Nuclear mRNA Degradation Pathway(s) Are Implicated in Xist Regulation and X Chromosome Inactivation

A critical step in X-chromosome inactivation (XCI), which results in the dosage compensation of X-linked gene expression in mammals, is the coating of the presumptive inactive X chromosome by the large noncoding Xist RNA, which then leads to the recruitment of other factors essential for the heterochromatinisation of the inactive X and its transcriptional silencing. In an approach aimed at identifying genes implicated in the X-inactivation process by comparative transcriptional profiling of female and male mouse gastrula, we identified the Eif1 gene involved in translation initiation and RNA degradation. We show here that female embryonic stem cell lines, silenced by RNA interference for the Eif1 gene, are unable to form Xist RNA domains upon differentiation and fail to undergo X-inactivation. To probe further an effect involving RNA degradation pathways, the inhibition by RNA interference of Rent1, a factor essential for nonsense-mediated decay and Exosc10, a specific nuclear component of the exosome, was analysed and shown to similarly impair Xist upregulation and XCI. In Eif1-, Rent1-, and Exosc10-interfered clones, Xist spliced form(s) are strongly downregulated, while the levels of unspliced form(s) of Xist and the stability of Xist RNA remain comparable to that of the control cell lines. Our data suggests a role for mRNA nuclear degradation pathways in the critical regulation of spliced Xist mRNA levels and the onset of the X-inactivation process

A Latent Pro-survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.National Institutes of Health (U.S.) (NIH grant RO1-GM34277)National Cancer Institute (U.S.) (NCI grant PO1-CA42063)National Cancer Institute (U.S.) (NCI Cancer Center Support (core) grant P30-CA14051

Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

MicroRNAs (miRNAs) are 19–22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer[superscript −/−] mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.Leukemia & Lymphoma Society of AmericaUnited States. Public Health Service (Grant R01-GM34277)United States. Public Health Service (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P01-CA42063)National Cancer Institute (U.S.) Cancer Center Support (Grant P30-CA14051

Xist localization and function: new insights from multiple levels

In female m ammals, one of the two X chromosomes in each cell is transcriptionally silenced in order to achieve dosage compensation between the genders in a process called X chromosome inactivation. The master regulator of this process is the long non-coding RNA Xist. During X-inactivation, Xist accumulates in cis on the future inactive X chromosome, triggering a cascade of events that provoke the stable silencing of the entire chromosome, with relatively few genes remaining active. How Xist spreads, what are its binding sites, how it recruits silencing factors and how it induces a specific topological and nuclear organization of the chromatin all remain largely unanswered questions. Recent studies have improved our understanding of Xist localization and the proteins with which it interacts, allowing a reappraisal of ideas about Xist function. We discuss recent advances in our knowledge of Xist-mediated silencing, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation

Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems

Conclusions: We compare for the first time the sRNA profile of four different tissues, including source, sink and conductive (phloem) tissues, in two plant-virus pathosystems. Our results indicate that antiviral silencing machinery in melon and cucumber acts mainly through DCL4. Upon infection, the total sRNA pattern in phloem remains unchanged in contrast to the rest of the analyzed tissues indicating a certain tissue-tropism to this polulation. Independently of the accumulation level of the vsRNAs both viruses were able to modulate the host sRNA pattern.We thank Dr A. Niehl for critical reading and helpful comments on the manuscript. This work was funded by a supporting program for the research from the Universidad Politecnica de Valencia (PAID-05-10), a grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica and the PROMETEO program 2011/003 from the Generalitat Valenciana. MCH is the recipient of a contract from JAE-DOC program of the CSIC, JAN is the recipient of a postdoctoral contract from the Ministerio de Educacion y Ciencia of Spain.Herranz Gordo, MDC.; Navarro Bohigues, JA.; Sommen, E.; Pallás Benet, V. (2015). Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems. BMC Genomics. 16:1-15. https://doi.org/10.1186/s12864-015-1327-5S11516Pumplin N, Voinnet O. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol. 2013;11(11):745–60.Brodersen P, Voinnet O. The diversity of RNA silencing pathways in plants. Trends Genet. 2006;22(5):268–80.Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009;10(2):94–108.Ciaudo C, Jay F, Okamoto I, Chen CJ, Sarazin A, Servant N, et al. RNAi-dependent and independent control of LINE1 accumulation and mobility in mouse embryonic stem cells. PLoS Genet. 2013;9(11):e1003791.Ding SW, Voinnet O. Antiviral immunity directed by small RNAs. Cell. 2007;130(3):413–26.Szittya G, Moxon S, Pantaleo V, Toth G, Rusholme Pilcher RL, Moulton V, et al. Structural and functional analysis of viral siRNAs. PLoS Pathog. 2010;6(4):e1000838.Donaire L, Wang Y, Gonzalez-Ibeas D, Mayer KF, Aranda MA, Llave C. Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology. 2009;392(2):203–14.Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell. 2009;136(4):669–87.Liu Q, Feng Y, Zhu Z. Dicer-like (DCL) proteins in plants. Funct Integr Genomics. 2009;9(3):277–86.Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, Green PJ, et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet. 2006;38(6):721–5.Margis R, Fusaro AF, Smith NA, Curtin SJ, Watson JM, Finnegan EJ, et al. The evolution and diversification of Dicers in plants. FEBS Lett. 2006;580(10):2442–50.Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science. 2006;313(5783):68–71.Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, et al. Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res. 2006;34(21):6233–46.Bouche N, Lauressergues D, Gasciolli V, Vaucheret H. An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J. 2006;25(14):3347–56.Moissiard G, Voinnet O. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci U S A. 2006;103(51):19593–8.Qu F, Ye X, Morris TJ. Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc Natl Acad Sci U S A. 2008;105(38):14732–7.Vaucheret H. Plant ARGONAUTES. Trends Plant Sci. 2008;13(7):350–8.Hutvagner G, Simard MJ. Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol. 2008;9(1):22–32.Voinnet O. Use, tolerance and avoidance of amplified RNA silencing by plants. Trends Plant Sci. 2008;13(7):317–28.Palauqui JC, Elmayan T, Pollien JM, Vaucheret H. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 1997;16(15):4738–45.Yoo BC, Kragler F, Varkonyi-Gasic E, Haywood V, Archer-Evans S, Lee YM, et al. A systemic small RNA signaling system in plants. Plant Cell. 2004;16(8):1979–2000.Buhtz A, Pieritz J, Springer F, Kehr J. Phloem small RNAs, nutrient stress responses, and systemic mobility. BMC Plant Biol. 2010;10:64.Buhtz A, Springer F, Chappell L, Baulcombe DC, Kehr J. Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J. 2008;53(5):739–49.Rodriguez-Medina C, Atkins CA, Mann AJ, Jordan ME, Smith PM. Macromolecular composition of phloem exudate from white lupin (Lupinus albus L.). BMC Plant Biol. 2011;11:36.Pallas V, Gomez G. Phloem RNA-binding proteins as potential components of the long-distance RNA transport system. Frontiers in Plant Science. 2013;4:130.Tournier B, Tabler M, Kalantidis K. Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J. 2006;47(3):383–94.Hamilton A, Voinnet O, Chappell L, Baulcombe D. Two classes of short interfering RNA in RNA silencing. EMBO J. 2002;21(17):4671–9.Voinnet O. MicroRNA and autophagy--C. elegans joins the crew. EMBO Rep. 2013;14(6):485–7.Dunoyer P, Schott G, Himber C, Meyer D, Takeda A, Carrington JC, et al. Small RNA duplexes function as mobile silencing signals between plant cells. Science. 2010;328(5980):912–6.Brosnan CA, Mitter N, Christie M, Smith NA, Waterhouse PM, Carroll BJ. Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proc Natl Acad Sci U S A. 2007;104(37):14741–6.Silva TF, Romanel EA, Andrade RR, Farinelli L, Osteras M, Deluen C, et al. Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus. BMC Mol Biol. 2011;12:40.Martinez G, Donaire L, Llave C, Pallas V, Gomez G. High-throughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem. Mol Plant Pathol. 2010;11(3):347–59.Donaire L, Barajas D, Martinez-Garcia B, Martinez-Priego L, Pagan I, Llave C. Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs. J Virol. 2008;82(11):5167–77.Qi X, Bao FS, Xie Z. Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PLoS One. 2009;4(3):e4971.Pantaleo V, Saldarelli P, Miozzi L, Giampetruzzi A, Gisel A, Moxon S, et al. Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine. Virology. 2010;408(1):49–56.Lin KY, Cheng CP, Chang BC, Wang WC, Huang YW, Lee YS, et al. Global analyses of small interfering RNAs derived from Bamboo mosaic virus and its associated satellite RNAs in different plants. PLoS One. 2010;5(8):e11928.Navarro B, Pantaleo V, Gisel A, Moxon S, Dalmay T, Bisztray G, et al. Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant-viroid interaction. PLoS One. 2009;4(11):e7686.Martin R, Arenas C, Daros JA, Covarrubias A, Reyes JL, Chua NH. Characterization of small RNAs derived from Citrus exocortis viroid (CEVd) in infected tomato plants. Virology. 2007;367(1):135–46.St-Pierre P, Hassen IF, Thompson D, Perreault JP. Characterization of the siRNAs associated with peach latent mosaic viroid infection. Virology. 2009;383(2):178–82.Di Serio F, Gisel A, Navarro B, Delgado S, de Alba AE M, Donvito G, et al. Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis. PLoS One. 2009;4(10):e7539.Li R, Gao S, Hernandez AG, Wechter WP, Fei Z, Ling KS. Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation. PLoS One. 2012;7(5):e37127.Hu Q, Hollunder J, Niehl A, Korner CJ, Gereige D, Windels D, et al. Specific impact of tobamovirus infection on the Arabidopsis small RNA profile. PLoS One. 2011;6(5):e19549.Hibi T, Furuki I. Melon Necrotic Spot Virus. In: CMI: AAB Descriptions of Plants Viruses N° 302. Kew, UK: Commonwealth Mycological Institute; 1985.Riviere CJ, Rochon DM. Nucleotide sequence and genomic organization of melon necrotic spot virus. J Gen Virol. 1990;71(Pt 9):1887–96.Diaz JA, Nieto C, Moriones E, Truniger V, Aranda MA. Molecular characterization of a Melon necrotic spot virus strain that overcomes the resistance in melon and nonhost plants. Mol Plant Microbe Interact. 2004;17(6):668–75.Navarro JA, Genoves A, Climent J, Sauri A, Martinez-Gil L, Mingarro I, et al. RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins. Virology. 2006;356(1–2):57–67.Genoves A, Navarro JA, Pallas V. A self-interacting carmovirus movement protein plays a role in binding of viral RNA during the cell-to-cell movement and shows an actin cytoskeleton dependent location in cell periphery. Virology. 2009;395(1):133–42.Genoves A, Navarro JA, Pallas V. The Intra- and intercellular movement of Melon necrotic spot virus (MNSV) depends on an active secretory pathway. Mol Plant Microbe Interact. 2010;23(3):263–72.Serra-Soriano M, Pallas V, Navarro JA. A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. Plant J. 2014;77(6):863–79.Genoves A, Navarro JA, Pallas V. Functional analysis of the five melon necrotic spot virus genome-encoded proteins. J Gen Virol. 2006;87(Pt 8):2371–80.Pallas V, Aparicio F, Herranz MC, Amari K, Sanchez-Pina MA, Myrta A, et al. Ilarviruses of Prunus spp.: a continued concern for fruit trees. Phytopathology. 2012;102(12):1108–20.Pallas V, Aparicio F, Herranz MC, Sanchez-Navarro JA, Scott SW. The molecular biology of ilarviruses. Adv Virus Res. 2013;87:139–81.Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods. 2007;3:12.Blevins T, Rajeswaran R, Aregger M, Borah BK, Schepetilnikov M, Baerlocher L, et al. Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Res. 2011;39(12):5003–14.Takeda A, Tsukuda M, Mizumoto H, Okamoto K, Kaido M, Mise K, et al. A plant RNA virus suppresses RNA silencing through viral RNA replication. EMBO J. 2005;24(17):3147–57.Andersson MG, Haasnoot PC, Xu N, Berenjian S, Berkhout B, Akusjarvi G. Suppression of RNA interference by adenovirus virus-associated RNA. J Virol. 2005;79(15):9556–65.Himeno M, Maejima K, Komatsu K, Ozeki J, Hashimoto M, Kagiwada S, et al. Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome. Virology. 2010;396(1):69–75.Aparicio F, Vilar M, Perez-Paya E, Pallas V. The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA. Virology. 2003;313(1):213–23.Ruiz-Ruiz S, Navarro B, Gisel A, Pena L, Navarro L, Moreno P, et al. Citrus tristeza virus infection induces the accumulation of viral small RNAs (21-24-nt) mapping preferentially at the 3′-terminal region of the genomic RNA and affects the host small RNA profile. Plant Mol Biol. 2011;75(6):607–19.Folimonova SY, Folimonov AS, Tatineni S, Dawson WO. Citrus tristeza virus: survival at the edge of the movement continuum. J Virol. 2008;82(13):6546–56.Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, et al. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology. 2009;388(1):1–7.Karyeija RF, Kreuze JF, Gibson RW, Valkonen JP. Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology. 2000;269(1):26–36.Melnyk CW, Molnar A, Bassett A, Baulcombe DC. Mobile 24 nt small RNAs direct transcriptional gene silencing in the root meristems of Arabidopsis thaliana. Curr Biol. 2011;21(19):1678–83.Gosalvez-Bernal B, Genoves A, Navarro JA, Pallas V, Sanchez-Pina MA. Distribution and pathway for phloem-dependent movement of Melon necrotic spot virus in melon plants. Mol Plant Pathol. 2008;9(4):447–61.Harper SJ, Cowell SJ, Robertson CJ, Dawson WO. Differential tropism in roots and shoots infected by Citrus tristeza virus. Virology. 2014;460–461:91–9.Andika IB, Kondo H, Tamada T. Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves. Mol Plant Microbe Interact. 2005;18(3):194–204.Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide. Cell. 2008;133(1):116–27.Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y. The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol. 2008;49(4):493–500.Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y. Rice MicroRNA effector complexes and targets. Plant Cell. 2009;21(11):3421–35.Xu Y, Huang L, Fu S, Wu J, Zhou X. Population diversity of rice stripe virus-derived siRNAs in three different hosts and RNAi-based antiviral immunity in Laodelphgax striatellus. PLoS One. 2012;7(9):e46238

Argonaute proteins: from structure to function in development and pathological cell fate determination

The highly conserved Argonaute protein family members play a central role in the regulation of gene expression networks, orchestrating the establishment and the maintenance of cell identity throughout the entire life cycle, as well as in several human disorders, including cancers. Four functional Argonaute proteins (AGO1–4), with high structure similarity, have been described in humans and mice. Interestingly, only AGO2 is robustly expressed during human and mouse early development, in contrast to the other AGOs. Consequently, AGO2 is indispensable for early development in vivo and in vitro. Here, we review the roles of Argonaute proteins during early development by focusing on the interplay between specific domains of the protein and their function. Moreover, we report recent works highlighting the importance of AGO posttranslational modifications in cancer

Editorial: The RNA Revolution in Embryonic Development and Cell Differentiation in Health and Disease

Non-coding RNAs (ncRNAs) and their RNA binding proteins (RBPs) are emerging as crucial molecular players involved in normal and pathological cell fate determination. Among them microRNA (miRNAs), acting as gene expression regulators at post-transcriptional level, are involved in several networks relevant for the regulation of stemness, pluripotency, and cell fate determination during embryogenesis and adult life. Interestingly, the relevance for comprehension of the generation of spatio-temporal specificity of miRNA, their levels and dynamics of expression, and how the animal miRNA repertoire has evolved and diversified, is reported by Dexheimer and Cochella. The understanding of these regulation mechanisms could really help us to understand the contribution of miRNAs to the embryonic development and cell differentiation. Of note, alteration of the molecular mechanisms involving the ncRNA and their effectors in addition to defects in RNA modification and editing may contribute to the pathological cell fate determination in cancer and degenerative diseases

An overview of the girafe package

The intent of package girafe is to facilitate the functional exploration of the alignments of multiple reads 1 from next-generation sequencing (NGS) data to a genome. It extends the functionality of the Bioconductor (Gentleman et al., 2004) packages Short