Maya Ameyar-Zazoua

The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation

Deciphering the mechanisms underlying skeletal muscle-cell differentiation in mammals is an important challenge. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recent observations have revealed the importance of small (20–25 base pair) non-coding RNAs (microRNAs or miRNAs) that are expressed in both lower organisms and in mammals. miRNAs modulate gene expression by affecting mRNA translation or stability. In lower organisms, miRNAs are essential for cell differentiation during development; some miRNAs are involved in maintenance of the differentiated state. Here, we show that miR-181, a microRNA that is strongly upregulated during differentiation, participates in establishing the muscle phenotype. Moreover, our results suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of mammalian skeletal-muscle differentiation. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype — even when they are not expressed in the corresponding fully differentiated tissue.

A Subset of the Histone H3 Lysine 9 Methyltransferases Suv39h1, G9a, GLP, and SETDB1 Participate in a Multimeric Complex

Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.

Argonaute proteins couple chromatin silencing to alternative splicing

Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.

Tandem affinity purification of miRNA target mRNAs (TAP-Tar)

MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs are generally poorly homologous to their target sequence, and target identification cannot be based solely on bioinformatics. Here, we describe a biochemical approach, based on tandem affinity purification, in which mRNA/miRNA complexes are sequentially pulled down, first via the Argonaute moiety and then via the miRNA. Our ‘TAP-Tar’ procedure allows the specific pull down of mRNA targets of miRNA. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.

siRNA as a route to new cancer therapies

The RNA interference (RNAi) gene-silencing mechanism is induced by do-uble-stranded RNA (dsRNA) and is highly sequence-specific. It is an extremely powerful tool for silencing gene expression invitro, and might be used as therapy in human pathologies such as cancer, viral infections and genetic disorders. RNAi was initially discovered in plants, but it has become clear that it is also conserved in animal species. Triggering of RNAi by the introduction of small dsRNA (or small interfering RNA) into living cells as a tool to inhibit the expression of specific genes holds the promise to selectively extinguish the expression of disease-associated genes in humans. On the other hand, RNAi technology will serve to elucidate the functions and interactions of the thousands of human genes in high-throughput systems and help in target validation technology.

Physical and Functional Interaction between Heterochromatin Protein 1α and the RNA-binding Protein Heterogeneous Nuclear Ribonucleoprotein U

By combining biochemical purification and mass spectrometry, we identified proteins associated with human heterochromatin protein 1α (HP1α) both in the nucleoplasm and in chromatin. Some of these are RNA-binding proteins, and among them is the protein heterogeneous nuclear ribonucleoprotein U (hnRNP U)/SAF-A, which is linked to chromatin organization and transcriptional regulation. Here, we demonstrate that hnRNP U is a bona fide HP1α-interacting molecule. More importantly, hnRNP U depletion reduces HP1α-dependent gene silencing and disturbs HP1α subcellular localization. Thus, our data demonstrate that hnRNP U is involved in HP1α function, shedding new light on the mode of action of HP1α and on the function of hnRNP U.

10-million-years AGO: argonaute on chromatin in yeast and human, a conserved mode of action?

Whereas in yeast the function and mode of action of nuclear RNAi are well documented, mammalian nuclear RNAi is a matter of debates. Several papers support a role for nuclear Argonaute in alternative splicing. However, the molecular mechanism remains elusive. Here, we discuss the human nuclear RNAi mechanism in light of what is known of the yeast process.

Small Regulatory RNAs and Skeletal Muscle Cell Differentiation

Until recently, RNA was considered to be merely a downstream effector of the “noble” genome, the latter having all the information and therefore occupying a position at the very heart of gene regulation, according to the “central dogma” of DNA transcribed into RNA translated into protein. Although we all knew that RNAs also have accessory functions, and that non-coding RNAs intervene at all stages of gene expression, these essential functions were considered to be mere “housekeepers,” and RNA was denied a regulatory role. This dogma was, however, “blown up” several years ago by the concomitant discovery of RNA interference and microRNAs in a model organism, the worm Caenorhabditis elegans. We now know that small regulatory RNAs are widely conserved in plants and animals, and that microRNAs and short interfering RNAs are not the only kinds of regulatory small RNAs that exist. Indeed, the variety of functions in which small non-coding RNAs have been shown to play essential roles has grown rapidly. Basically, they are involved in controlling large genetic programs or large regions of cell genomes, and they participate in determining what is called cell fate, or the balance between cell proliferation, differentiation and death. This equilibrium is strictly controlled under normal conditions, and its deregulation leads to oncogenesis. One of our main interests is the function of microRNAs in mammalian skeletal muscle. We describe here the high-throughput screening strategy used in our laboratory to identify and validate the microRNAs and their specific targets which are essential for muscle cell differentiation.