Association of the mouse infertility factor DAZL1 with actively translating polyribosomes

The DAZ (Deleted in AZoospermia) gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.     

Caged oligonucleotides for bidirectional photomodulation of let-7 miRNA in zebrafish embryos

Many biological functions of microRNA (miRNA) have been identified in the past decade. However, a single miRNA can regulate multiple gene targets, thus it has been a challenge to elucidate the specific functions of each miRNA in different locations and times. New chemical tools make it possible to modulate miRNA activity with higher spatiotemporal resolution. Here, we describe light-activated (caged) constructs for switching let-7 miRNA ‘on’ or ‘off’ with 365 nm light in developing zebrafish embryos.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors

Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with let-7 precursors. Previous studies have described seemingly inconsistent Lin28 binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining the binding mechanism of Lin28 to the terminal loop of pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, we investigate Lin28 binding to TL-let-7g variants and short RNA fragments and identify three independent binding sites for Lin28 on TL-let-7g. We then determine that Lin28 assembles in a stepwise manner on TL-let-7g to form a stable 1:3 complex. We show that the cold-shock domain (CSD) of Lin28 is responsible for remodelling the terminal loop of TL-let-7g, whereas the NCp7-like domain facilitates the initial binding of Lin28 to TL-let-7g. This stable binding of multiple Lin28 molecules to the terminal loop of pre-let-7g extends to other precursors of the let-7 family, but not to other pre-miRNAs tested. We propose a model for stepwise assembly of the 1:1, 1:2 and 1:3 pre-let-7g/Lin28 complexes. Stepwise multimerization of Lin28 on pre-let-7 is required for maximum inhibition of Dicer cleavage for a least one member of the let-7 family and may be important for orchestrating the activity of the several factors that regulate let-7 biogenesis.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Cellular stress induced alterations in microRNA let-7a and let-7b expression are dependent on p53

Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53(-/-) colon cancer cell line (HCT116) or ATM(-/-) human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53(+/+) cells but not HCT116 p53(-/-) cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.     

miRNA let-7e targeting MMP9 is involved in adipose-derived stem cell differentiation toward epithelia

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.     

Human let-7 stem-loop precursors harbor features of RNase III cleavage products

The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21–23 nt long mature RNAs. These precursors are 60–80 nt long, they fold into a characteristic stem–loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, we have characterized the precise 5′ and 3′ ends of the let-7 precursors in human cells. We show that they harbor a 5′-phosphate and a 3′-OH and that, remarkably, they contain a 1–4 nt 3′ overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, our results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway.     

Genomic DNA Copy-Number Alterations of the let-7 Family in Human Cancers

In human cancer, expression of the let-7 family is significantly reduced, and this is associated with shorter survival times in patients. However, the mechanisms leading to let-7 downregulation in cancer are still largely unclear. Since an alteration in copy-number is one of the causes of gene deregulation in cancer, we examined copy number alterations of the let-7 family in 2,969 cancer specimens from a high-resolution SNP array dataset. We found that there was a reduction in the copy number of let-7 genes in a cancer-type specific manner. Importantly, focal deletion of four let-7 family members was found in three cancer types: medulloblastoma (let-7a-2 and let-7e), breast cancer (let-7a-2), and ovarian cancer (let-7a-3/let-7b). For example, the genomic locus harboring let-7a-3/let-7b was deleted in 44% of the specimens from ovarian cancer patients. We also found a positive correlation between the copy number of let-7b and mature let-7b expression in ovarian cancer. Finally, we showed that restoration of let-7b expression dramatically reduced ovarian tumor growth in vitro and in vivo. Our results indicate that copy number deletion is an important mechanism leading to the downregulation of expression of specific let-7 family members in medulloblastoma, breast, and ovarian cancers. Restoration of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of cancer.     

Human cytomegalovirus UL38 protein blocks apoptosis

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.     

miRNA let-7e Modulates the Wnt Pathway and Early Nephrogenic Markers in Mouse Embryonic Stem Cell Differentiation

This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3β^P) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3β^P and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.     

The type II poly(A)-binding protein PABP-2 genetically interacts with the let-7 miRNA and elicits heterochronic phenotypes in Caenorhabditis elegans

The type II poly(A)-binding protein PABP2/PABPN1 functions in general mRNA metabolism by promoting poly(A) tail formation in mammals and flies. It also participates in poly(A) tail shortening of specific mRNAs in flies, and snoRNA biogenesis in yeast. We have identified Caenorhabditis elegans pabp-2 as a genetic interaction partner of the let-7 miRNA, a widely conserved regulator of animal stem cell fates. Depletion of PABP-2 by RNAi suppresses loss of let-7 activity, and, in let-7 wild-type animals, leads to precocious differentiation of seam cells. This is not due to an effect on let-7 biogenesis and activity, which remain unaltered. Rather, PABP-2 levels are developmentally regulated in a let-7-dependent manner. Moreover, using RNAi PABP-2 can be depleted by >80% without significantly impairing larval viability, mRNA levels or global translation. Thus, it unexpectedly appears that the bulk of PABP-2 is dispensable for general mRNA metabolism in the larva and may instead have more restricted, developmental functions. This observation may be relevant to our understanding of why the phenotypes associated with human PABP2 mutation in oculopharyngeal muscular dystrophy (OPMD) seem to selectively affect only muscle cells.     

A Genome-Wide Survey of Sexually Dimorphic Expression of Drosophila miRNAs Identifies the Steroid Hormone-Induced miRNA let-7 as a Regulator of Sexual Identity

MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.     

Role of ribosomal protein RPS2 in controlling let-7a expression in human prostate cancer

We discovered that an inverse relationship exists in the expression of ras/c-myc and ribosomal protein RPS2 with pre-let-7a-1/let-7a/let-7f miRNA and prostate tumor cell malignancy. Nonmalignant IBC-10a cells expressed low levels of ras/RPS2 and elevated pre-let-7a-1/let-7a/let-7f miRNA, whereas the reverse occurred in malignant PCa-20a and PC-3ML cells. Stable transfection of IBC-10a cells with pBABE.ras and pBABE.RPS2 induced ras, c-myc, and RPS2 expression, whereas the levels of let-7a/let-7f miRNA dropped to near zero. Conversely, in pBABE.pre-let-7a-1 transfected PCa-20a and PC-3ML clones, let-7a/let-7f increased whereas ras, RPS2, and c-myc dropped greater than 5-fold. Electrophoretic mobility shift assays, antibody "supershift" assays and immunoprecipitation assays revealed that RPS2 specifically binds pre-let-7a-1 to block RNA processing. Immunoflourescent studies and Northern blots confirmed that RPS2 complexes with pre-let-7a-1 (i.e., in episomal structures) to block processing to let-7a/let-7f, indicating RPS2 may prevent let-7a miRNA expression to indirectly promote oncogene expression. Functional studies further showed that the colony-forming ability (CFA) and invasive activities of IBC-10a cells were significantly enhanced in pBABE-ras.IBC-10a and pBABE-RPS2-IBC-10a clones. Conversely, with the "knockdown" of ras and RPS2 in malignant PC-3ML cells (i.e., in pLKO.TRC.shRNA.ras.PC3-ML, pLKO.TRC.shRNA.RPS2.PC-3ML transfected cells), there was both a loss of these functions and a loss of tumorigenesis in SCID mice. Likewise, with the overexpression of let-7a/let-7f in pBABE.pre-let-7a-1.PC-3ML clones (and PCa-20a clones), CFAs, invasive activities in vitro, and tumorigenesis in vivo were significantly reduced. These results show for the first time that RPS2 blocks pre-let-7a-1 processing to enable ras and c-myc expression and the transformation of primary tumor cells.     

NMR structure of the let-7 miRNA interacting with the site LCS1 of lin-41 mRNA from Caenorhabditis elegans

We have determined the 3D structure of a 34-nt RNA construct, herein named LCS1co, which mimics the interaction of let-7 microRNA (miRNA) to one of its complementary binding sites, LCS1, in the 3′-untranslated region of lin-41 mRNA by solution-state NMR spectroscopy. let-7 miRNAs control the timing of development of the nematode Caenorhabditis elegans and are highly conserved in mammals. The sequence and structure of the two conserved let-7 complementary sites, LCS1 and LCS2, in the 3′-untranslated region of lin-41 mRNA are important for a proper downregulation of lin-41. The high-resolution NMR structure reveals details of the binding of let-7 miRNA to lin-41 mRNA which involves formation of a complex with non-canonical structural elements within the seed region. LCS1co exhibits a stem-loop structure with two stems, an asymmetric internal loop and an adenine bulge. Comparison with the NMR solution-state structure of the let-7:lin-41 complex involving the LCS2-binding site shows that conformational freedom of the asymmetric internal loop of LCS1co correlates with a smaller bend between the upper and lower stems in comparison to the well-defined asymmetric loop of LCS2co.     

Down-regulation of circulating microRNA let-7a in Egyptian smokers

Altered miRNAs were associated with cigarette smoking. The study aimed to examine the gene expression level of plasma let-7a among healthy smokers and compared it with the non-smokers. Forty subjects were recruited for the present study and classified into 21 smokers and 19 non-smokers, age, and sex were matched. The software that used to design functional primers was MIRprimer. Quantitative real-time PCR was employed to compare the relative expression of plasma let-7a. Results showed that the level of let-7a was down-regulated in smokers to 0.34fold (p?=?0.006) that of the non-smokers. Plasma let-7a showed an area under curve (AUC) of 0.749 with sensitivity 43% and specificity 100%. In conclusion, plasma let-7a was significantly down-regulated in the smokers, and it might be considered a candidate biomarker to discriminate between smokers and non-smokers.