Penaeus monodon is protected against gill-associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

Gill-associated virus (GAV) is a nidovirus that commonly infects Penaeus monodon (black tiger shrimp) in eastern Australia, causing morbidity and mortalities in the acute stage of disease. Here we explored the possibility of inhibiting GAV replication and disease using double-stranded (ds)RNAs expressed in bacteria and delivered either orally or by muscle injection. To enhance potential RNA interference (RNAi) responses, 5 long dsRNAs were used that targeted open reading frame 1a/1b (ORF1a/b) gene regions and thus only the genomic length RNA. To examine oral delivery, P. monodon were fed pellets incorporating a pool of formalin-fixed bacteria containing the 5 GAV-specific dsRNAs before being injected with a minimal lethal GAV dose. Feeding with the pellets continued post-challenge but did not reduce mortality accumulation and elevation in GAV loads. In contrast, muscle injection of the dsRNAs purified from bacteria was highly effective at slowing GAV replication and protecting shrimp against acute disease and mortalities. In synergy with these data, dsRNA targeted to P. monodon beta-actin mRNA caused 100% mortality following injection, whilst its oral delivery caused no mortality. Findings confirm that injected dsRNA can mount effective RNAi responses in P. monodon to endogenous shrimp mRNA and exogenous viral RNAs, but when delivered orally in bacteria as a feed component, the same dsRNAs are ineffective. The efficacy of the RNAi response against GAV provided by injection of dsRNAs targeted to multiple genome sites suggests that this strategy might have general applicability in enhancing protection against other shrimp single-stranded (ss)RNA viruses, particularly in hatcheries or breeding programs where injection-based delivery systems are practical.     

Molecular characterization and gene functional analysis of Dicer‐2 gene from Nilaparvata lugens (Hemiptera: Geometroidea)

Abstract Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a serious rice pest in many countries of Asia, causes a great loss in rice production every year. RNA interference (RNAi) is a powerful technology for gene function study in insects and a potential tool for pest control. As a core component of RNAi pathway, Dicer‐2 (Dcr‐2) protein determines the production of small interfering RNA (siRNA) and is crucial for the efficiency of RNAi. In this study, the full‐length complementary DNA (cDNA) of N. lugens Dcr‐2 (NlDcr‐2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of NlDcr‐2 gene. The complete Dcr‐2 cDNA of N. lugens was 4 971 bp in length with an open reading frame (ORF) of 1,656 amino acids. Phylogenetic and protein domain analysis showed that the predicted NlDcr‐2 protein was similar to Tribolium castaneum. In the RNAi experiment, the messenger RNA level of NlDcr‐2 was significantly reduced by NlDcr‐2 double‐stranded RNA (dsRNA) (dsDcr‐2). Fifty‐five per cent decrease of NlDcr‐2 was found after 4 days of unremitting feeding. No significant effect was observed on the development of N. lugens after dsRNA ingestion.     

Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon)

We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection.     

Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage

The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.     

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.     

Isolation and characterization of cDNAs encoding Ars2 and Pasha homologues, two components of the RNA interference pathway in Litopenaeus vannamei

The RNA interference (RNAi) is an evolutionarily conserved protective mechanism in eukaryotes against parasitic foreign nucleic acids. Previous studies demonstrated that the RNAi mechanism is important for shrimp antiviral immunity. Here, we report the identification and functional analysis of two key components of the shrimp RNAi activity: Litopenaeus vannamei arsenite resistance gene 2 (LvArs2) and partner of drosha (LvPasha). The full-length cDNA of LvArs2 was 3470 bp, including a 5′ untranslated region (UTR) of 167 bp, a 3′ UTR of 639 bp, and an open reading frame (ORF) of 2664 bp that encoded 887 amino acid residues with an estimated molecular mass of 102.5 kDa. The full-length cDNA of LvPasha was 2654 bp, including a 5′ UTR of 99 bp, a 3′ UTR of 560 bp, and an ORF of 1995 bp that encoded 664 amino acid residues with an estimated molecular mass of 74.2 kDa. Co-immunoprecipitation demonstrated that LvArs2 interacted with L. vannamei Dicer2 (LvDcr2) and LvPasha in Drosophila Schneider 2 (S2) cells, suggesting that LvArs2 may be involved in regulation of the miRNA/siRNA pathways in L.vannamei. Subcellular localization assays demonstrated both LvArs2 and LvPasha proteins mainly presented in the nucleus. After Poly(C-G) stimulation, the expression of LvArs2 was suppressed and expression of LvPasha was enhanced in shrimp gills. These results suggest that LvArs2 and LvPasha may participate in the defense against RNA viruses in crustacea.     

The contributions of dsRNA structure to Dicer specificity and efficiency

Dicer processes long double-stranded RNA (dsRNA) and pre-microRNAs to generate the functional intermediates (short interfering RNAs and microRNAs) of the RNA interference pathway. Here we identify features of RNA structure that affect Dicer specificity and efficiency. The data presented show that various attributes of the 3' end structure, including overhang length and sequence composition, play a primary role in determining the position of Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA). We also demonstrate that siRNA end structure affects overall silencing functionality. Awareness of these new features of Dicer cleavage specificity as it is related to siRNA functionality provides a more detailed understanding of the RNAi mechanism and can shape the development of hairpins with enhanced functionality.     

Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila

Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.     

Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs

Many classes of small RNA (sRNA) involved in RNA silencing are generated by double-stranded RNA (dsRNA) processing. Although principles of sRNA biogenesis have emerged, newly identified classes of sRNAs have features that suggest additional biogenesis mechanisms. Tetrahymena thermophila expresses one such class, comprising sRNAs of 23 and 24 nucleotides (nt) with an absolute strand bias in accumulation. Here we demonstrate sRNA production by the T. thermophila Dicer Dcr2 and the RNA-dependent RNA polymerase Rdr1, which purifies as a multisubunit RNA-dependent RNA polymerase complex (RDRC). Dcr2 and RDRC interact, stimulating Dcr2 activity. Moreover, Dcr2 specificity is influenced by RDRC beyond this physical interaction, as Dcr2 generates discrete 23- and 24-nt sRNAs only from dsRNA with a 5'-triphosphate. These findings suggest that sRNA strand bias arises from Dcr2 processing polarity, conferred by physical and functional coupling of RDRC and Dicer enzymes.     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

A Tudor staphylococcal nuclease from Penaeus monodon: cDNA cloning and its involvement in RNA interference

RNA interference (RNAi) plays an important role in an antiviral defense in shrimp. RNAi technology has been extensively used for inhibition of viral replication and studying gene function. However, the mechanism of shrimp RNAi pathway is still poorly understood. In this study, we identified and characterized an additional protein in the RNAi pathway, Tudor staphylococcal nuclease from Penaeus monodon (PmTSN). The full-length cDNA of PmTSN is 2897 bp, with an open reading frame encoding a putative protein of 889 amino acids. Phylogenetic analysis and domain structure comparison revealed that PmTSN is more closely related to vertebrate TSN by sharing the amino acid sequence identity of 57% with TSN of zebrafish. This represents a new type of TSN proteins by exhibiting the four tandem repeat of staphylococcal nuclease-like domain (SN), followed by a Tudor and a partially truncated C-terminal SN domain. Knockdown of PmTSN by dsRNA targeting SN3 domain resulted in the impairment of dsRNA targeting PmRab7 gene to silence PmRab7 expression. In addition, the efficiency of dsRNA targeting YHV-protease gene inhibiting yellow head virus replication was decreased in the PmTSN-knockdown shrimps. Our results imply that PmTSN is involved in dsRNA-mediated gene silencing in shrimp and thus we identified the additional protein involved in shrimp RNAi pathway.     

Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia

In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3′-to-5′ and 5′-to-3′ transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.     

Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi

The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.     

High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon

A novel leucine-rich repeat (LRR) cDNA has been cloned from hemocytes of the black tiger shrimp Penaeus monodon by 5' rapid amplification of cDNA ends. The full-length of P. monodon LRR (PmLRR) consisted of 2604 bp with a 1686-bp open reading frame, encoding 561 amino acids. The deduced protein contained a high proportion of leucine residues (17%) and had significant homology to LRR-containing proteins from bacteria to humans. Sixteen tandem LRR motifs of 23-24 amino acids in length occurred in the primary sequence. The computed 3D structure revealed a horseshoe shape consisting of alternately repeated strand and helical domains. Such structures are generally considered to mediate protein - protein interactions and to our knowledge, this is the first report of an LRR protein from a crustacean. PmLRR expression was tissue-specific (i.e. highest in hemocytes, intestine and lymphoid organ) suggesting that it may play some roles in shrimp defense against pathogens. A preliminary test suggested that PmLRR may be down-regulated after viral injection.     

Characterization of Argonaute2 gene from black tiger shrimp (Penaeus monodon) and its responses to immune challenges

Argonaute2 binds to a short guide RNA (microRNA or short interfering RNA) and guides RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. Here we identified and characterized Argonaute2 from black tiger shrimp Penaeus monodon (designated as PmAgo2). The full-length cDNA of PmAgo2 contained a 5′ untranslated region (UTR) of 106 bp, an open reading frame (ORF) of 2616 bp and a 3′ UTR of 123 bp. The predicted PmAgo2 protein is 99.4 KDa with the theoretical isoelectric point of 9.54. PmAgo2 shared the highest similarity of amino acid with Marsupenaeus japonicus Argonaute2 and Litopenaeus vannamei Argonaute2, at 69.0% and 68.5%, respectively. Phylogenic analysis showed PmAgo2 clustered with shrimp Argonaute2, and closed to the group of insects. Real-time quantitative PCR showed that PmAgo2 was widely expressed in almost all examined tissues except eyestalk, with high expression in lymph and haemocyte. mRNA expression also revealed that PmAgo2 was significantly up-regulated by Staphylococcus aureus and White Spot Syndrome Virus (WSSV) in hepatopancreas. Furthermore, our study also confirmed that dsRNA and ssRNA homologous poly (I:C) and R848 activated the expression of PmAgo2. The result indicated that PmAgo2 responded to both bacterial infection and viral infection, especially, it may induce an ssRNA-mediated RNAi with other core members of siRNA pathway in black tiger shrimp.     

Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos

RNA interference (RNAi) refers to the selective degradation of mRNA induced by double-stranded RNA (dsRNA), first discovered in Caenorhabditis elegans. Homology-dependent silencing phenomena related to RNAi have been observed in many species from all eukaryotic kingdoms. RNAi and related mechanisms share several conserved components. The hallmark of these phenomena is the presence of short dsRNA molecules (21-25 bp long), termed short interfering RNA (siRNA), which are generated from dsRNA by the activity of Dicer, a specific type III RNAse. These molecules serve as a template for the recognition and cleavage of the cognate mRNA. As it is beyond the scope of a single review to cover all aspects of RNAi, this review will focus on certain steps of the pathway relevant to mammals and on the use of long dsRNA to specifically silence genes in mammalian cells permissive to this technique, such as oocytes and early embryos.     

凡纳滨对虾MT基因序列及其在卵巢发育和低温胁迫中的表达分析

在低温处理仔虾全长cDNA文库的筛选测序中, 获得凡纳滨对虾(Litopenaeus vannmei)金属硫蛋白基因全长cDNA序列, 该序列含有425个碱基, 包含177 bp开放阅读框, 上游98 bp的非编码区及下游150 bp 的非编码区, 编码58个氨基酸, 其中半胱氨酸含量丰富, 富含金属硫蛋白典型的Cys-X(1-3)-Cys 结构。多序列比对表明, 凡纳滨对虾MT蛋白序列与美洲螯龙虾(Homarus americanus)MT蛋白序列具最高同源性72.4%。Real-time PCR结果表明, 凡纳滨对虾MT基因在卵巢组织中呈优势表达, 在不同发育期的卵巢中的表达量都很高, 在低温处理凡纳滨对虾肝胰腺组织中上调表达。实验所得结果为研究凡纳滨对虾金属硫蛋白基因在生殖发育和低温应激中的功能提供了参考。