microRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidence supports the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1), an epigenetic repressor of skeletal myogenesis in mouse. Genome-wide identification of potential down-stream targets of YY1 by combining computational prediction with expression profiling data reveals a large number of putative miRNA targets of YY1 during skeletal myoblasts differentiation into myotubes with muscle miRs ranking on top of the list. The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both C2C12 myogenic differentiation and injury-induced muscle regeneration. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. Together, these results identify a novel regulatory circuit required for skeletal myogenesis and reinforce the idea that regulatory circuitries involving miRNAs and TFs are prevalent mechanisms.

BACKGROUND Computational methods for microRNA target prediction are a fundamental step to understand the miRNA role in gene regulation, a key process in molecular biology. In this paper we present miREE, a novel microRNA target prediction tool. miREE is an ensemble of two parts entailing complementary but integrated roles in the prediction. The Ab-Initio module leverages upon a genetic algorithmic approach to generate a set of candidate sites on the basis of their microRNA-mRNA duplex stability properties. Then, a Support Vector Machine (SVM) learning module evaluates the impact of microRNA recognition elements on the target gene. As a result the prediction takes into account information regarding both miRNA-target structural stability and accessibility. RESULTS The proposed method significantly improves the state-of-the-art prediction tools in terms of accuracy with a better balance between specificity and sensitivity, as demonstrated by the experiments conducted on several large datasets across different species. miREE achieves this result by tackling two of the main challenges of current prediction tools:(1) The reduced number of false positives for the Ab-Initio part thanks to the integration of a machine learning module (2) the specificity of the machine learning part, obtained through an innovative technique for rich and representative negative records generation. The validation was conducted on experimental datasets where the miRNA:mRNA interactions had been obtained through (1) direct validation where even the binding site is provided, or through (2) indirect validation, based on gene expression variations obtained from high-throughput experiments where the specific interaction is not validated in detail and consequently the specific binding site is not provided. CONCLUSIONS The coupling of two parts: a sensitive Ab-Initio module and a selective machine learning part capable of recognizing the false positives, leads to an improved balance between sensitivity and specificity. miREE obtains a reasonable trade-off between filtering false positives and identifying targets. miREE tool is available online at http://didattica-online.polito.it/eda/miREE/

MicroRNAs (miRNAs) influence many biological processes, including cancer. They do so by posttranscriptionally repressing target mRNAs to which they have sequence complementarity. Although it has been postulated that miRNAs can regulate other miRNAs, this has never been shown experimentally to our knowledge. Here, we demonstrate that miR-107 negatively regulates the tumor suppressor miRNA let-7 via a direct interaction. miR-107 was found to be highly expressed in malignant tissue from patients with advanced breast cancer, and its expression was inversely correlated with let-7 expression in tumors and in cancer cell lines. Ectopic expression of miR-107 in human cancer cell lines led to destabilization of mature let-7, increased expression of let-7 targets, and increased malignant phenotypes. In contrast, depletion of endogenous miR-107 dramatically increased the stability of mature let-7 and led to downregulation of let-7 targets. Accordingly, miR-107 expression increased the tumorigenic and metastatic potential of a human breast cancer cell line in mice via inhibition of let-7 and upregulation of let-7 targets. By mutating individual sites within miR-107 and let-7, we found that miR-107 directly interacts with let-7 and that the internal loop of the let-7/miR-107 duplex is critical for repression of let-7 expression. Altogether, we have identified an oncogenic role for miR-107 and provide evidence of a transregulational interaction among miRNAs in human cancer development.

Bacterial sRNAs are a class of small regulatory RNAs involved in regulation of expression of a variety of genes. Most sRNAs act in trans via base-pairing with target mRNAs, leading to repression or activation of translation or mRNA degradation. To date, more than 1,000 sRNAs have been identified. However, direct targets have been identified for only approximately 50 of these sRNAs. Computational predictions can provide candidates for target validation, thereby increasing the speed of sRNA target identification. Although several methods have been developed, target prediction for bacterial sRNAs remains challenging. Here, we propose a novel method for sRNA target prediction, termed sTarPicker, which was based on a two-step model for hybridization between an sRNA and an mRNA target. This method first selects stable duplexes after screening all possible duplexes between the sRNA and the potential mRNA target. Next, hybridization between the sRNA and the target is extended to span the entire binding site. Finally, quantitative predictions are produced with an ensemble classifier generated using machine-learning methods. In calculations to determine the hybridization energies of seed regions and binding regions, both thermodynamic stability and site accessibility of the sRNAs and targets were considered. Comparisons with the existing methods showed that sTarPicker performed best in both performance of target prediction and accuracy of the predicted binding sites. sTarPicker can predict bacterial sRNA targets with higher efficiency and determine the exact locations of the interactions with a higher accuracy than competing programs. sTarPicker is available at http://ccb.bmi.ac.cn/starpicker/.

MicroRNAs (miRNAs) are short, non-coding sequences that control gene expression via translational regulation. Through interactions with the 3'-untranslated region of messenger RNA, miRNAs trigger translational repression and play a key role in developmental timing. Furthermore, many miRNA groups have now been shown to regulate various processes in tumorigenesis, including angiogenesis and metastasis. These links highlight the importance of microRNA research in further understanding cancer development. This review article summarizes the current state of microRNA research, with a focus on the roles of microRNAs in various cancer types. Up to date knowledge of the structure and biogenesis pathway of microRNA are also reviewed.

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

Many computational microRNA target prediction tools are focused on several key features, including complementarity to 5'seed of miRNAs and evolutionary conservation. While these features allow for successful target identification, not all miRNA target sites are conserved and adhere to canonical seed complementarity. Several studies have propagated the use of energy features of mRNA:miRNA duplexes as an alternative feature. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement. We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide sites of AGO protein interaction. This trend is confirmed on datasets that assay the effect of miRNAs on mRNA and protein expression changes, and a simple linear regression model leads to significant correlation of predicted versus observed expression change. Compared to 6-mer seed matches as baseline, application of our energy-based model leads to ∼3-5-fold enrichment on highly down-regulated targets, and allows for prediction of strictly imperfect targets with enrichment above baseline. In conclusion, our results indicate significant promise for energy-based miRNA target prediction that includes a broader range of targets without having to use conservation or impose stringent seed match rules.

MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis, as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize to perivascular α-SMA(-) stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA targets of both the heart and other organs.

MicroRNAs (miRNAs) are a class of non-coding regulatory RNAs of ~22 nucleotides in length. miRNAs regulate gene expression post-transcriptionally, primarily by associating with the 3' untranslated region (UTR) of their regulatory target mRNAs. Recent work has begun to reveal roles for miRNAs in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Many miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related disorders. We have previously reported that the expression of muscle-specific miR-1 and miR-133 is induced during skeletal muscle differentiation and miR-1 and miR-133 play central regulatory roles in myoblast proliferation and differentiation in vitro. In this study, we measured the expression of miRNAs in the skeletal muscle of mdx mice, an animal model for human muscular dystrophy. We also generated transgenic mice to overexpress miR-133a in skeletal muscle. We examined the expression of miRNAs in the skeletal muscle of mdx mice. We found that the expression of muscle miRNAs, including miR-1a, miR-133a and miR-206, was up-regulated in the skeletal muscle of mdx mice. In order to further investigate the function of miR-133a in skeletal muscle in vivo, we have created several independent transgenic founder lines. Surprisingly, skeletal muscle development and function appear to be unaffected in miR-133a transgenic mice. Our results indicate that miR-133a is dispensable for the normal development and function of skeletal muscle.

BACKGROUND Left ventricular (LV) reverse remodelling after valve replacement in aortic stenosis (AS) has been classically linked to the hydraulic performance of the replacement device, but myocardial status at the time of surgery has received little attention. OBJECTIVE To establish predictors of LV mass (LVM) regression 1 year after valve replacement in a surgical cohort of patients with AS based on preoperative clinical and echocardiographic parameters and the myocardial gene expression profile at surgery. METHODS Transcript levels of remodelling-related proteins and regulators were determined in LV intraoperative biopsies from 46 patients with AS by RT-PCR. Using multiple linear regression analysis, an equation was developed (adjusted R²=0.73; p<0.0001) that included positive [preoperative LVM, microRNA-133a, serum response factor (SRF, which is known to be a transactivator of miR-133) and age] and negative [body mass index (BMI), Wolf-Hirschhorn syndrome candidate-2 (WHSC2, which is a target for repression by miR-133a), β-myosin heavy chain, myosin light chain-2, diabetes mellitus, and male gender] independent predictors of LVM reduction. RESULTS Aortic valve area gain or the reduction in transvalvular gradient maintained no significant relationships with the dependent variable. Logistic regression analysis identified microRNA-133a as a significant positive predictor of LVM normalisation, whereas β-myosin heavy chain and BMI constituted negative predictors. CONCLUSIONS Hypertrophy regression 1 year after pressure overload release is related to the preoperative myocardial expression of remodelling-related genes, in conjunction with the patient's clinical background. In this scenario, miR-133 emerges as a key element of the reverse remodelling process. Postoperative improvement of valve haemodynamics does not predict the degree of hypertrophy regression or LVM normalisation. These results led us to reconsider the current reverse remodelling paradigm and (1) to include criteria of hypertrophy reversibility in the decision algorithm used to decide timing for the operation; and (2) to modify other prevailing factors (overweight, diabetes, etc) known to maintain LV hypertrophy.

MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

MicroRNAs (miRNAs) are genomically encoded small non-coding RNAs that regulate flow of genetic information by controlling translation or stability of mRNAs. Recent recognition that many miRNAs are expressed in a tissue-specific manner during development of organisms, from worms to humans, has revealed a novel mechanism by which the proteome is regulated during the dynamic events of cell-lineage decisions and morphogenesis. Advances in the understanding of miRNA biogenesis, target recognition and participation in regulatory networks demonstrate a role for miRNAs in lineage decisions of progenitor cells and organogenesis. Future discoveries in this area are likely to reveal developmental-regulation and disease mechanisms related to miRNAs.

A variety of immunosuppressive drugs are currently used in patients with allo-grafts or autoimmune diseases. Though the effects of rapamycin (RPM) and other immunosuppressant on the CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) were studied, their impact on Ag-specific Tregs during immune response was not well defined. In our studies, we adoptively transferred TCR-transgenic CD4(+)KJ1-26(+) T cells, CD4(+)KJ1-26(+)CD25(-) naïve T cells or CD4(+)KJ1-26(+)CD25(+) Tregs into syngeneic BALB/c mice. 24h later, we treated the recipients with OVA immunization and immunosuppressant including rapamycin (RPM), fingolimod (FTY720), cyclosporin A (CsA), mycophenolate mofetil (MMF), leflunomide (LEF), cyclophosphamide (Cy) or none, respectively. The levels and function of CD4(+)KJ1-26(+)CD25(+)Foxp3(+) Tregs in draining lymph nodes (dLNs) and spleens were determined at different time points. Significantly higher percentage and cell number of Ag-specific CD4(+)KJ1-26(+)CD25(+)Foxp3(+) Tregs were observed in OVA immunized mice treated with RPM or FTY720 compared with mice that received OVA immunization alone. Furthermore, RPM augmented the population of functional iTregs in dLNs and spleens whereas inhibited nTregs during immune response. In contrast to RPM and FTY720, MMF, LEF, CsA, and Cy markedly decreased the levels of Ag-specific CD4(+)KJ1-26(+)CD25(+)Foxp3(+) Tregs during immune response. Thus, different immunosuppressive drugs have distinct effects on the Ag-specific CD4(+)CD25(+)Foxp3(+) Tregs during immune response. The stronger inhibiting effects of MMF, LEF, CsA and Cy on CD4(+)CD25(+)Foxp3(+) Tregs than on T effectors may block the host immune tolerance potentiality.

OBJECTIVE: To provide the anatomical basis for the feasibility and clinical practice of lengthened sacroiliac screw fixation, by measuring various related indicators of the safe insertion regions of S1 and S2 lengthened sacroiliac screws. METHODS: A total of 66 healthy pelvises of adults were scanned by 64-slice spiral CT and the length, width and height of the safe insertion regions for S1 and S2 lengthened sacroiliac screw were measured. The safe screw entrance point locations were described with a quantitative method. The indicators were recorded by descriptive statistics and the statistics of left and right sides, segments of S1 and S2, and different layers (including top, middle and bottom parts) of S1 and S2 were compared. RESULTS: The lengths of ilium within the safe insertion regions for lengthened screws are more than 16 mm. The width and height of the safe insertion region of S1 and S2 are almost all more than 7.3 mm. Generally, the width and height of S1 are larger than those of S2. The reference ranges of the best/safest entrance point locations of lengthened sacroiliac screws are as follows-S1: 42.21-63.69 mm in front of posterior superior iliac spine, 32.77-53.75 mm above the highest point of the greater sciatic notch; S2: 22.68-54.28 mm in front of posterior superior iliac spine, 14.06-33.70 mm above the highest point of the greater sciatic notch. CONCLUSION:(1) There is anatomical feasibility for the placements of S1 and S2 lengthened sacroiliac screws.(2) φ 7.3-mm partial thread cannulated screw (thread length 16 mm) and φ 6.5-mm partial thread cancellous screw(thread length 16 mm) can be used as lengthened sacroiliac lag screw.(3) The safe insertion space of S1 is larger than that of S2.(4) There is safe space for placement of at least one piece of lengthened sacroiliac screw in both S1 and S2.(5) The best/safest entrance points of S1 and S2 can be approximately located with anatomical landmarks.

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.

The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.

Background. Adverse maternal environments may predispose the offspring to metabolic syndrome in adulthoods, but the underlying mechanism has not been fully understood. Methods. Maternal hyperglycemia was induced by streptozotocin (STZ) injection while control (CON) rats received citrate buffer. Litters were adjusted to eight pups per dam and then weaned to standard diet. Since 13 weeks old, a subset of offspring from STZ and CON dams were switched to high fat diet (HFD) for another 13 weeks. Glucose and insulin tolerance tests (GTT and ITT) and insulin secretion assay were performed; serum levels of lipids and leptin were measured. Hepatic fat accumulation and islet area were evaluated through haematoxylin and eosin staining. Results. STZ offspring exhibited lower survival rate, lower birth weights, and growth inhibition which persisted throughout the study. STZ offspring on HFD showed more severe impairment in GTT and ITT, and more profound hepatic steatosis and more severe hyperlipidemia compared with CON-HFD rats. Conclusions. Offspring from diabetic dams would be prone to exhibit low birth weight and postnatal growth inhibition, but could maintain normal glucose tolerance and insulin sensitivity. HFD accelerates development of insulin resistance in the offspring of diabetic dams mainly via a compensatory response of islets.

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin β4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

Carbon nanotube (CNT) and graphene hybrid is an attractive candidate for field emission (FE) because of its unique properties, such as high conductivity, large aspect ratio of CNT and numerous sharp edges of graphene. We report here a vapor-solid (VS) growth of few-layer graphene (FLG, less than 10 layers) on CNTs (FLG/CNT) and Si wafers using a radio frequency sputtering deposition system. Based on SEM, TEM and Raman spectrum analyses, a defect nucleation mechanism of the FLG growth was proposed. The FE measurements indicate that the FLG/CNT hybrids have low turn-on (0.956 V/μm) and threshold field (1.497 V/μm), large field enhancement factor (~4398), and good stability. Excellent FE properties of the FLG/CNT hybrids make them attractive candidates as high-performance field emitters.

Silicon photonics has emerged as the premier candidate for the photonic systems-on-chip (SoC). The scheme based on the silicon Mach-Zehnder modulator (MZM) to generate photonic ultra-wideband (UWB) signals is helpful to the integration of the UWB system with other optical networks on a single silicon-based chip. In this paper, according to the influence of the nonlinear free carrier dispersion (FCD) effect and the free carrier absorption (FCA) effect, the performance of two typical UWB generation schemes is numerically analyzed. The double side-band UWB (DSB-UWB) generation scheme needs the DC reverse bias which increases the complexity of the modulator and there is a residual chirp resulting from the FCD effect even the push-pull operation is adopted. The quasi single-sideband UWB (QSSB-UWB) generation scheme doesn't have these problems. However there is the asymmetric amplitude peak in the generated UWB signal. The property of the large singal modulation is also investigated to improve the signal-to-noise ratio (SNR).

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F-actin binding ability, located to the C-terminal half of the protein, which promotes stabilization of F-actin in the cell thus releasing myocardin-related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor. Initial attempts to purify the protein only resulted in heavily degraded samples that showed distinct bands on SDS gels, suggesting the presence of stable domains. Using a combination of combinatorial domain hunting and sequence analysis, a set of potential domains was identified. The C-terminal half of the protein actually contains two independent F-actin binding domains. The most C-terminal fragment (294-375), named actin binding domain 2 (ABD2), is independently folded while a proximal fragment called ABD1 (193-296) binds to F-actin with higher affinity than ABD2 (K(D) 2.21 ± 0.47 μM vs. 10.61 ± 0.7 μM), but is not structured by itself in solution. NMR interaction experiments show that it binds and folds in a cooperative manner to F-actin, justifying the label of domain. The architecture of the MS1 C-terminus suggests that ABD1 alone could completely fulfill the F-actin binding function opening up the intriguing possibility that ABD2, despite its high level of conservation, could have developed other functions. Proteins 2011; © 2011 Wiley Periodicals, Inc.

Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

Enteroviral infection can lead to dilated cardiomyopathy (DCM), which is a major cause of cardiovascular mortality worldwide. However, the pathogenetic mechanisms have not been fully elucidated. Serum response factor (SRF) is a cardiac-enriched transcription regulator controlling the expression of a variety of target genes, including those involved in the contractile apparatus and immediate early response, as well as microRNAs that silence the expression of cardiac regulatory factors. Knockout of SRF in the heart results in downregulation of cardiac contractile gene expression and development of DCM. The goal of this study is to understand the role of SRF in enterovirus-induced cardiac dysfunction and progression to DCM. Here we report that SRF is cleaved following enteroviral infection of mouse heart and cultured cardiomyocytes. This cleavage is accompanied by impaired cardiac function and downregulation of cardiac-specific contractile and regulatory genes. Further investigation by antibody epitope mapping and site-directed mutagenesis demonstrates that SRF cleavage occurs at the region of its transactivation domain through the action of virus-encoded protease 2A. Moreover, we demonstrate that cleavage of SRF dissociates its transactivation domain from DNA-binding domain, resulting in the disruption of SRF-mediated gene transactivation. In addition to loss of functional SRF, finally we report that the N-terminal fragment of SRF cleavage products can also act as a dominant-negative transcription factor, which likely competes with the native SRF for DNA binding. Our results suggest a mechanism by which virus infection impairs heart function and may offer a new therapeutic strategy to ameliorate myocardial damage and progression to DCM.

Approximately 20,000 genes are encoded in our genome, one tenth of which are thought to be transcription factors. Considering the complexity and variety of cell types generated during development, many transcription factors likely play multiple roles. Uncovering the versatile roles of Sox6 in vertebrate development sheds some light on how an organism efficiently utilizes the limited resources of transcription factors. The structure of the Sox6 gene itself may dictate its functional versatility. First, Sox6 contains no known regulatory domains; instead, it utilizes various cofactors. Second, Sox6 has a long 3'-UTR that contains multiple microRNA targets, thus its protein level is duly adjusted by cell type-specific microRNAs. Just combining these two characteristics alone makes Sox6 extremely versatile. To date, Sox6 has been reported to regulate differentiation of tissues of mesoderm, ectoderm, and endoderm origins, making Sox6 a truly multifaceted transcription factor.

In skeletal muscle differentiation, muscle-specific genes are regulated by two groups of transcription factors, the MyoD and MEF2 families, which work together to drive the differentiation process. Here, we show that ERK5 regulates muscle cell fusion through Klf transcription factors. The inhibition of ERK5 activity suppresses muscle cell fusion with minimal effects on the expression of MyoD, MEF2, and their target genes. Promoter analysis coupled to microarray assay reveals that Klf-binding motifs are highly enriched in the promoter regions of ERK5-dependent upregulated genes. Remarkably, Klf2 and Klf4 expression are also upregulated during differentiation in an ERK5-dependent manner, and knockdown of Klf2 or Klf4 specifically suppresses muscle cell fusion. Moreover, we show that Sp1 transcription factor links ERK5 to Klf2/4, and that nephronectin, a Klf transcriptional target, is involved in muscle cell fusion. Therefore, an ERK5/Sp1/Klf module plays a key role in the fusion process during skeletal muscle differentiation.

Serum response factor (SRF) regulates certain microRNAs that play a role in cardiac and skeletal muscle development. However, the role of SRF in the regulation of microRNA expression and microRNA biogenesis in cardiac hypertrophy has not been well established. In this report, we employed two distinct transgenic mouse models to study the impact of SRF on cardiac microRNA expression and microRNA biogenesis. Cardiac-specific overexpression of SRF (SRF-Tg) led to altered expression of a number of microRNAs. Interestingly, downregulation of miR-1, miR-133a and upregulation of miR-21 occurred by 7 days of age in these mice, long before the onset of cardiac hypertrophy, suggesting that SRF overexpression impacted the expression of microRNAs which contribute to cardiac hypertrophy. Reducing cardiac SRF level using the antisense-SRF transgenic approach (Anti-SRF-Tg) resulted in the expression of miR-1, miR-133a and miR-21 in the opposite direction. Furthermore, we observed that SRF regulates microRNA biogenesis, specifically the transcription of pri-microRNA, thereby affecting the mature microRNA level. The mir-21 promoter sequence is conserved among mouse, rat and human; one SRF binding site was found to be in the mir-21 proximal promoter region of all three species. The mir-21 gene is regulated by SRF and its cofactors, including myocardin and p49/Strap. Our study demonstrates that the downregulation of miR-1, miR-133a, and upregulation of miR-21 can be reversed by one single upstream regulator, SRF. These results may help to develop novel therapeutic interventions targeting microRNA biogenesis.

MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

MicroRNAs are involved in several aspects of cardiac hypertrophy, including cardiac growth, conduction, and fibrosis. However, their effects on the regulation of the cardiomyocyte cytoskeleton in this pathological process are not known. Here, with microRNA microarray and small RNA library sequencing, we show that microRNA-1 (miR-1) is the most abundant microRNA in the human heart. By applying bioinformatic target prediction, a cytoskeleton regulatory protein twinfilin-1 was identified as a potential target of miR-1. Overexpression of miR-1 not only reduced the luciferase activity of the reporter containing the 3' untranslated region of twinfilin-1 mRNA, but also suppressed the endogenous protein expression of twinfilin-1, indicating that twinfilin-1 is a direct target of miR-1. miR-1 was substantially downregulated in the rat hypertrophic left ventricle and phenylephrine-induced hypertrophic cardiomyocytes, and accordingly, the protein level of twinfilin-1 was increased. Furthermore, overexpression of miR-1 in hypertrophic cardiomyocytes reduced the cell size and attenuated the expression of hypertrophic markers, whereas silencing of miR-1 in cardiomyocytes resulted in the hypertrophic phenotype. In accordance, twinfilin-1 overexpression promoted cardiomyocyte hypertrophy. Taken together, our results demonstrate that the cytoskeleton regulatory protein twinfilin-1 is a novel target of miR-1, and that reduction of miR-1 by hypertrophic stimuli induces the upregulation of twinfilin-1, which in turn evokes hypertrophy through the regulation of cardiac cytoskeleton.

In this issue, Gillespie et al.(Gillespie et al. 2009. J. Cell Biol. doi:10.1083/jcb.200907037) demonstrate that the mitogen-activated protein kinase isoform p38-gamma plays a crucial role in blocking the premature differentiation of satellite cells, a skeletal muscle stem cell population. p38-gamma puts the brakes on skeletal muscle differentiation by promoting the association of the transcription factor MyoD with the histone methyltransferase, KMT1A, which act together in a complex to repress the premature expression of the gene encoding the myogenic transcription factor Myogenin.

microRNAs have recently opened new pathways to explain gene expression and disease biology in many scenarios, including cardiac diseases. microRNAs are endogenous small non-coding RNAs that mediate post-transcriptional repression or messenger RNA degradation. By annealing to inexactly complementary sequences in the 3' untranslated region of the target messenger RNA, protein level is down-regulated. Several microRNAs appear to act cooperatively through multiple target sites in one gene and, conversely, most microRNAs can target several genes. miR-133 and miR-1 are specifically expressed in cardiac and skeletal muscle and control myogenesis, cardiac development, cardiac performance and cardiomyocyte hypertrophy (mainly by tuning transcription factors and other growth-related targets). They also modulate the expression of certain cardiac ion channels and related proteins with proarrhythmic effect. Besides them, other microRNAs have been shown to exert influence on the myocardial growth, the electrical balance and the angiogenesis processes that take place in the heart. Bioinformatics is a useful tool to identify potential targets of a given microRNA, although there is still substantial concern about their reliability. Experimental manipulation of microRNAs has provided a tantalizing basis to speculate that future research on microRNAs may yield important progress in the prevention of sudden cardiac death and in the treatment of cardiac heart failure. However, the final effect of the blockage of microRNAs in vivo remains unclear, since each of them can target hundreds of genes with different intensity. The era of the microRNAs in cardiovascular diseases has just started.