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. Using this integrative approach, combined with molecular validation data, we present evidence that < 10% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identify TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

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. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unusual mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1’s double-stranded RNA binding domain (dsRBD), which is dispensable for binding of a subset of targets in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

The development of in vitro models, which accurately recapitulate early embryonic development, is one of the fundamental challenges in stem cell research. Most of the currently employed approaches involve the culture of embryonic stem cells (ESCs) on 2D surfaces. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic 3D microenvironments, as in an early embryo. In this paper, novel 3D synthetic microstructure arrays, fabricated by two‐photon polymerization photolithography, are utilized to mimic tissue‐specific architecture, enabling cell‐to‐matrix interaction and cell‐to‐cell communication in vitro. Mouse ESCs (mESCs) are able to grow and proliferate on these structures and maintain their pluripotent state. Furthermore, the 3D microstructure arrays are integrated into a microscopy slide allowing the evaluation of the expression of key pluripotency factors at the single cell level. Comparing 2D and 3D surfaces, mESCs grown in serum + leukemia inhibitory factor on 3D microstructures exhibit a stronger signal intensity of three pluripotency markers—homeobox protein NANOG, octamer‐binding transcription factor 4, and estrogen‐related receptor beta (ESRRB)—and more homogenous expression of NANOG and ESRRB, than cells cultivated in 2i medium, demonstrating that 3D microstructures capture naïve pluripotency in vitro. Thus, the slide affords a novel and unique tool to model and study early development.

The development of in vitro models, which accurately recapitulate early embryonic development, is one of the fundamental challenges in stem cell research. Most of the currently employed approaches involve the culture of embryonic stem cells (ESCs) on two-dimensional (2D) surfaces. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic three-dimensional (3D) microenvironments, as in an early embryo. In this paper, novel 3D synthetic scaffold arrays, fabricated by two-photon polymerization photolithography, are utilized to mimic tissue-specific architecture, enabling cell-to-matrix interaction and cell-to-cell communication in vitro . Mouse ESCs (mESCs) are able to grow and proliferate on these structures and maintain their pluripotent state. Furthermore, the 3D microscaffold arrays are integrated into a microscopy slide allowing the evaluation of the expression of key pluripotency factors at the single-cell level. Comparing 2D and 3D surfaces, mESCs grown in serum+LIF on 3D microscaffolds exhibit a stronger and more homogenous expression of NANOG and OCT4 pluripotency factors, than cells cultivated in 2i media, demonstrating that 3D microscaffolds capture naive pluripotency in vitro . Thus, the slide affords a novel and unique tool to model and study mammalian early development with greater physiological relevance than conventional 2D cultures. ### Competing Interest Statement The authors have declared no competing interest.

Posttranscriptional repression by microRNA (miRNA) occurs through transcript destabilization or translation inhibition. Whereas RNA degradation explains most miRNA-dependent repression, transcript decay occurs co-translationally, raising questions regarding the requirement of target translation to miRNA-dependent transcript destabilization. To assess the contribution of translation to miRNA-mediated RNA destabilization, we decoupled these two molecular processes by dissecting the impact of miRNA loss of function on cytosolic long noncoding RNAs (lncRNAs). We show, that despite interacting with miRNA loaded RNA-induced silencing complex (miRISC), the steady state abundance and degradation rates of these endogenously expressed non-translated transcripts are minimally impacted by miRNA loss. To validate the requirement of translation for miRNA-dependent decay, we fused a miRISC bound lncRNA, whose levels are unaffected by miRNAs, to the 3’end of a protein-coding gene reporter and show that this results in its miRNA-dependent transcript destabilization. Furthermore, analysis of the few lncRNAs whose levels are regulated by miRNAs revealed these tend to associate with translating ribosomes and are likely misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. Our analyses reveal the strict requirement of translation for miRNA-dependent transcript destabilization and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.

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. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and 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). The N-terminal basic domain of TRF2 binds to TERRA-like RNA and enables TRF2 to promote efficient RNA invasion. TRF1, through its N-terminal acidic domain, counteracts TRF2-mediated RNA invasion but not ssDNA invasion. In vivo, when TRF1 is depleted or replaced with a variant lacking the acidic domain, TRF2 induces formation of telR loops, which in turn cause telomere loss. Hence, uncontrolled TRF2 threatens telomere integrity, and TRF1 directly supports end protection by suppressing harmful telR loops.

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. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.

Introduction The alternative lengthening of telomeres (ALT) mechanism was first observed in the model organism S. cerevisiae. Interestingly, this mechanism is necessary for the viability of some tumor cells. Unfortunately, its molecular underpinnings are not yet completely understood.Objective Here, we combine carefully designed non-targeted mass spectrometry-based metabolomics experiments with a bioinformatics approach to characterize the ALT positive phenotype observed in yeast at the metabolomics level.Methods We profiled the metabolome using mass spectrometry in yeast strains that have lost telomerase expression, as well as that in pre-senescence and the rescued states. To dissect unwanted technical variation from biologically relevant variation between these states, we used a two-step normalization strategy, i. e., first, an empirical Bayesian framework; and next, we corrected for secondorder technical effects.Results Our results show that ALT-positive yeast strains present two different types of metabolic responses to the genetically-induced telomerase dysfunction: (i) systemic and (ii) specific. The key-difference between these responses is that the systemic response lasts even after the yeast strains have been genetically rescued, while the specific response does not. Interestingly, these metabolic changes can be associated with generic stress responses (e. g., DNA damage) as well as specific responses like accelerated aging of early telomerase-inactivation.Conclusions A mass spectrometry-based metabolomics approach reveals two distinct types of metabolomics response to telomerase dysfunction in yeast. By identifying these changes in protein (e. g., ARG7, and ARG1), and metabolite (e. g., dATP, and dDTP) amounts, we complement the available information on ALT at the genome-wide level.

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. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres.

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). Here we present the deep sequencing of the >p‐containing transcriptomes of mpn1Δ fission yeast and PN cells. While in yeast U6 seems to be the only substrate of Mpn1, human Mpn1 also processes U6atac snRNA. PN cells bear unstable U6atac species with aberrantly long and oligoadenylated 3′ ends. Our data corroborate the link between Mpn1 and snRNA stability suggesting that PN could derive from pre‐mRNA splicing aberrations.

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. In cay1∆ mutants, the cellular levels of the telomeric factor Rap1 are drastically diminished due to defects in rap1+ pre‐mRNA splicing and Rap1 protein stability. cay1∆ cells accumulate histone H3 acetylated at lysine 9 at telomeres, which become transcriptionally desilenced, are over‐elongated by telomerase and cause chromosomal aberrations in the cold. Overexpressing Rap1 in cay1+ deleted cells significantly reverts all telomeric defects. Additionally, cay1∆ mutants accumulate unprocessed Tf2 retrotransposon RNA through Rap1‐independent mechanisms. Thus, Cay1 plays crucial roles in cells by ultimately harmonizing expression of transcripts originating from seemingly unrelated genomic loci.

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. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA-DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely.

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. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

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. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading‐strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis. UPF1, a helicase/ATPase with roles in mRNA surveillance that is also found at human telomeres, directly interacts with telomerase and a shelterin component, and involved selectively in telomere leading‐strand replication.

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. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organisms.

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. Finally, the identified promoter CpG dinucleotides are methylated in vivo, and cytosine methylation negatively regulates TERRA abundance. The existence of subtelomeric promoters, driving TERRA transcription from independent chromosome ends, supports the idea that TERRA exerts fundamental functions in the context of telomere biology.