CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets

Link to original article

Summary: MicroRNAs (miRNAs) are ~20- to 22-nt long endogenous RNA sequences that play a critical role in the regulation of gene expression in eukaryotic genomes. Confident identification of miRNA targets is vital to understand their functions.

Currently available computational algorithms for miRNA target prediction have diverse degrees of sensitivity and specificity and as a consequence each predicted target generally requires experimental confirmation. miRNAs and other small RNAs that direct endonucleolytic cleavage of target mRNAs produce diagnostic uncapped, polyadenylated mRNA fragments.

Degradome sequencing [also known as PARE (parallel analysis of RNA ends) and GMUCT (genome-wide mapping of uncapped transcripts)] samples the 5'-ends of uncapped mRNAs and can be used to discover in vivo miRNA targets independent of computational predictions.

Here, we describe a generalizable computational pipeline, CleaveLand, for the detection of cleaved miRNA targets from degradome data. CleaveLand takes as input degradome sequences, small RNAs and an mRNA database and outputs small RNA targets. CleaveLand can thus be applied to degradome data from any species provided a set of mRNA transcripts and a set of query miRNAs or other small RNAs are available.

Availability: The code and documentation for CleaveLand is freely available under a GNU license at http://www.bio.psu.edu/people/faculty/Axtell/AxtellLab/Software.html

Contact: mja18@psu.edu

(Via Bioinformatics - recent issues.)

Conservation and implications of eukaryote transcriptional regulatory regions across multiple species

Link to original article

Background: Increasing evidence shows that whole genomes of eukaryotes are almost entirely transcribed into both protein coding genes and an enormous number of non-protein-coding RNAs (ncRNAs). Therefore, revealing the underlying regulatory mechanisms of transcripts becomes imperative. However, for a complete understanding of transcriptional regulatory mechanisms, we need to identify the regions in which they are found. We will call these transcriptional regulation regions, or TRRs, which can be considered functional regions containing a cluster of regulatory elements that cooperatively recruit transcriptional factors for binding and then regulating the expression of transcripts.

Results: We constructed a hierarchical stochastic language (HSL) model for the identification of core TRRs in yeast based on regulatory cooperation among TRR elements. The HSL model trained based on yeast achieved comparable accuracy in predicting TRRs in other species, e.g., fruit fly, human, and rice, thus demonstrating the conservation of TRRs across species. The HSL model was also used to identify the TRRs of genes, such as p53 or OsALYL1, as well as microRNAs. In addition, the ENCODE regions were examined by HSL, and TRRs were found to pervasively locate in the genomes.

Conclusions: Our findings indicate that 1) the HSL model can be used to accurately predict core TRRs of transcripts across species and 2) identified core TRRs by HSL are proper candidates for the further scrutiny of specific regulatory elements and mechanisms. Meanwhile, the regulatory activity taking place in the abundant numbers of ncRNAs might account for the ubiquitous presence of TRRs across the genome. In addition, we also found that the TRRs of protein coding genes and ncRNAs are similar in structure, with the latter being more conserved than the former.

(Via BMC Genomics - Latest articles.)

MicroRNA target prediction by expression analysis of host genes

Link to original article

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by inducing RNA cleavage or translational inhibition. Most human miRNAs are intragenic and are transcribed as part of their hosting transcription units.

We hypothesized that the expression profiles of miRNA host genes and of their targets are inversely correlated and devised a novel procedure, HOCTAR (Host gene Oppositely Correlated TARgets), which ranks predicted miRNA target genes based on their anti-correlated expression behaviour relative to their respective miRNA host genes. HOCTAR is the first tool for systematic miRNA target prediction that utilizes the same set of microarray experiments to monitor the expression of both miRNAs (through their host genes) and candidate targets.

We applied the procedure to 178 human intragenic miRNAs and found that it performs better than currently available prediction softwares in pinpointing previously validated miRNA targets. The high-scoring HOCTAR predicted targets were enriched in Gene Ontology categories, which were consistent with previously published data, as in the case of miR-106b and miR-93. By means of overexpression and loss-of-function assays, we also demonstrated that HOCTAR is efficient in predicting novel miRNA targets and we identified, by microarray and qRT-PCR procedures, 34 and 28 novel targets for miR-26b and miR-98, respectively.

Overall, we believe that the use of HOCTAR significantly reduces the number of candidate miRNA targets to be tested compared to the procedures based solely on target sequence recognition. Finally, our data further confirm that miRNAs have a significant impact on the mRNA levels of most of their targets.

(Via GR-in-Advance.)

Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect

Link to original article

Short interfering RNA (siRNA) may down-regulate many unintended genes whose transcripts possess complementarity to the siRNA seed region, which contains 7 nt. The capability of siRNA to induce this off-target effect was highly correlated with the calculated melting temperature or standard free-energy change for formation of protein-free seed duplex, indicating that thermodynamic stability of seed duplex formed between the seed and target is one of the major factor in determining the degree of off-target effects.

Furthermore, unlike intended gene silencing (RNA interference), off-target effect was completely abolished by introduction of a G:U pair into the seed duplex, and this loss in activity was completely recovered by a second mutation regenerating Watson-Crick pairing, indicating that seed duplex Watson-Crick pairing is also essential for off-target gene silencing. The off-target effect was more sensitive to siRNA concentration compared to intended gene silencing, which requires a near perfect sequence match between the siRNA guide strand and target mRNA.

(Via Nucleic Acids Research - current issue.)

Protein kinase C epsilon is important for migration of neuroblastoma cells

Link to original article

Background: Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility.

Methods: PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot.

Results: Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Go6976 inhibited migration while an inhibitor of PKC isoforms did not have an effect. Downregulation of PKC epsilon, but not of PKC alpha or PKC delta, with siRNA led to a suppression of both basal and TPA-stimulated migration.

Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKC epsilon. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS.

Conclusions: PKC epsilon is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKC epsilon but they may be involved in TPA-mediated migration.

(Via BioMed Central - Latest articles.)

Genome-Wide siRNA-Based Functional Genomics of Pigmentation Identifies Novel Genes and Pathways That Impact Melanogenesis in Human Cells

Link to original article

by Anand K. Ganesan, Hsiang Ho, Brian Bodemann, Sean Petersen, Jayavani Aruri, Shiney Koshy, Zachary Richardson, Lu Q. Le, Tatiana Krasieva, Michael G. Roth, Pat Farmer, Michael A. White

Author Summary

Aberrant pigment regulation correlates with skin disorders, opthalmologic disorders, and neurologic disorders. While extensive studies have identified regulators of mouse coat color, the regulation of human skin phenotypic variation is less well understood. To give a broader picture of the molecular regulators of melanogenesis in human cells, we used a genome-wide siRNA functional genomics approach to identify 92 novel regulators of melanin production in heavily pigmented MNT-1 melanoma cells.

Our screen identified several genes that converge to regulate tyrosinase, the rate-limiting step in pigment production, in both MNT-1 cells and primary melanocytes. Some of the identified genes were selectively active in different genetic backgrounds, suggesting that they may regulate human phenotypic variation. Small molecule inhibition of a family of novel pigment regulators was sufficient to impair pigment production in melanocytes.

Additionally, our screen identified molecular machinery known to support autophagosome biosynthesis as putative regulators of melanogenesis. In vitro co-localization studies and autophagy-deficient mice provided evidence that normal melanogenesis requires the same molecular machinery used by the autophagy pathway.

Taken together, these results illustrate the utility of genome wide siRNA screening approaches for identifying genes, novel pharmacologic agents, and pathways that regulate differentiated cellular phenotypes.

(Via PLoS Genetics: Archived Table of Contents.)

Modular control of endothelial sheet migration

Link to original article

Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood.

Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell-cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space.

In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell-cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate.

Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell-cell coordination.

(Via Genes & Development current issue.)

Most mammalian mRNAs are conserved targets of microRNAs

Link to original article

MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2-7), particularly those in 3' untranslated regions (3'UTRs), are preferentially conserved. Here we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3'UTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites.

The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the 'offset 6mer,' to be detected. In total, >45,000 miRNA target sites within human 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3' end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (P_CT) correlating with experimental measurements of repression.

Our expanded set of target predictions (including conserved 3'-compensatory sites), are available at the TargetScan website, which displays the P_CT for each site and each predicted target.

(Via GR-in-Advance.)

Regulation of neural macroRNAs by the transcriptional repressor REST

Link to original article:

The essential transcriptional repressor REST (repressor element 1-silencing transcription factor) plays central roles in development and human disease by regulating a large cohort of neural genes. These have conventionally fallen into the class of known, protein-coding genes; recently, however, several noncoding microRNA genes were identified as REST targets.

Given the widespread transcription of messenger RNA-like, noncoding RNAs ('macroRNAs'), some of which are functional and implicated in disease in mammalian genomes, we sought to determine whether this class of noncoding RNAs can also be regulated by REST. By applying a new, unbiased target gene annotation pipeline to computationally discovered REST binding sites, we find that 23% of mammalian REST genomic binding sites are within 10 kb of a macroRNA gene. These putative target genes were overlooked by previous studies.

Focusing on a set of 18 candidate macroRNA targets from mouse, we experimentally demonstrate that two are regulated by REST in neural stem cells. Flanking protein-coding genes are, at most, weakly repressed, suggesting specific targeting of the macroRNAs by REST. Similar to the majority of known REST target genes, both of these macroRNAs are induced during nervous system development and have neurally restricted expression profiles in adult mouse.

We observe a similar phenomenon in human: the DiGeorge syndrome-associated noncoding RNA, DGCR5, is repressed by REST through a proximal upstream binding site. Therefore neural macroRNAs represent an additional component of the REST regulatory network. These macroRNAs are new candidates for understanding the role of REST in neuronal development, neurodegeneration, and cancer.

(Via In Advance.)

Position dependent mismatch discrimination on DNA microarrays - experiments and model

Link to original article

Background: The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.

Results: We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results.

Conclusions: Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.

(Via BMC Bioinformatics - Latest articles.)

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts

Link to original article:

Authors: Thomas Thum, Carina Gross, Jan Fiedler, Thomas Fischer, Stephan Kissler, Markus Bussen, Paolo Galuppo, Steffen Just, Wolfgang Rottbauer, Stefan Frantz, Mirco Castoldi, Jürgen Soutschek, Victor Koteliansky, Andreas Rosenwald, M. Albert Basson, Jonathan D. Licht, John T. R. Pena, Sara H. Rouhanifard, Martina U. Muckenthaler, Thomas Tuschl, Gail R. Martin, Johann Bauersachs & Stefan Engelhardt

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear.

Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK-MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK-MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK-MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction.

These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

(Via Nature.)

High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA

Link to original article

MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods.

Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step.

The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.

(Via Nucleic Acids Research - current issue.)

Annotation of Mammalian Primary microRNAs

Link to original article:

Background: MicroRNAs (miRNAs) are important regulators of gene expression and have been implicated in development, differentiation and pathogenesis. Hundreds of miRNAs have been discovered in mammalian genomes. Approximately 50% of mammalian miRNAs are expressed from introns of protein-coding genes; the primary transcript (pri-miRNA) is therefore assumed to be the host transcript. However, very little is known about the structure of pri-miRNAs expressed from intergenic regions. Here we annotate transcript boundaries of miRNAs in human, mouse and rat genomes using various transcription features. The 5' end of the pri-miRNA is predicted from transcription start sites, CpG islands and 5' CAGE tags mapped in the upstream flanking region surrounding the precursor miRNA (pre-miRNA). The 3' end of the pri-miRNA is predicted based on the mapping of polyA signals, and supported by cDNA/EST and ditags data. The predicted pri-miRNAs are also analyzed for promoter and insulator-associated regulatory regions.

Results: We define sets of conserved and non-conserved human, mouse and rat pre-miRNAs using bidirectional BLAST and synteny analysis. Transcription features in their flanking regions are used to demarcate the 5' and 3' boundaries of the pri-miRNAs. The lengths and boundaries of primary transcripts are highly conserved between orthologous miRNAs. A significant fraction of pri-miRNAs have lengths between 1 and 10kb, with very few introns. We annotate a total of 59 pri-miRNA structures, which include 82 pre-miRNAs. 36 pri-miRNAs are conserved in all 3 species. In total, 18 of the confidently annotated transcripts express more than one pre-miRNA. The upstream regions of 54% of the predicted pri-miRNAs are found to be associated with promoter and insulator regulatory sequences.

Conclusions: Little is known about the primary transcripts of intergenic miRNAs. Using comparative data, we are able to identify the boundaries of a significant proportion of human, mouse and rat pri-miRNAs. We confidently predict the transcripts including a total of 77, 58 and 47 human, mouse and rat pre-miRNAs respectively. Our computational annotations provide a basis for subsequent experimental validation of predicted pri-miRNAs.

(Via BioMed Central - Latest articles.)

Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression

Link to original article:

"We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA.

Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression.

The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF."

(Via Proceedings of the National Academy of Sciences recent issues.)

[ARTICLE] Deadenylation is a widespread effect of miRNA regulation

Link to original article:

miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. In animal cells, degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion.

To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1, or NOT1. We show that ~60% of AGO1 targets are regulated by CAF1 and/or NOT1, indicating that deadenylation is a widespread effect of miRNA regulation. However, neither a poly(A) tail nor mRNA circularization are required for silencing, because mRNAs whose 3' ends are generated by a self-cleaving ribozyme are also silenced in vivo.

We show further that miRNAs trigger mRNA degradation, even when binding by 40S ribosomal subunits is inhibited in cis. These results indicate that miRNAs promote mRNA decay by altering mRNP composition and/or conformation, rather than by directly interfering with the binding and function of ribosomal subunits.

(Via In Advance.)

[Research Papers] Chromatin structure analyses identify miRNA promoters

Link to original article:

Although microRNAs (miRNAs) are key regulators of gene expression in normal human physiology and disease, transcriptional regulation of miRNAs is poorly understood, because most miRNA promoters have not yet been characterized. We identified the proximal promoters of 175 human miRNAs by combining nucleosome mapping with chromatin signatures for promoters.

We observe that one-third of intronic miRNAs have transcription initiation regions independent from their host promoters and present a list of RNA polymerase II- and III-occupied miRNAs. Nucleosome mapping and linker sequence analyses in miRNA promoters permitted accurate prediction of transcription factors regulating miRNA expression, thus identifying nine miRNAs regulated by the MITF transcription factor/oncoprotein in melanoma cells.

Furthermore, DNA sequences encoding mature miRNAs were found to be preferentially occupied by positioned-nucleosomes, and the 3' end sites of known genes exhibited nucleosome depletion. The high-throughput identification of miRNA promoter and enhancer regulatory elements sheds light on evolution of miRNA transcription and permits rapid identification of transcriptional networks of miRNAs.

(Via Genes & Development current issue.)

Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-{kappa}B pathway

Link to original article:

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-kappa-B (NF-kappa-B). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen.

We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-kappa-B binding sites in the miR-155 promoter; both sites recruited NF-kappa-B complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-kappa-B sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-kappa-B binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation.

Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target.

(Via Nucleic Acids Research - current issue.)

Analysis of regulatory network topology reveals functionally distinct classes of microRNAs

Link to original article:

MicroRNAs (miRNAs) negatively regulate the expression of target genes at the post-transcriptional level. Little is known about the crosstalk between miRNAs and transcription factors (TFs). Here we provide data suggesting that the interaction patterns between TFs and miRNAs can influence the biological functions of miRNAs. From this global survey, we find that a regulated feedback loop, in which two TFs regulate each other and one miRNA regulates both of the factors, is the most significantly overrepresented network motif.

Mathematical modeling shows that the miRNA in this motif stabilizes the feedback loop to resist environmental perturbation, providing one mechanism to explain the robustness of developmental programs that is contributed by miRNAs. Furthermore, on the basis of a network motif profile analysis, we demonstrate the existence of two classes of miRNAs with distinct network topological properties.

The first class of miRNAs is regulated by a large number of TFs, whereas the second is regulated by only a few TFs. The differential expression level of the two classes of miRNAs in embryonic developmental stages versus adult tissues suggests that the two classes may have fundamentally different biological functions. Our results demonstrate that the TFs and miRNAs extensively interact with each other and the biological functions of miRNAs may be wired in the regulatory network topology.

(Via Nucleic Acids Research - current issue.)

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

Link to original article:

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.

(Via Nucleic Acids Research - current issue.)

Posttranscriptional Regulation of miRNAs Harboring Conserved Terminal Loops

Posttranscriptional Regulation of miRNAs Harboring Conserved Terminal Loops: "Gracjan Michlewski, Sonia Guil, Colin A. Semple, Javier F. Cáceres."

(Via Molecular Cell.)

Summary: We recently found that hnRNP A1, a protein implicated in many aspects of RNA processing, acts as an auxiliary factor for the Drosha-mediated processing of a microRNA precursor, pri-miR-18a. Here, we provide the mechanism by which hnRNP A1 regulates this event. We show that hnRNP A1 binds to the loop of pri-miR-18a and induces a relaxation at the stem, creating a more favorable cleavage site for Drosha. We found that approximately 14% of all pri-miRNAs have highly conserved loops, which we predict act as landing pads for trans-acting factors influencing miRNA processing. In agreement, we show that 2O-methyl oligonucleotides targeting conserved loops (LooptomiRs) abolish miRNA processing invitro. Furthermore, we present evidence to support an essential role of conserved loops for pri-miRNA processing. Altogether, these data suggest the existence of auxiliary factors for the processing of specific miRNAs, revealing an additional level of complexity for the regulation of miRNA biogenesis.

Embryonic stem cell–specific microRNAs regulate the G1-S transition and promote rapid proliferation

Link to original article: "

Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation

Nature Genetics 40, 1478 (2008). doi:10.1038/ng.250

Authors: Yangming Wang, Scott Baskerville, Archana Shenoy, Joshua E Babiarz, Lauren Baehner & Robert Blelloch

Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1-S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells. Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

"

(Via Nature Genetics.)

11p Microdeletion including WT1 but not PAX6, presenting with cataract, mental retardation, genital abnormalities and seizures: case report

Link to original article

"WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation) and Potocki-Shaffer syndrome are rare contiguous gene deletion syndromes caused by deletions of the 11p14-p12 chromosome region. We present a patient with mental retardation, unilateral cataract, bilateral ptosis, genital abnormalities, seizures and a dysmorphic face. Cytogenetic analysis showed a deletion on 11p that was further characterized using FISH and MLPA analyses. The deletion (11p13-p12) located in the area between the deletions associated with the WAGR and Potocki-Shaffer syndromes had a maximum size of 8.5 Mb and encompasses 44 genes. Deletion of WT1 explains the genital abnormalities observed. As PAX6 was intact the cataract observed cannot be explained by a deletion of this gene. Seizures have been described in Potocki-Shaffer syndrome while mental retardation has been described in both WAGR and Potocki-Shaffer syndrome. Characterization of this patient contributes further to elucidate the function of the genes in the 11p14-p12 chromosome region."

(Via BioMed Central - Latest articles.)

Sall2 is a novel p75NTR-interacting protein that links NGF signalling to cell cycle progression and neurite outgrowth

By screening a fetal brain two-hybrid library with the death domain of the p75 neurotrophin receptor (NTR), we identified the Sall2 transcription factor as a novel interacting protein. Sall2 is a unique member of the Sall gene family, which is believed to be a tumour suppressor. Here, we show that Sall2 contains a p75NTR interaction domain not found in other Sall proteins and that p75NTR/Sall2 complexes co-immunoprecipitate from brain lysates. NGF dissociates p75NTR/Sall2 complexes and activates TrkA, which has an obligate function in the nuclear translocation of Sall2. NGF also increases Sall2 expression and this is mediated by p75NTR, but may not require TrkA. Depletion of Sall2 from cells decreases the expression and activity of p21(WAF1/CIP1), as well as the ability of NGF to induce growth arrest and the development of neurites. Overexpression of Sall2 activates p21(WAF1/CIP1), induces growth arrest, and promotes neurite outgrowth independently of NGF. These data establish Sall2 as a link between NTRs and transcriptional events that regulate the growth and development of neuronal cells. Pubmed link

Genomic copy number determination in cancer cells from SNP microarrays based on quantitative genotyping corrected for aneuploidy

[METHODS] Microarrays are frequently used to profile genome-wide copy number (CN) aberrations. While generally robust for detecting CN variants in germline DNA, the methods used to derive CN from signal intensity values have been suboptimal when applied to cancer genomes. The complexity of genomic aberrations in cancer makes it more difficult to discriminate between signal and noise, and measuring CN as a discrete variable does not account for tumor heterogeneity. Furthermore, standard normalization approaches detect CN changes relative to the overall DNA content, which is often not diploid in cancer. We propose an algorithm that uses the degree of allelic imbalance as well as probe intensity, with a correction for aneuploidy, for a quantitative CN assessment and scoring of allelic ratios. This algorithm results in a more precise definition of CN and allelic aberration in the cancer genome, which is essential for translational efforts focused on using these tools for molecular diagnostics and for the discovery of therapeutic targets.

Link to original article

Chromosomal instability mediated by non-B DNA: Cruciform conformation and not DNA sequence is responsible for recurrent translocation in humans

Chromosomal aberrations have been thought to be random events. However, recent findings introduce a new paradigm in which certain DNA segments have the potential to adopt unusual conformations that lead to genomic instability and nonrandom chromosomal rearrangement. One of the best-studied examples is the palindromic AT-rich repeat (PATRR), which induces recurrent constitutional translocations in humans. Here, we established a plasmid-based model that promotes frequent intermolecular rearrangements between two PATRRs in HEK293 cells. In this model system, the proportion of PATRR plasmid that extrudes a cruciform structure correlates to the levels of rearrangement. Our data suggest that PATRR-mediated translocations are attributable to unusual DNA conformations that confer a common pathway for chromosomal rearrangements in humans.

Link to original article

Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites

Background: Gene rearrangements such as chromosomal translocations have been shown to contribute to cancer development. Human chromosomal fragile sites are regions of the genome especially prone to breakage, and have been implicated in various chromosome abnormalities found in cancer. However, there has been no comprehensive and quantitative examination of the location of fragile sites in relation to all chromosomal aberrations.

Results: Using up-to-date databases containing all cancer-specific recurrent translocations, we have examined 444 unique pairs of genes involved in these translocations to determine the correlation of translocation breakpoints and fragile sites in the gene pairs. We found that over half (52%) of translocation breakpoints in at least one gene of these gene pairs are mapped to fragile sites. Among these, we examined the DNA sequences within and flanking three randomly selected pairs of translocation-prone genes, and found that they exhibit characteristic features of fragile DNA, with frequent AT-rich flexibility islands and the potential of forming highly stable secondary structures.

Conclusions: Our study is the first to examine gene pairs involved in all recurrent chromosomal translocations observed in tumor cells, and to correlate the location of more than half of breakpoints to positions of known fragile sites. These results provide strong evidence to support a causative role for fragile sites in the generation of cancer-specific chromosomal rearrangements.

Link to original article

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis ...

Background: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones.

Methods: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays.

Results: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients.

Conclusions: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype–phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Link to original article

Unbalanced chromosome 1 abnormalities in four infants with Down syndrome and acute megakaryocytic leukemia

Background: Children with Down syndrome have an increased risk of childhood acute leukemia, especially acute megakaryoblastic leukemia (AMKL) also called acute myeloid leukemia (AML) type M7. Here four yet unreported infants with such malignancies are reported.

Results: An unbalanced translocation involving chromosome 1 was identified by GTG banding in all cases. These were characterized in more detail by molecular cytogenetic approaches and hints were found on a critical region in 1q31~32. Additional molecular analysis revealed in three of the four cases mutations in exon 2 of the GATA binding protein 1 (globin transcription factor 1), located in Xp11.23.

Conclusions: Our results corroborate that abnormalities of chromosome 1 are common in DS-associated AMKL. Whether this chromosomal region contains gene(s) involved in hematopoietic malignant transformation remains to be determined.

Link to original article