The solution structure of Dead End bound to AU-rich RNA reveals an unusual mode of tandem RRM-RNA recognition required for mRNA regulation.

Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1's tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA.

Global and precise identification of functional miRNA targets in mESCs by integrative analysis.

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Previous works suggest that up to 60% of mammalian transcripts might be subject to miRNA-mediated regulation, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrate transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles.

Translation is required for miRNA-dependent decay of endogenous transcripts.

Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization remains unknown.

FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops.

Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells.

Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells.

In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich single-stranded DNA (ssDNA) overhang into the duplex telomeric array. The other shelterin factor, TRF1, is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA, leading to formation of telomeric RNA-DNA hybrids (telR loops).

TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation.

Human Mpn1 promotes post-transcriptional processing and stability of U6atac.

Mpn1 is an exoribonuclease that modifies the spliceosomal small nuclear RNA (snRNA) U6 by trimming its oligouridine tail and introducing a cyclic phosphate group (>p). Mpn1 deficiency induces U6 3' end misprocessing, accelerated U6 decay and pre-mRNA splicing defects. Mutations in the human MPN1 gene are associated with the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN).

Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels.

The telomeric transcriptome comprises multiple long non-coding RNAs generated by transcription of linear chromosome ends. In a screening performed in Schizosaccharomyces pombe, we identified factors modulating the cellular levels of the telomeric transcriptome. Among these factors, Cay1 is the fission yeast member of the conserved family of Cactins, uncharacterized proteins crucial for cell growth and survival.

RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells.

A fraction of cancer cells maintain telomeres through the telomerase-independent, 'Alternative Lengthening of Telomeres' (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA-DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells.

Mpn1, mutated in poikiloderma with neutropenia protein 1, is a conserved 3'-to-5' RNA exonuclease processing U6 small nuclear RNA.

Clericuzio-type poikiloderma with neutropenia (PN) is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA) posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3' end polyuridine tails that are longer than those in normal cells and lack a terminal 2',3' cyclic phosphate group.

The telomeric transcriptome of Schizosaccharomyces pombe.

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species.

Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication.

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5'-to-3' helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation.

Cell-cell propagation of NF-κB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection.

The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection.

CpG-island promoters drive transcription of human telomeres.

The longstanding dogma that telomeres, the heterochromatic extremities of linear eukaryotic chromosomes, are transcriptionally silent was overturned by the discovery that DNA-dependent RNA polymerase II (RNAPII) transcribes telomeric DNA into telomeric repeat-containing RNA (TERRA). Here, we show that CpG dinucleotide-rich DNA islands, shared among multiple human chromosome ends, promote transcription of TERRA molecules. TERRA promoters sustain cellular expression of reporter genes, are located immediately upstream of TERRA transcription start sites, and are bound by active RNAPII in vivo.

Humanization of yeast to produce complex terminally sialylated glycoproteins.

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation.

Intact {alpha}-1,2-endomannosidase is a typical type II membrane protein.

Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc(1-3)Man), from N-glycosylation intermediates with terminal glucose residues. To date it is the only characterized member of this class of endomannosidic enzymes. Although this protein has been demonstrated to localize to the Golgi lumenal membrane, the mechanism by which this occurs has not yet been determined.

Production of complex human glycoproteins in yeast.

We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2).