Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR-34a as a Regulator of Growth Factor Signaling

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division

Com o diabo no corpo: os terríveis papagaios do Brasil colônia

Desde a Antiguidade, papagaios, periquitos e afins (Psittacidae) fascinaram os europeus por seu vivo colorido e uma notável capacidade de interação com seres humanos. A descoberta do Novo Mundo nada faria além de acrescentar novos elementos ao tráfico de animais exóticos há muito estabelecido pelos europeus com a África e o Oriente. Sem possuir grandes mamíferos, a América tropical participaria desse comércio com o que tinha de mais atrativo, essencialmente felinos, primatas e aves - em particular os papagaios, os quais eram embarcados em bom número. Contudo, a julgar pelos documentos do Brasil colônia, esses voláteis podiam inspirar muito pouca simpatia, pois nenhum outro animal - exceto as formigas - foi tantas vezes mencionado como praga para a agricultura. Além disso, alguns psitácidas mostravam-se tão loquazes que inspiravam a séria desconfiança de serem animais demoníacos ou possessos, pois só três classes de entidades - anjos, homens e demônios - possuíam o dom da palavra. Nos dias de hoje, vários representantes dos Psittacidae ainda constituem uma ameaça para a agricultura, enquanto os indivíduos muito faladores continuam despertando a suspeita de estarem possuídos pelo demônio. Transcendendo a mera curiosidade, essa crença exemplifica o quão intrincadas podem ser as relações do homem com o chamado “mundo natural”, revelando um universo mais amplo e multifacetado do que se poderia supor a princípio. Nesse sentido, a existência de aves capazes de falar torna essa relação ainda mais complexa e evidencia que as dificuldades de estabelecer o limite entre o animal e o humano se estendem além dos primatas e envolvem as mais inusitadas espécies zoológicas.Since ancient times, parrots and their allies (Psittacidae) have fascinated Europeans by their striking colors and notable ability to interact with human beings. The discovery of the New World added new species to the international exotic animal trade, which for many centuries had brought beasts to Europe from Africa and the Orient. Lacking large mammals, tropical America participated in this trade with its most appealing species, essentially felines, primates and birds - especially parrots - which were shipped in large numbers. It should be noted, however, that at times these birds were not well liked. In fact, according to documents from colonial Brazil, only the ants rank higher than parrots as the animals most often mentioned as agricultural pests. On the other hand, some of these birds were so chatty that people suspected them to be demonic or possessed animals, since only three classes of beings - angels, men and demons - have the ability to speak. Nowadays, several Psittacidae still constitute a threat to agriculture, and the suspicion that extremely talkative birds were demon possessed has also survived. More than a joke or a mere curiosity, this belief exemplifies how intricate man’s relationships with the “natural world” may be. In this sense, the existence of birds that are able to speak adds a further twist to these relationships, demonstrating that the problem of establishing a boundary between the animal and the human does not only involve primates, but also includes some unusual zoological species

Regioselective synthesis of 2-Aryl-5-cyano-1-(2-hydroxyaryl)-1H-imidazole-4-carboxamides self-assisted by a 2-Hydroxyaryl group

The reactivity of the diaminomaleonitrile-based imines containing hydroxyphenyl substituents with diverse aromatic aldehydes has been explored for the synthesis of novel highly substituted nitrogen heterocycles, which are considered privileged scaffolds in drug discovery. We report here a simple and efficient method for the regiocontrolled synthesis of a variety of 2-aryl-5cyano-1-(2-hydroxyaryl)-1H-imidazole-4-carboxamides from 2-hydroxybenzylidene imines and aromatic aldehydes. Computational studies on the reaction path revealed that the regioselectivity of the reaction toward the formation of imidazole derivatives instead of 1,2-dihydropyrazines, most likely via a diaza-Cope rearrangement, is driven by the 2-hydroxyaryl group in the scaffold. The latter group promotes the intramolecular abstraction and protonation process in the cycloadduct intermediate, triggering the evolution of the reaction toward the formation of imidazole derivatives.Financial support from Axencia Galega de Innovación (2020-PG067, CG-B), Programa NORTE 2020─CCDR-N (ref. NORTE-08-5369-FSE-000033, MFP), the Spanish Ministry of Science and Innovation (PID2019-105512RB-I00, CG-B), the Xunta de Galicia [ED431C 2021/29 and the Centro singular de investigación de Galicia accreditation 2019–2022 (ED431G 2019/03), CG-B], and the European Regional Development Fund (ERDF) is gratefully acknowledged. All authors thank the Centro de Supercomputación de Galicia (CESGA) for the use of the Finis Terrae computer

Intact p53-Dependent Responses in miR-34–Deficient Mice

<div><p>MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34–deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation <em>in vitro</em>, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34–deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc–initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.</p> </div

An allelic series of miR-17 ∼ 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron.

Polycistronic microRNA (miRNA) clusters are a common feature of vertebrate genomes. The coordinated expression of miRNAs belonging to different seed families from a single transcriptional unit suggests functional cooperation, but this hypothesis has not been experimentally tested. Here we report the characterization of an allelic series of genetically engineered mice harboring selective targeted deletions of individual components of the miR-17 ∼ 92 cluster. Our results demonstrate the coexistence of functional cooperation and specialization among members of this cluster, identify a previously undescribed function for the miR-17 seed family in controlling axial patterning in vertebrates and show that loss of miR-19 selectively impairs Myc-driven tumorigenesis in two models of human cancer. By integrating phenotypic analysis and gene expression profiling, we provide a genome-wide view of how the components of a polycistronic miRNA cluster affect gene expression in vivo. The reagents and data sets reported here will accelerate exploration of the complex biological functions of this important miRNA cluster

Targeted deletion of miR-34a and miR-34b∼c.

<p>(A) Targeting and screening strategy for the generation of constitutive and conditional miR-34a KO alleles. The restriction sites used for the Southern blot screening are indicated (S = SphI, E = EcoRI). The gray bar with an asterisk represents a genomic region absent in the 129SvJae strain but present in the C57BL/6 strain, which results in two distinct sizes in digestions. (B) Targeting and screening strategy for the generation of miR-34b∼c KO allele (H = HindIII, S = SpeI). (C) Genotyping by tail genomic PCR showing germline transmission of the miR-34a deleted and floxed alleles (upper panel), and the miR-34b∼c deleted allele (lower panel). (D) Northern blotting (upper panel) on total RNA extracted from the testes of mice with the indicated genotypes. Probes specific for miR-34a and miR-34c were used. Complete loss of miR-34a and miR-34c expression was further confirmed in MEFs by qPCR (lower panel). Representative pictures of miR-34a^−/− (E), miR-34b∼c^−/− (F), and miR-34^TKO/TKO (G) males at 4 weeks of age. The table below each picture summarizes the expected and observed frequencies of mice of each genotype as obtained from heterozygous inter-crosses. For the miR-34^TKO allele (G), double heterozygous mice were inter-crossed.</p

MiR-34 expression in wild-type and p53^−/− mouse tissues.

<p>(A) Sequence alignment of mouse miR-34a, miR-34b and miR-34c. Differing nucleotides are colored in blue. The seed sequences are in bold. (B–D) MiR-34a and miR-34c expression as detected by qPCR (B,C) and by Northern blotting (D) in tissues of wild-type and p53^−/− mice.</p

Response to p53 activation in miR-34^TKO/TKO mouse embryonic fibroblasts (MEFs).

<p>(A) MiR-34a and miR-34c expression in serially-passaged wild-type MEFs, as measured by qPCR. Error bars indicate 1 standard deviation (SD). (B) Cumulative population doublings of wild-type, miR-34^TKO/TKO and p53^−/− MEFs. Error bars indicate 1 SD. (C) Growth curves of wild-type and miR-34^TKO/TKO MEFs. Error bars indicate 1 SD. (D) Immunoblots of p53, p21 and Mdm2 in wild-type (W) and miR-34^TKO/TKO (K) MEFs treated with 0.2 µg/ml doxorubicin for the indicated time. (E) Expression of selected p53 targets in total RNA from doxorubicin-treated MEFs. Cells were treated with 0.2 µg/ml doxorubicin for 12 hours (Dox) or left untreated (U). Expression of the indicated genes was determined by qPCR. Error bars represent 1 SD. (F) Immunoblots showing p53 activation in three wild-type and three miR-34^TKO/TKO MEF lines. Cells were left untreated or treated with 0.2 µg/ml doxorubicin for 12 hours. (G) Time course of miR-34a and miR-34c expression in wild-type and p53^−/− cells treated with 0.2 µg/ml doxorubicin. MicroRNA expression was determined by qPCR. Error bars indicate 1 SD. (H, I) Cell cycle distribution of wild-type and miR-34^TKO/TKO MEFs. Asynchronously growing MEFs of the indicated genotype were treated with increasing doses of doxorubicin for 16 hours (H), or with 0.2 µg/ml doxorubicin for increasing time (I). Error bars indicate 1 SD. (J) Upper panel: cell cycle distribution of wild-type, miR-34^TKO/TKO, and p53^−/− MEFs after 72 hours in starvation medium (gray histogram). Starved cells were released in complete medium containing colcemid and mock-treated (light blue histogram) or exposed to 20 Gy irradiation (red histogram). Cells were analyzed by 7-AAD staining at the indicated time after release in complete medium. Lower panel: percentages of irradiated and untreated cells in G1 and G2-M phases after 24 hours in complete medium. Experiments were performed on three independent wild-type and three independent miR-34^TKO/TKO MEF lines. (K) Immunoblot detection of predicted miR-34 targets on three independent wild-type and three independent miR-34^TKO/TKO MEF lines.</p