predictors may use thermodynamic, evolutionary, probabilistic or sequence-based features. We developed an open-source software library, miRmap, which for the first time comprehensively covers all four approaches using 11 predictor features, 3 of which are novel. This allowed us to examine feature correlations and to compare their predictive power in an unbiased way using high-throughput experimental data from immunopurification, transcriptomics, proteomics and polysome fractionation experiments. Overall, target site accessibility appears to be the most predictive feature. Our novel feature based on PhyloP, which evaluates the significance of negative selection, is the best performing predictor in the evolutionary category. We combined all the features into an integrated model that almost doubles the predictive power of TargetScan. miRmap is freely available from http://cegg.unige.ch/mirma

miRmap web: comprehensive microRNA target prediction online

MicroRNAs (miRNAs) posttranscriptionally repress the expression of protein-coding genes. Based on the partial complementarity between miRNA and messenger RNA pairs with a mandatory so-called ‘seed' sequence, many thousands of potential targets can be identified. Our open-source software library, miRmap, ranks these potential targets with a biologically meaningful criterion, the repression strength. MiRmap combines thermodynamic, evolutionary, probabilistic and sequence-based features, which cover features from TargetScan, PITA, PACMIT and miRanda. Our miRmap web application offers a user-friendly and feature-rich resource for browsing precomputed miRNA target predictions for model organisms, as well as for predicting and ranking targets for user-submitted sequences. MiRmap web integrates sorting, filtering and exporting of results from multiple queries, as well as providing programmatic access, and is available at http://mirmap.ezlab.or

miROrtho: computational survey of microRNA genes

MicroRNAs (miRNAs) are short, non-protein coding RNAs that direct the widespread phenomenon of post-transcriptional regulation of metazoan genes. The mature ∼22-nt long RNA molecules are processed from genome-encoded stem-loop structured precursor genes. Hundreds of such genes have been experimentally validated in vertebrate genomes, yet their discovery remains challenging, and substantially higher numbers have been estimated. The miROrtho database (http://cegg.unige.ch/mirortho) presents the results of a comprehensive computational survey of miRNA gene candidates across the majority of sequenced metazoan genomes. We designed and applied a three-tier analysis pipeline: (i) an SVM-based ab initio screen for potent hairpins, plus homologs of known miRNAs, (ii) an orthology delineation procedure and (iii) an SVM-based classifier of the ortholog multiple sequence alignments. The web interface provides direct access to putative miRNA annotations, ortholog multiple alignments, RNA secondary structure conservation, and sequence data. The miROrtho data are conceptually complementary to the miRBase catalog of experimentally verified miRNA sequences, providing a consistent comparative genomics perspective as well as identifying many novel miRNA genes with strong evolutionary suppor

linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease

UPF1 regulates mRNA stability by sensing poorly translated coding sequences

Summary: Post-transcriptional mRNA regulation shapes gene expression, yet how cis-elements and mRNA translation interface to regulate mRNA stability is poorly understood. We find that the strength of translation initiation, upstream open reading frame (uORF) content, codon optimality, AU-rich elements, microRNA binding sites, and open reading frame (ORF) length function combinatorially to regulate mRNA stability. Machine-learning analysis identifies ORF length as the most important conserved feature regulating mRNA decay. We find that Upf1 binds poorly translated and untranslated ORFs, which are associated with a higher decay rate, including mRNAs with uORFs and those with exposed ORFs after stop codons. Our study emphasizes Upf1’s converging role in surveilling mRNAs with exposed ORFs that are poorly translated, such as mRNAs with long ORFs, ORF-like 3′ UTRs, and mRNAs containing uORFs. We propose that Upf1 regulation of poorly/untranslated ORFs provides a unifying mechanism of surveillance in regulating mRNA stability and homeostasis in an exon-junction complex (EJC)-independent nonsense-mediated decay (NMD) pathway that we term ORF-mediated decay (OMD)

Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation

Arginine, a semiessential amino acid implicated in diverse cellular processes, is a substrate for two arginases-Arg1 and Arg2-having different expression patterns and functions. Although appropriately regulated Arg1 expression is critical for immune responses, this has not been documented for Arg2. We show that Arg2 is the dominant enzyme in dendritic cells (DCs) and is repressed by microRNA-155 (miR155) during their maturation. miR155 is known to be strongly induced in various mouse and human DC subsets in response to diverse maturation signals, and miR155-deficient DCs exhibit an impaired ability to induce Ag-specific T cell responses. By means of expression profiling studies, we identified Arg2 mRNA as a novel miR155 target in mouse DCs. Abnormally elevated levels of Arg2 expression and activity were observed in activated miR155-deficient DCs. Conversely, overexpression of miR155 inhibited Arg2 expression. Bioinformatic and functional analyses confirmed that Arg2 mRNA is a direct target of miR155. Finally, in vitro and in vivo functional assays using DCs exhibiting deregulated Arg2 expression indicated that Arg2-mediated arginine depletion in the extracellular milieu impairs T cell proliferation. These results indicate that miR155-induced repression of Arg2 expression is critical for the ability of DCs to drive T cell activation by controlling arginine availability in the extracellular environment

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing.

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species

Integration of microRNA miR-122 in hepatic circadian gene expression

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBα as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparβ/δ and the peroxisome proliferator-activated receptor α (PPARα) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control