ABSTRACT
The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.
Subject(s)
Bombyx , RNA, Small Interfering , Bombyx/genetics , Bombyx/metabolism , Animals , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , Argonaute Proteins/metabolism , Argonaute Proteins/genetics , Piwi-Interacting RNAABSTRACT
PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.
Subject(s)
Bombyx , Animals , Bombyx/genetics , Larva/genetics , Sex Differentiation , Piwi-Interacting RNA , DrosophilaABSTRACT
The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte-specific factor 1 (Gtsf1), an evolutionarily conserved zinc finger protein, promotes catalysis by PIWI proteins. Many animals have multiple Gtsf1 paralogs; however, their respective roles in the piRNA pathway are not fully understood. Here, we dissected the roles of Gtsf1 and its paralog Gtsf1-like (Gtsf1L) in the silkworm piRNA pathway. We found that Gtsf1 and Gtsf1L preferentially bind the two silkworm PIWI paralogs, Siwi and BmAgo3, respectively, and facilitate the endonuclease activity of each PIWI protein. This orthogonal activation effect was further supported by specific reduction of BmAgo3-bound Masculinizer piRNA and Siwi-bound Feminizer piRNA, the unique piRNA pair required for silkworm feminization, upon depletion of Gtsf1 and Gtsf1L, respectively. Our results indicate that the two Gtsf paralogs in silkworms activate their respective PIWI partners, thereby facilitating the amplification of piRNAs.
ABSTRACT
Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins have diverse biological functions and many retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, the PIWI proteins-a specialized class of Argonaute proteins-use 21-35 nucleotide PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis1. The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalysed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2) and MIWI22-4 (PIWIL4). The piRNA-guided endoribonuclease activities of MIWI and MILI are essential for the production of functional sperm5,6. piRNA-directed silencing in mice and insects also requires GTSF1, a PIWI-associated protein of unknown function7-12. Here we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 is thus an example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.
Subject(s)
Argonaute Proteins , Intracellular Signaling Peptides and Proteins , RNA Cleavage , RNA, Small Interfering , Animals , Argonaute Proteins/classification , Argonaute Proteins/metabolism , Biocatalysis , Female , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , RNA, Small Interfering/metabolismABSTRACT
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.
Subject(s)
Endoribonucleases/genetics , Gene Expression Profiling/methods , Insect Proteins/genetics , RNA, Small Interfering/genetics , RNA/genetics , Animals , Base Sequence , Bombyx , Cell Line , Endoribonucleases/metabolism , Female , Insect Proteins/metabolism , Male , Mice, Inbred C57BL , Mutation , Phosphatidic Acids/chemistry , RNA/chemistry , RNA/metabolism , RNA Interference , RNA, Small Interfering/metabolism , RNA-Seq/methods , Testis/metabolismABSTRACT
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.
Subject(s)
Bombyx , Drosophila Proteins , Animals , Argonaute Proteins/genetics , Bombyx/genetics , Bombyx/metabolism , DNA Transposable Elements , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Germ Cells/metabolism , Mice , RNA, Small Interfering/genetics , RibonucleoproteinsABSTRACT
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.
Subject(s)
Amino Acid Motifs , Consensus Sequence , Insect Proteins/chemistry , Insect Proteins/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Phospholipase D/chemistry , Phospholipase D/metabolism , RNA, Small Interfering/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Base Sequence , Bombyx , Cell Line , Cell-Free System , Gene Knockout Techniques , Insect Proteins/genetics , Methylation , Mice , RNA Helicases/metabolismABSTRACT
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.
Subject(s)
Exoribonucleases/genetics , Germ Cells/growth & development , Meiosis/genetics , RNA, Small Interfering/genetics , Ribonucleases/metabolism , Animals , Gene Silencing , Germ Cells/metabolism , Male , Mice , Mitochondrial Proteins/genetics , Mutation , Phospholipase D/genetics , Retroelements/genetics , Ribonucleases/genetics , Spermatogenesis/genetics , Testis/growth & developmentABSTRACT
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.
ABSTRACT
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.
Subject(s)
Arginine/metabolism , Argonaute Proteins/metabolism , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolism , Animals , Argonaute Proteins/chemistry , Argonaute Proteins/genetics , Crystallography, X-Ray , Epigenesis, Genetic , HEK293 Cells , Humans , Male , Methylation , Models, Molecular , Mutation , Protein Binding , Protein Processing, Post-Translational , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Signal Transduction , Structure-Activity RelationshipABSTRACT
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 â¼35-40-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.
Subject(s)
Bombyx/enzymology , Bombyx/genetics , Insect Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolism , Animals , Bombyx/metabolism , Mitochondria/metabolismABSTRACT
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.
Subject(s)
Adenine/metabolism , Argonaute Proteins/metabolism , RNA, Small Interfering/metabolism , Uridine/metabolism , Animals , Bombyx/genetics , Bombyx/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Mice , Models, Genetic , Molecular Sequence Data , Peptide Initiation Factors/metabolismABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Gene Expression Regulation, Developmental , Genome, Helminth/genetics , RNA, Small Interfering/biosynthesis , AnimalsABSTRACT
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.
Subject(s)
Argonaute Proteins/genetics , Exoribonucleases/metabolism , MicroRNAs/metabolism , Amino Acid Sequence , Argonaute Proteins/metabolism , Gene Expression , HEK293 Cells , HeLa Cells , Humans , K562 Cells , MicroRNAs/chemistry , MicroRNAs/genetics , Molecular Sequence Data , TransfectionABSTRACT
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.
Subject(s)
Argonaute Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Insect Proteins/metabolism , RNA, Small Interfering/metabolism , Animals , Argonaute Proteins/genetics , Benzoquinones/pharmacology , Bombyx/cytology , Cell Line , Female , Gene Expression Regulation , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/genetics , Immunoprecipitation , Insect Proteins/genetics , Lactams, Macrocyclic/pharmacology , Methylation , MicroRNAs/genetics , MicroRNAs/metabolism , Ovary/cytology , RNA, Small Interfering/geneticsABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/metabolism , DNA Helicases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinases/metabolism , Stress, Physiological , Animals , Humans , Molecular Chaperones/metabolismABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , DNA Helicases/metabolism , HSP90 Heat-Shock Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-ets/metabolism , ATPases Associated with Diverse Cellular Activities , Blotting, Western , HeLa Cells , Humans , Immunoprecipitation , Mass Spectrometry , Signal TransductionABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , Nonsense Mediated mRNA Decay , Telomerase/metabolism , Trans-Activators/metabolism , 14-3-3 Proteins/chemistry , Binding Sites , HEK293 Cells , HeLa Cells , Humans , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases , Protein Structure, Tertiary , RNA Helicases , Telomerase/chemistry , Threonine/physiology , Trans-Activators/chemistryABSTRACT
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.