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1.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
2.
EMBO Rep ; 22(7): e51342, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33973704

RESUMO

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposable elements and safeguard fertility in germ cells. Many protein factors required for piRNA biogenesis localize to perinuclear ribonucleoprotein (RNP) condensates named nuage, where target silencing and piRNA amplification are thought to occur. In mice, some of the piRNA factors are found in discrete cytoplasmic foci called processing bodies (P-bodies). However, the dynamics and biological significance of such compartmentalization of the piRNA pathway remain unclear. Here, by analyzing the subcellular localization of functional mutants of piRNA factors, we show that piRNA factors are actively compartmentalized into nuage and P-bodies in silkworm cells. Proper demixing of nuage and P-bodies requires target cleavage by the PIWI protein Siwi and ATP hydrolysis by the DEAD-box helicase BmVasa, disruption of which leads to promiscuous overproduction of piRNAs deriving from non-transposable elements. Our study highlights a role of dynamic subcellular compartmentalization in ensuring the fidelity of piRNA biogenesis.


Assuntos
Bombyx , Proteínas de Drosophila , Animais , Proteínas Argonauta/genética , Bombyx/genética , Bombyx/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Camundongos , RNA Interferente Pequeno/genética , Ribonucleoproteínas
3.
Nature ; 578(7794): 311-316, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996847

RESUMO

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.


Assuntos
Motivos de Aminoácidos , Sequência Consenso , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Fosfolipase D/química , Fosfolipase D/metabolismo , RNA Interferente Pequeno/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Sistema Livre de Células , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Metilação , Camundongos , RNA Helicases/metabolismo
4.
EMBO Rep ; 19(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29444933

RESUMO

PIWI-interacting RNAs (piRNAs) are germ cell-specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single-stranded RNAs to generate 5' termini of precursor piRNAs (pre-piRNAs) that are consecutively loaded into PIWI-family proteins. Subsequently, these pre-piRNAs are trimmed at their 3'-end by an exonuclease called Trimmer. Recently, poly(A)-specific ribonuclease-like domain-containing 1 (PNLDC1) was identified as the pre-piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post-natal pre-piRNAs. In addition, piRNA trimming defects in embryonic and post-natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post-meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1-mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.


Assuntos
Exorribonucleases/genética , Células Germinativas/crescimento & desenvolvimento , Meiose/genética , RNA Interferente Pequeno/genética , Ribonucleases/metabolismo , Animais , Inativação Gênica , Células Germinativas/metabolismo , Masculino , Camundongos , Proteínas Mitocondriais/genética , Mutação , Fosfolipase D/genética , Retroelementos/genética , Ribonucleases/genética , Espermatogênese/genética , Testículo/crescimento & desenvolvimento
5.
DNA Res ; 2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29360973

RESUMO

Bombyx mori macula-like virus (BmMLV) is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmMLV is currently characterized as an unclassified maculavirus. BmMLV accumulates at extremely high levels in cell lines derived from the silkworm, Bombyx mori, but it does not lead to lethality and establishes persistent infections. It is unknown how this insect maculavirus replicates and establishes persistent infections in insect cells. Here, we showed that BmMLV p15, which is located on a subgenomic fragment and is not found in plant maculaviruses, is highly expressed in BmMLV-infected silkworm cells and that p15 protein is required to establish BmMLV infections in silkworm cells. We also showed that two distinct small RNA-mediated pathways maintain BmMLV levels in BmMLV-infected silkworm cells, thereby allowing the virus to establish persistent infection. Virus-derived siRNAs and piRNAs were both produced as the infection progressed. Knockdown experiments demonstrated that the exogenous RNAi pathway alone or RNAi and piRNA pathways function cooperatively to silence BmMLV RNA and that both pathways are important for normal growth of BmMLV-infected silkworm cells. On the basis of our study, we propose a mechanism of how a plant virus-like insect virus can establish persistent infections in insect cells.

6.
Proc Natl Acad Sci U S A ; 114(47): 12483-12488, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29118143

RESUMO

The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.


Assuntos
Arginina/metabolismo , Proteínas Argonauta/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Cristalografia por Raios X , Epigênese Genética , Células HEK293 , Humanos , Masculino , Metilação , Modelos Moleculares , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Relação Estrutura-Atividade
7.
Cell ; 164(5): 962-73, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26919431

RESUMO

PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3' ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3'-5' exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ∼35-40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.


Assuntos
Bombyx/enzimologia , Bombyx/genética , Proteínas de Insetos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Bombyx/metabolismo , Mitocôndrias/metabolismo
9.
Mol Cell ; 56(5): 708-16, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25453759

RESUMO

PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. We find that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3.


Assuntos
Adenina/metabolismo , Proteínas Argonauta/metabolismo , RNA Interferente Pequeno/metabolismo , Uridina/metabolismo , Animais , Bombyx/genética , Bombyx/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Fatores de Iniciação de Peptídeos/metabolismo
10.
Genes Dev ; 28(7): 665-71, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24696451

RESUMO

The PIWI-interacting RNA (piRNA) pathway protects animal germline cells from transposable elements and other genomic invaders. Although the genome defense function of piRNAs has been well established, the mechanisms of their biogenesis remain poorly understood. In this issue of Genes & Development, three groups identify novel factors required for piRNA biogenesis in Caenorhabditis elegans. These works greatly expand our understanding of the piRNA pathway in worms, highlighting both its shared and its unique properties.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma Helmíntico/genética , RNA Interferente Pequeno/biossíntese , Animais
11.
Cell Rep ; 5(3): 715-26, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24209750

RESUMO

MicroRNAs (miRNAs) are typically generated as ~22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form an RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3' arm of the pre-miR-451 precursor hairpin by Ago2. The 3' end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A)-specific ribonuclease (PARN) as the enzyme responsible for the 3'-5' exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage.


Assuntos
Proteínas Argonauta/genética , Exorribonucleases/metabolismo , MicroRNAs/metabolismo , Sequência de Aminoácidos , Proteínas Argonauta/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células K562 , MicroRNAs/química , MicroRNAs/genética , Dados de Sequência Molecular , Transfecção
12.
RNA ; 19(7): 896-901, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23681506

RESUMO

PIWI-interacting RNAs (piRNAs) defend the genome against transposon activity in animal gonads. The Hsp90 chaperone machinery has been implicated in the piRNA pathway, but its exact role remains obscure. Here, we examined the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on the piRNA pathway. In the silkworm ovary-derived BmN4 cells, 17-AAG treatment reduced the level of piRNAs and PIWI proteins. In vitro, the 5'-nucleotide preference upon precursor piRNA loading was compromised by 17-AAG, whereas 3'-end trimming and 2'-O-methylation were unaffected. Our data highlight a role of Hsp90 in accurate loading of precursor piRNAs into PIWI proteins.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Insetos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/genética , Benzoquinonas/farmacologia , Bombyx/citologia , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Imunoprecipitação , Proteínas de Insetos/genética , Lactamas Macrocíclicas/farmacologia , Metilação , MicroRNAs/genética , MicroRNAs/metabolismo , Ovário/citologia , RNA Interferente Pequeno/genética
13.
Nucleus ; 3(1): 29-43, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22540023

RESUMO

Proteins of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family are activated by various cellular stresses, including DNA damage, premature termination codon and nutritional status, and induce appropriate cellular responses. The importance of PIKK functions in the maintenance of genome integrity, accurate gene expression and the proper control of cell growth/proliferation is established. Recently, ATPase associated diverse cellular activities (AAA+) proteins RUVBL1 and RUVBL2 (RUVBL1/2) have been shown to be common regulators of PIKKs. The RUVBL1/2 complex regulates PIKK-mediated stress responses through physical interactions with PIKKs and by controlling PIKK mRNA levels. In this review, the functions of PIKKs in stress responses are outlined and the physiological significance of the integrated regulation of PIKKs by the RUVBL1/2 complex is presented. We also discuss a putative "PIKK regulatory chaperone complex" including other PIKK regulators, Hsp90 and the Tel2 complex.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Estresse Fisiológico , Animais , Humanos , Chaperonas Moleculares/metabolismo
14.
Nucleic Acids Res ; 40(3): 1251-66, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21965535

RESUMO

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors.


Assuntos
Proteínas de Transporte/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Telomerase/metabolismo , Transativadores/metabolismo , Proteínas 14-3-3/química , Sítios de Ligação , Células HEK293 , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases , RNA Helicases , Telomerase/química , Treonina/fisiologia , Transativadores/química
15.
Cancer Sci ; 103(1): 50-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21951644

RESUMO

Heat shock protein 90 (Hsp90), a conserved molecular chaperone for a specific set of proteins critical for signal transduction including several oncogenic proteins, has been recognized as a promising target for anticancer therapy. Hsp90 inhibition also sensitizes cancer cells to DNA damage. However, the underlying mechanisms are not fully understood. Here, we provide evidence that Hsp90 is a general regulator of phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins, central regulators of stress responses including DNA damage. Inhibition of Hsp90 causes a reduction of all PIKK and suppresses PIKK-mediated signaling. In addition, Hsp90 forms complexes with RUVBL1/2 complex and Tel2 complex, both of which have been shown to interact with all PIKK and control their abundance and functions. These results suggest that Hsp90 can form multiple complexes with the RUVBL1/2 complex and Tel2 complex and function in the regulation of PIKK, providing additional rationale for the effectiveness of Hsp90 inhibition for anticancer therapy, including sensitization to DNA damage.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Western Blotting , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Transdução de Sinais
16.
Nat Struct Mol Biol ; 18(10): 1153-8, 2011 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-21926993

RESUMO

Drosophila melanogaster has two Dicer proteins with specialized functions. Dicer-1 liberates miRNA-miRNA* duplexes from precursor miRNAs (pre-miRNAs), whereas Dicer-2 processes long double-stranded RNAs into small interfering RNA duplexes. It was recently demonstrated that Dicer-2 is rendered highly specific for long double-stranded RNA substrates by inorganic phosphate and a partner protein R2D2. However, it remains unclear how Dicer-1 exclusively recognize pre-miRNAs. Here we show that fly Dicer-1 recognizes the single-stranded terminal loop structure of pre-miRNAs through its N-terminal helicase domain, checks the loop size and measures the distance between the 3' overhang and the terminal loop. This unique mechanism allows fly Dicer-1 to strictly inspect the authenticity of pre-miRNA structures.


Assuntos
Proteínas de Drosophila/metabolismo , MicroRNAs/genética , RNA Helicases/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , MicroRNAs/química , RNA Helicases/genética , Ribonuclease III/genética , Especificidade por Substrato
17.
Mol Cell ; 43(6): 1015-22, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21925389

RESUMO

PIWI-interacting RNAs (piRNAs) are 23-30 nucleotides small RNAs that act with PIWI proteins to silence transposon activity in animal gonads. In contrast to microRNAs and small interfering RNAs, the biogenesis of piRNAs, including how 3' ends are formed, remains largely unknown. Here, by using lysate from BmN4, a silkworm ovary-derived cell line, we have developed a cell-free system that recapitulates key steps of piRNA biogenesis: loading of long single-stranded precursor RNAs into PIWI proteins with 5'-nucleotide bias, followed by Mg(2+)-dependent 3' to 5' exonucleolytic trimming and 2'-O-methylation at 3' ends. Importantly, 3' end methylation is tightly coupled with trimming and yet is not a prerequisite for determining the mature piRNA length. Our system provides a biochemical framework for dissecting piRNA biogenesis.


Assuntos
Bombyx/genética , Processamento de Terminações 3' de RNA , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/fisiologia
18.
Genes Dev ; 25(2): 153-64, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21245168

RESUMO

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.


Assuntos
Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosfatidilinositol 3-Quinases/genética , Proteínas Quinases/genética , Multimerização Proteica/fisiologia , Estrutura Quaternária de Proteína , Proteínas Serina-Treonina Quinases , RNA Helicases , Proteínas Recombinantes/metabolismo , Transativadores/metabolismo
19.
Sci Signal ; 3(116): ra27, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20371770

RESUMO

Phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins play essential roles in DNA-based and RNA-based processes, such as the response to DNA damage, messenger RNA (mRNA) quality control, transcription, and translation, where they contribute to the maintenance of genome integrity and accurate gene expression. The adenosine triphosphatases associated with diverse cellular activities (AAA+) family proteins RuvB-like 1 (RUVBL1) and RUVBL2 are involved in various cellular processes, including transcription, RNA modification, DNA repair, and telomere maintenance. We show that RUVBL1 and RUVBL2 associate with each PIKK family member. We also show that RUVBL1 and RUVBL2 control PIKK abundance at least at the mRNA level. Knockdown of RUVBL1 or RUVBL2 decreased PIKK abundance and impaired PIKK-mediated signaling. Analysis of SMG-1, a PIKK family member involved in nonsense-mediated mRNA decay (NMD), revealed an essential role for RUVBL1 and RUVBL2 in NMD. RUVBL1 and RUVBL2 associated with SMG-1 and the messenger ribonucleoproteins in the cytoplasm and promoted the formation of mRNA surveillance complexes during NMD. Thus, RUVBL1 and RUVBL2 regulate PIKK functions on two different levels: They control the abundance of PIKKs, and they stimulate the formation of PIKK-containing molecular complexes, such as those involved in NMD.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Estabilidade de RNA/fisiologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Western Blotting , Caenorhabditis elegans , DNA Complementar/genética , Escherichia coli , Humanos , Imunoprecipitação , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases , Estabilidade de RNA/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas/metabolismo
20.
Genes Dev ; 23(9): 1091-105, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19417104

RESUMO

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed "SURF" that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.


Assuntos
Códon sem Sentido/metabolismo , Regulação Enzimológica da Expressão Gênica , Complexos Multienzimáticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Subunidades Proteicas/metabolismo , Estabilidade de RNA/fisiologia , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/metabolismo , Glutationa/análogos & derivados , Glutationa/metabolismo , Células HeLa , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Ribossomos/metabolismo
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