their size. Here, we differentiate the major types of siRNAs according to the molecules that trigger their production, a classification scheme that best captures the biological distinctions among small silencing RNAs.
siRNAs derived from exogenous agents
Early examples of RNAi were triggered by exogenous dsRNA. In these cases, long, exogenous dsRNA is cleaved into double-stranded siRNAs by Dicer (Dcr), a dsRNA-specific RNase III family ribonuclease 7 (FIG. 1). siRNA duplexes produced by Dicer comprise two ~21 nt strands,each bearing a 5′ phosphate and 3′ hydroxyl group, paired so as to leave 3′, two-nucleotide overhangs 5,8,9. The strand that directs silencing is called the guide, whereas the other strand,which is ultimately destroyed, is the passenger. Target regulation by siRNAs is mediated by the RNA-induced silencing complex (RISC), the generic name for an Argonaute-small RNA complex 6. In addition to an Argonaute protein and a small RNA guide, RISC may also contain auxiliary proteins that extend or modify its function, for example, proteins that re-direct the target mRNA to a site of general mRNA degradation 10.
While mammals and C. elegans each have a single Dicer that makes both miRNAs and
siRNAs 11–14, Drosophila has two Dicers: Dcr-1 makes miRNAs, whereas Dcr-2 is specialized for siRNA production 15. The fly RNAi pathway defends against viral infection, and Dicer specialization may reduce competition between pre-miRNAs and viral dsRNAs for Dicer.Alternatively, Dcr-2 and Ago2 specialization might reflect the evolutionary pressure on the siRNA pathway to counter rapidly evolving viral strategies to escape RNAi. In fact, dcr-2 and ago2 are among the most rapidly evolving Drosophila genes 16. C. elegans may achieve similar specialization with a single Dicer by using the double-stranded RNA-binding protein, RDE-4,as the gatekeeper for entry into the RNAi pathway 17. However, no natural virus infection has been documented in C. elegans 18. In contrast, mammals may not use the RNAi pathway to respond to viral infection, having evolved an elaborate, protein-based immune system 19–21.The relative thermodynamic stabilities of the 5′ ends of the two siRNA strands in the duplex determines the identity of the guide and passenger strands 22–24. In flies, this thermodynamic difference is sensed by the dsRNA-binding protein R2D2, the partner of Dcr-2 and a component of the RISC Loading Complex (RLC)25,26. The RLC recruits Argonaute2 (Ago2), to which it transfers the siRNA duplex. Ago2 can then cleave the passenger strand as if it were a target RNA 27–31. (Ago2 always cleaves its RNA target at the phosphodiester bond that lies between the nucleotides paired to guide nucleotides 10 and 11 (Refs. 8,9). Release of the passenger strand after its cleavage converts pre-RISC to RISC: mature RISC contains only single-stranded guide RNA. In flies, the guide strand is 2′-O -methylated at its 3′ end by the S -adenosyl methionine-dependent methyltransferase, Hen1, completing RISC assembly 32,33. In plants,both miRNAs and siRNAs are terminally methylated, which is crucial for their stability 34–36.
Plants exhibit a surprising diversity of small RNA types and the proteins that generate them.The diversification of RNA silencing pathways in plants may reflect the need of a sessile organism to cope with biotic and abiotic stress. The number of RNA silencing proteins can vary enormously among animals, too, with C. elegans producing 27 distinct Argonaute proteins compared to five in flies. Phylogenetic data suggest that nearly all of these “extra” C.elegans Argonautes act in the secondary siRNA pathway, perhaps because endogenous,
secondary siRNAs are so plentiful in worms 37. Arabidopsis thaliana has four Dicer-like (DCL)proteins and 10 Argonautes, with both unique and redundant functions. In plants, inverted repeat transgenes or co-expressed sense and antisense transcripts produce two sizes of siRNAs,21 nt and 24 nt 38,39. The 21 nt siRNAs are produced by DCL4, but in the absence of DCL4,DCL2 can substitute, making 22 nt siRNAs 40–44. The DCL4-produced 21-mers typically
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associate with AGO1 and guide mRNA cleavage. The 24-mers associate with AGO4 (major)and AGO6 (surrogate), and promote the formation of repressive chromatin 45.
In plants, exogenous sources of siRNAs are not confined to dsRNAs. Single-stranded sense transcripts from tandemly repeated or highly expressed single-copy transgenes are converted to dsRNA by RDR6, a member of the RNA-dependent RNA Polymerase (RdRP) family,transcribe single-stranded RNA from an RNA template 46 (Box 1). RDR6 and RDR1, also convert viral single-stranded RNA into dsRNA, initiating an anti-viral RNAi response 47. The resulting dsRNA is cleaved by Dicer into siRNAs that are terminally 2′-O -methylated by HEN136. Why plants RNAs expressed from transgenes are converted by RDR6 into dsRNA,but abundant, endogenous mRNAs are not, is poorly understood. Recent evidence that some housekeeping exonucleases compete with plant RNA silencing pathways for aberrant RNAs suggests that substandard RNA transcripts—e.g. those lacking a 5′ cap or 3′ poly(A) tail—act as substrates for RdRPs. Highly expressed transgenes might overwhelm normal RNA quality control pathways, escape destruction, and be converted to dsRNA by RdRPs 47–49.
Endogenous siRNAs (endo-siRNAs)
The first endo-siRNAs were detected in plants and C. elegans 38,50,51. Plants too produce a variety of endo-siRNAs, and, the recent discovery of endo-siRNAs in flies and mammals suggests that endo-siRNAs are ubiquitous among higher eukaryotes.
Plant endo-siRNAs
In plants, cis -acting siRNAs (casiRNAs) originate from transposons, repetitive elements, and tandem repeats such as 5S rRNA genes, and comprise the bulk of endo-siRNAs 44 (FIG. 2).CasiRNAs are predominantly 24 nt long and methylated by HEN1. Their accumulation requires DCL3 and the RNA polymerases, RDR2 and POL IV, and either AGO6 (primarily) or AGO4,which act redundantly 44,51–60. CasiRNAs promote heterochromatin formation by directing DNA methylation and histone modification of the loci from which they originate 38,44,51,52,61–63.
Another class of plant endo-siRNAs illustrates how distinct small RNA pathways interact.Trans -acting siRNAs (tasiRNAs) are endo-siRNAs generated by the convergence of the miRNA and siRNA pathways in plants 64–68(FIG. 2). miRNA-directed cleavage of certain transcripts recruits the RdRP enzyme, RDR6. RDR6 then copies the cleaved transcript into dsRNA, which DCL4 dices into tasiRNAs that are phased. This phasing suggests that DCL4begins dicing precisely at the miRNA cleavage site, making a tasiRNA every 21 nt 68. The site of miRNA cleavage is critical, because in determining the entry point for Dicer, it establishes the target specificity of the tasiRNAs produced. One of the determinants that seems to
predispose a transcript to produce tasiRNAs after its cleavage by a miRNA is the presence of a second miRNA or siRNA complementary site on the transcript. Of special mention is the TAS3 locus, whose RNA transcript has two binding sites for miR-390. Only one of these sites is efficiently cleaved by miR-390, but binding of the miRNA to both appears to be required to initiate conversion of the TAS3 transcript to dsRNA by RDR669,70.
Natural antisense transcript-derived siRNAs (natsiRNAs) are produced in response to stress in plants 71,72(FIG. 2). They are generated from a pair of convergently transcribed RNAs:typically, one transcript is expressed constitutively, whereas the complementary RNA is transcribed only when the plant is subject to environmental stress, such as high salt. Production of 24 nt siRNAs from region of overlap of the two transcripts requires DCL2 and/or DCL1,RDR6, and SGS3 (SUPPRESSOR OF GENE SILENCING3, probably an RNA-binding protein)73 and Pol IV 71,72. The 24 nt natsiRNAs then direct cleavage of one of the mRNAs of the pair, and in one such case, trigger the DCL1-dependent production of 21 nt secondary
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siRNAs 72. In addition to natsiRNAs, “long” siRNAs (lsiRNAs) in Arabidopsis also originate from NAT pairs and are stress-induced. Unlike natsi-RNAs, lsiRNAs are 30–40 nts long and require DCL1, DCL4, AGO7, RDR6 and POL IV for their production 74.
Animal endo-siRNAs
Plant and worm endo-siRNAs are typically produced through the action of RdRPs (Box 1).The genomes of flies and mammals seem not to encode such RdRP proteins, so the recent discovery of endo-siRNAs in flies and mice was unexpected.
The first mammalian endo-siRNAs to be reported corresponded to the LINE-1 retrotransposon and were detected in cultured human cells 75. Full length LINE-1 (L1) contains both sense and antisense promoters in its 5′ untranslated region (UTR) that could, in principle, drive bi-directional transcription of L1, producing overlapping, complementary transcripts to be processed into siRNAs by Dicer, but the precise mechanism by which transposons trigger siRNA production in mammals remains unknown.
More recently, endogenous siRNAs have been detected in Drosophila somatic and germ cells and in mouse oocytes. High throughput sequencing of small RNAs from germ-line and somatic tissues of Drosophila and of Ago2 immunoprecipitates revealed a small RNA population that could readily be distinguished from miRNAs and piRNAs 76–81. These small RNAs are nearly always exactly 21 nt long, are present in both sense and antisense orientations, have modified 3′ ends, and, unlike miRNAs and piRNAs, are not biased toward beginning with uracil.Production of the 21-mers requires Dcr-2, although in the absence of Dcr-2 a remnant of the endo-siRNA population inexplicably persists.
Fly endo-siRNAs derive from transposons, heterochromatic sequences, intergenic regions,long RNA transcripts with extensive structure, and, most interestingly, from mRNAs (FIG. 3).Expression of transposon mRNAs increases in both dcr-2 and ago2 mutants, implicating an endogenous RNAi pathway in the silencing of transposons in flies, as reported previously for C. elegans 82,83. siRNAs derived from mRNAs are >10 times more likely to come from regions predicted to produce overlapping, convergent transcripts than expected by chance 76,suggesting that endo-siRNAs originate from endogenous dsRNA formed when these complementary transcripts pair.
A subset of fly endo-siRNAs derive from “structured loci” whose RNA transcripts can fold into long, intramolecularly paired hairpins 77–79. Accumulation of these siRNAs requires Dcr-2and the dsRNA-binding protein Loquacious (Loqs)—typically considered the partner of Dcr-1,the dicer that produces miRNA—rather than R2D284, the usual partner of Dcr-2 (FIG. 1). While surprising, a role for Loqs in the biogenesis of endo-siRNAs from structured loci was anticipated by the earlier finding that Loqs plays a role in the production of siRNAs from transgenes designed to produce long, intramolecularly paired inverted repeat transcripts so as to trigger RNAi in flies 85.
Endo-siRNAs have also been identified in mouse oocytes 86,87. As in flies, mouse endo-siRNAs are 21 nt long, Dicer-dependent, and derived from a variety of genomic sources (FIG. 3). The mouse endo-siRNAs were bound to Ago2, the sole mammalian Ago protein thought to mediate target cleavage, although it is not known if they also associate with any of the other three mouse Ago proteins. (Mammalian Ago2 is not, however, the ortholog of fly Ago2, whose sequence is considerably diverged from other Ago proteins.)
A subset of mouse oocyte endo-siRNAs maps to regions of protein-coding genes capable of pairing to their cognate pseudogenes and to regions of pseudogenes capable of forming
inverted-repeat structures (FIG. 3). Pseudogenes can no longer encode proteins, yet they drift
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from their ancestral sequence more slowly than would be expected if they were simply junk.Perhaps some pseudogene sequences are under evolutionary selection to retain the ability to produce antisense transcripts that can pair with their cognate genes so as to produce endo-siRNAs 88.
A key challenge for the future will be to understand the biological function of endo-siRNAs,especially those that can pair with protein-coding mRNAs. Do they regulate mRNA
expression? Can endo-siRNAs act like miRNAs, tuning the expression of large numbers of genes?
miRNAs
The first microRNA, lin-4, was identified in a screen for genes required for post-embryonic development in C. elegans 89,90. The lin-4 locus produces a 22 nt RNA that is partially complementary to sequences in the 3′ UTR of its regulatory target, the lin-14 mRNA 89,91.miRNA binding to partially complementary sites in mRNA 3′ UTRs is now considered a hallmark of animal miRNA regulation. In 2001, tens of miRNAs were identified in humans,flies, and worms by small RNA cloning and sequencing, establishing miRNAs as a new class of small silencing RNAs 92–94. miRBase (Release 12.0), the registry that coordinates miRNA naming, now lists 1,638 distinct miRNAs in plants and 6,930 in animals and their viruses 95.
miRNA Biogenesis
miRNAs derive from precursor transcripts called pri-miRNAs, which are typically transcribed by Polymerase II 96–99. Several miRNAs are present as clusters in the genome and likely derive from a common pri-miRNA transcript. Liberating a 20–24 nt miRNA from its pri-miRNA requires the sequential action of two RNase III endonucleases, assisted by their double-stranded RNA-binding domain (dsRBD) partner proteins (FIG. 1). First, the pri-miRNA is processed in the nucleus into a 60–70 nt long pre-miRNA by Drosha, acting with its dsRBD partner,called DGCR8 in mammals and Pasha in flies 96,100–104. The resulting pre-miRNA has a hairpin structure: a loop flanked by base-paired arms that form a stem. Pre-miRNAs have a two nt overhang at their 3′ ends and a 5′ phosphate group, reflecting their production by an RNase III.The nuclear export protein, Exportin-5, carries the pre-miRNA to the cytoplasm bound to Ran GTP, a GTPase that moves RNA and proteins through the nuclear pore 105–108.
In the cytoplasm, Dicer and its dsRBD partner protein, TRBP (mammals) or Loqs (flies),cleaves the pre-miRNA 7,11–13,85,109–112. Drosha and Dicer differ in that Dicer—like
Argonaute proteins, but unlike Drosha—contains a PAZ domain, presumably allowing it to bind the two-nucleotide, 3′ overhanging end left by Drosha. Dicer cleavage generates a duplex containing two strands, the miRNA and miRNA*, corresponding to the two sides of the base of the stem. These roughly correspond to the guide and passenger strands of an siRNA, and similar thermodynamic criteria influence the choice of miRNA versus miRNA*22,23. miRNAs can arise from either arm of the pre-miRNA stem, and some pre-miRNAs produce mature miRNAs from both arms, whereas others show such pronounced asymmetry that the miRNA*is rarely detected even in high throughput sequencing experiments 113.
In flies, worms and mammals, a few pre-miRNAs are produced by the nuclear pre-mRNA splicing pathway instead of Drosha processing 114–118. These pre-miRNA-like introns,
“mirtrons,” are spliced out of mRNA precursors whose sequence suggests they encode proteins.The spliced introns first accumulate as lariat products that require 2′-5′ debranching by the lariat debranching enzyme. Debranching yields an authentic pre-miRNA, which can then enter the standard miRNA biogenesis pathway.
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In plants, DCL1 fills the roles of both Drosha and Dicer, converting pri-miRNAs to miRNA/miRNA* duplexes 44,119–121. DCL1, assisted by its dsRBD partner HYL1, converts pri-miRNAs to miRNA/miRNA* duplexes in the nucleus, after which the miRNA/miRNA*duplex is thought to be exported to the cytoplasm by HASTY, an Exportin-5 homolog
(HASTY mutants develop precociously, hence their name)65,121–123. Unlike animal miRNAs,plant miRNAs are 2′-O -methylated at their 3′ ends by HEN134,119,124. HEN1 protects plant miRNAs from 3′ uridylation, thought to be a signal for degradation 36. HEN1 likely acts before miRNAs are loaded into AGO1, because both miRNA* and miRNA strands are modified in plants 34.
Target regulation by miRNAs
The mechanism by which a miRNA regulates its mRNA target reflects both the specific Argonaute protein into which the small RNA is loaded and the extent of complementarity between the miRNA and the mRNA 125–127. A few miRNAs in flies and mammals are nearly fully complementary to their mRNA targets; these direct endonucleolytic cleavage of the mRNA 128–132. Such extensive complementarity is considered the norm in plants, as target cleavage was thought to be the main mode of target regulation in plants 39,62,133. However, in flies and mammals, most miRNAs pair with their targets through only a limited region of sequence at the 5′ end of the miRNA, the “seed”; these repress translation and direct
degradation of their mRNA targets 134–139. The “seed” region of all small silencing RNAs contributes most of the energy for target binding 140,141. Thus, the seed is the primary specificity determinant for target selection. The small size of the seed means that a single miRNA can regulate many—even hundreds—of different genes 142,143. Intriguingly, recent data suggests that the nuclear transcriptional history of an mRNA influences if a miRNA represses its translation at the initiation or the elongation step 144.
Since plant miRNAs are highly complementary to their mRNA targets, they can direct mRNA target cleavage. Nonetheless, AGO1-loaded plant miRNAs can also block translation,
suggesting a common mechanism between plant and animal miRNAs, despite the absence of specific miRNAs shared between the two kingdoms 145.
Functions of miRNAs
Like transcription factors, miRNAs regulate diverse cellular pathways, and are widely believed to regulate most biological processes, in both plants and animals, ranging from housekeeping functions to responses to environmental stress. Covering this vast body of work is beyond the scope of this review; the cited reviews provide valuable insight 146–148.
The study of miRNA pathway mutants provided early evidence for the influence of miRNAs on biological processes in both plants and animals. Loss of Dicer or miRNA-associated Argonaute proteins is nearly always lethal in animals, and such mutants show severe
developmental defects in both plants and animals. In Drosophila, dcr-1 mutant germ-line stem cell clones divide slowly; in Arabidopsis , embryogenesis is abnormal in dcl1 mutants; in C.elegans, dcr-1 mutants display defects in germ-line development and embryonic
morphogenesis; zebrafish lacking both maternal and zygotic Dicer are similarly defective in embryogenesis; and mice lacking Dicer die as early embryos, apparently devoid of stem cells 14,119,149–152. Loss of Dicer in mouse embryonic fibroblasts causes increased DNA damage and consequently, the up-regulation of p19Arf and p53 signaling that induces premature senescence 153.
Many miRNAs function in specific biological processes, in specific tissues, and at specific times 154. The importance of small silencing RNAs goes far beyond the RNA silencing field:
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long-standing questions about the molecular basis of pluripotency, tumorogenesis, apoptosis,cell identity, etc. are finding answers in small RNAs 146,155.
piRNAs: the longest small RNAs
piRNAs function in the germ line
Piwi-interacting RNAs (piRNAs) are the most recently discovered class of small RNAs, and,as their name suggests, they bind to the Piwi clade of Argonaute proteins. (Animal Argonaute proteins can be subdivided by sequence relatedness into Ago and Piwi sub-families.) The Piwi clade comprises Piwi, Aubergine (Aub) and Ago3 in flies, MILI, MIWI and MIWI2 in mice,and HILI, HIWI1, HIWI2 and HIWI3 in humans.
piRNAs were first proposed to ensure germ line stability by repressing transposons when Aravin and colleagues discovered in flies a class of longer small RNAs (~25–30 nt) associated with silencing of repetitive elements 156. Later, these “repeat associated small interfering RNAs”—subsequently renamed piRNAs—were found to be distinct from siRNAs: they bind Piwi proteins and do not require Dcr-1 or Dcr-2 for their production, unlike miRNAs and siRNAs 33,157,158. Moreover, they are 2′-O -methylated at their 3′ termini, unlike miRNAs, but like siRNAs in flies 32,157,159–161.
High throughput sequencing of vertebrate piRNAs revealed a class of piRNAs unrelated to repetitive sequences, hence their name change 87,162–168. Mammalian piRNAs can be divided into pre-pachytene and pachytene piRNAs, according to the stage of meiosis at which they are expressed in developing spermatocytes. Like piRNAs in flies, pre-pachytene piRNAs
predominantly correspond to repetitive sequences and are implicated in silencing transposons,such as L1 and IAP 165. In male mice, gametic methylation patterns are established when germ cells arrest their cell cycle 14.5 days postcoitum, resuming cell division 2–3 days after birth 169,170. Both MILI and MIWI2 are expressed during this period, and miwi2 and mili deficient mice lose DNA methylation marks on transposons 171. The pre-pachytene piRNAs,which bind MIWI2 and MILI, may serve as guides to direct DNA methylation of transposons.In contrast to pre-pachytene piRNAs, the pachytene piRNAs mainly arise from unannotated regions of the genome, not transposons, and their function remains unknown 165.
Three recent studies report that the previously discovered germ-line “21U” RNAs in C.
elegans are piRNAs 113,172–174. These small RNAs were initially identified by high throughput sequencing 113. They are precisely 21 nt long, begin with a uridine 5′-monophosphate, and are 3′ modified. They bind Piwi-Related Gene-1 (PRG-1), a C. elegans Piwi protein. Each 21U-RNA may be transcribed separately, as all are flanked by a common upstream motif. Like piRNAs in Drosophila , the 21U-RNAs are required for maintenance of the germ line and fertility, and like Drosophila Aub and other piRNA pathway components, PRG-1 is found in specialized granules, P granules, associated with germ-line function, in a cytoplasmic,perinuclear ring called “nuage.” Worm piRNAs resemble pachytene piRNAs in mammals:their targets and functions are largely unknown.
Biogenesis
piRNA sequences are stunningly diverse, with more than 1.5 million distinct piRNAs identified thus far in flies, but collectively they map to a few hundred genomic clusters 77,79,81,158,175–177. The best studied cluster is the flamenco locus. flamenco was identified genetically as a repressor of the gypsy, ZAM and Idefix transposons 33,178–182. Unlike siRNAs, flamenco piRNAs are mainly antisense, suggesting that piRNAs arise from long, single-stranded
precursor RNAs. In fact, disruption of flamenco by insertion of a P-element near the 5′ end of the locus blocks the production of distal piRNAs up to 168 kbp away. Thus, an enormously
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long, single-stranded RNA transcript appears to be the source of those piRNAs that derive from the flamenco locus 176.
The current model for piRNA biogenesis was inferred from the sequences of piRNAs bound to Piwi, Aubergine and Ago3176,183. piRNAs bound to Piwi and Aubergine are typically antisense to transposon mRNAs, whereas Ago3 is loaded with piRNAs corresponding to the transposon mRNAs themselves (FIG. 1). Moreover, the first 10 nucleotides of antisense
piRNAs are frequently complementary to the sense piRNAs found in Ago3. This unexpected sequence complementarity has been proposed to reflect a feed-forward amplification mechanism—”piRNA ping-pong”—that is activated only upon transposon mRNA transcription (FIG. 4)176,183. A similar amplification loop has been inferred from high throughput piRNA sequencing in vertebrates, implying its conservation through
evolution 162,171. Many aspects of the ping-pong model remain speculative. Why Ago3 appears to bind only sense piRNAs derived from transposon mRNAs is unknown. An untested idea is that different forms of RNA Pol II transcribe primary piRNA transcripts and transposon mRNAs and that the specialized RNA Pol II that transcribes the primary piRNA precursor recruits Piwi and Aub, but not Ago3. How the 3′ ends of piRNAs are made is also not known.
piRNA Function and Regulation
Piwi family proteins are indispensable for germ-line development in many, perhaps all,
animals; but they have thus far been most extensively studied in Drosophila . Piwi is restricted to the nucleoplasm of Drosophila germ cells and adjacent somatic cells. Piwi is required to maintain germ line stem cells and to promote their division; the protein is required in both the somatic niche cells that support germ-line stem cells and in the stem cells themselves 184,185.In the male germ line, Aub is required for the silencing of the repetitive Stellate locus, which would otherwise cause male sterility. Expression of Stellate is controlled by the related,repetitive Suppressor of Stellate locus, the source of antisense piRNAs that act through Aub to repress Stellate 156,157,186.
aub was originally identified because it is required for specification of the embryonic
axes 187. The loss of anterior-posterior and dorsal-ventral patterning in embryos from mothers lacking Aub is an indirect consequence of the double-stranded DNA breaks that occur in the oocyte in its absence 188. The breaks appear to activate a DNA-damage checkpoint that disrupts patterning of the oocyte and, consequently, of the embryo. The defects in patterning, but not in silencing repetitive elements, are rescued by mutations that bypass the DNA damage
signaling pathway, suggesting the breaks are caused by transposition. That activation of a DNA damage checkpoint should inappropriately reorganize embryonic polarity was most
unexpected, but further underscores the vital role piRNAs play in germ line development.
piRNAs outside the germ line?
The role of piRNAs in the fly soma is hotly debated. Piwi and Aub are required to silence tandem arrays of white , a gene required to produce red eye pigment 189. It is not understood if piRNAs are produced in the soma as well as in the germ line, or if piRNAs present during germ-line development deposit long-lived chromatin marks that exert their effects days later.Both piRNAs and endo-siRNAs repress transposons in the germ line, where mutations caused by transposition, of course, would propagate to the next generation. siRNAs—that is, the RNAi pathway—likely provide a rapid response to the introduction of a new transposon into the germ line, a challenge not dissimilar to a viral infection. In contrast, the piRNA system appears to provide a more robust, permanent solution to the acquisition of a transposon. In the soma,however, endo-siRNAs are the predominant transposon-derived small RNA class, and their loss in dcr-2 and ago2 mutants increases transposon expression 76,77,79,81. Somatic piRNA-
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like small RNAs have been observed in ago2 mutant flies 76. Perhaps, in the absence of endo-siRNAs, piRNAs are produced somatically and resume transposon surveillance. Such a model implies significant cross-talk between the piRNA and endo-siRNA–generating machineries.
Intertwined pathways
The RNAi, miRNA and piRNA pathways were initially believed to be independent and distinct.However, the lines distinguishing them continue to fade. These pathways interact and rely on each other at several levels, competing for and sharing substrates, effector proteins and cross-regulating each other.
Competition for substrates during loading
Both the siRNA and miRNA pathways load dsRNA duplexes containing a 19 bp double-stranded core flanked by 2 nt 3′ overhangs. An siRNA duplex contains guide and passenger strands and is complementary throughout its core; a miRNA/miRNA* duplex contains
mismatches, bulges and G:U wobble pairs. In Drosophila , biogenesis of small RNA duplexes is uncoupled from its loading into Ago1 or Ago2190,191. Instead, loading is governed by the structure of the duplex: duplexes bearing bulges and mismatches are sorted into the miRNA pathway and hence loaded into Ago1; duplexes with greater double-stranded character partition into Ago2, the Argonaute protein associated with RNAi.
The partitioning of small RNAs between Ago1 and Ago2 also has implications for target regulation. Ago1 primarily represses translation whereas Ago2 represses by target cleavage,reflecting the faster rate of target cleavage by Ago2 compared to Ago1191. Sorting creates competition between the two pathways for substrates 190,191. In Drosophila loading of a small RNA duplex into one pathway decreases its association with other pathway.
Different double-stranded RNA precursors require distinct combinations of proteins to produce small silencing RNAs. For example, Drosophila endo-siRNAs derived from structured loci require Loqs, rather than R2D277–79. We presume that under some circumstances the endo-siRNA and miRNA pathways might therefore compete for Loqs. The endo-siRNA and RNAi pathways likely also compete for shared components.
In contrast to Drosophila , plants load small RNAs into Argonautes according to the identity of the 5′ nt of the small RNA 69,192. AGO1 is the main effector Argonaute for miRNAs, and the majority of miRNAs begin with uracil; and AGO4 is the major effector of the
heterochromatic pathway and is predominantly loaded with small RNAs beginning with an adenosine 193. AGO2 and AGO5, however have no characterized function in plants 193.Changing the 5 nt from A to U shifts the loading bias of a plant small RNA from AGO2 to AGO1, and vice versa. Similarly, Arabidopsis AGO4 binds small RNAs that begin with adenosine, while AGO5 prefers cytosine.
Aub- and Piwi-bound piRNAs typically begin with U, whereas those bound to Ago3 show no 5′ nucleotide bias. It remains to be determined if this reflects a 5′ nucleotide preference like the situation for the plant AGOs or some feature of an as-yet-discovered piRNA loading machinery that sorts piRNAs between Piwi proteins.
Cross talk
Small RNA pathways are often entangled. TasiRNA biogenesis in Arabidopsis is a classic example of such cross talk between pathways. miRNA-directed cleavage of tasiRNA-generating transcripts initiates tasiRNA production and subsequent regulation of tasiRNA targets 64–68. In C. elegans , at least one piRNA has been implicated in initiating endo-siRNA production 172,173, and in flies, the endo-siRNA pathway may repress expression of piRNAs
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in the soma 76. Moreover, small RNA levels may be buffered by negative feedback loops in which small RNAs from one pathway alter the expression levels of RNA silencing proteins that act in the same or in other RNA silencing pathways.194–198.
Conclusions
Despite our growing understanding of the mechanism and function of small RNAs, their evolutionary origins remain obscure. siRNAs are present in all three eukaryotic kingdoms,plants, animals, and fungi, and provide anti-viral defense in at least plants and animals. Thus,the siRNA machinery was present in the last common ancestor of plants, animals and fungi.In contrast, miRNAs have only been found in land plants, the unicellular green alga,
Chlamydomonas reinhardtii , and metazoan animals, but not in unicellular choanoflagellates or fungi 199–201. Deep sequencing experiments have found no miRNAs shared by plants and animals, suggesting that miRNA genes, unlike the miRNA protein machinery, arose
independently at least twice in evolution. Finally, piRNAs appear to be the youngest major small RNA family, having been found only in metazoan animals 201. While Dicer proteins have been identified only in eukaryotes, Argonaute proteins can also be found in eubacteria and archea, raising the prospect that small nucleic-acids may have served as guides for proteins at the very dawn of cellular life, and though the machinery might be ancient, the small RNA guides diversified over time to acquire specialized roles.
The history of small silencing RNAs makes predicting the future particularly daunting, as new discoveries have come at a breakneck pace, with each new small RNA mechanism or function forcing a re-evaluation of cherished models and “facts.” Several longstanding but unanswered questions, however, are worth highlighting. First, does RNAi—in the sense of an siRNA-guided defense against external nucleic acid threats such as viruses—exist in mammals?Second, how do miRNAs repress gene expression? Do several parallel mechanisms co-exist in vivo, or will the current, apparently contradictory, models for miRNA-directed translational repression and mRNA decay ultimately be unified in a larger mechanistic scheme? Third, can miRNA regulated genes ever be identified by computation alone, or will computational predictions ultimately give way to high-throughput experimental methods for associating individual miRNA species with their regulatory targets? Will network analysis uncover themes in miRNA-target relationships that reveal why miRNA-regulation is so widespread in animals?Fourth, how are piRNAs made? The feed-forward amplification “ping-pong” model is
appealing, but likely underestimates the complexity of piRNA biogenesis mechanisms? We do not yet know how piRNA 3′ ends are generated. Nor do we have a coherent model for how long, antisense transcripts from piRNA clusters are fragmented into piRNAs. Finally, will the increasing number of examples of small RNAs carrying epigenetic information across generations 3,202 ultimately force us to reexamine our Mendelian view of inheritance?
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简单的英语情景对话 C:l'm walking that way. Let me lead you the way.C:我正朝那边去。 让我给你带路吧!A:Thank you very much.A:非常感谢。 C:You're welcome.C:不用谢简单的英语情景对话2：A:Excuse me. I'm afraid I got lost. Where am I on this map?A:对不起，我恐怕是迷路了，请问我在地图上的什么位置?B:We are here, bus station, we are in the heart of the city.B:我们在这里，汽车站，我们现在在市中心。 A:Oh! Can I go from here to the Zhongshan Park?A:哦!我能否从这里到中山公园呢?B:Head straight up the street about two blocks then turn right.B:顺着这条街一直走过两个街区，然后向右转。 简单的英语情景对话3：A:Excuse me. I wonder if you could help me. l'm looking for the Oriental Pearl Theater.A:对不起，打扰一下，不知您能否帮我，我在找东方之珠剧院。 B:Boy, you are lost. lt's across town.B:哦，你迷路了。 它在城市的那头。 A:Oh! What bad luck! How can I get to the Theater?A:哦!太糟糕了!那我怎么去剧院呢?B:You can take a No. 12 bus here and then transfer to a No. 23 bus to get there.B:您可以在此乘坐12路公共汽车，然后换乘23路公共汽车到那里。 简单的英语情景对话4：A:l'm sorry, but I didn't catch what you said.A:对不起，我没听清你说的话。
生活情景会话参考答案
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生活情景会话 1．小张和同寝的小李约好早起去超市买东西，可是第二天早起时小张明显感到自己身体不适，约会可能要被取消了。假设你作为小张，如何与小李说明自己的情况，从而使小李欣然接受，又不因这次失约而损害到你们的感情呢？ 参考答案：“小李啊，实在不好意思，今早起床我忽然感觉身体不舒服，本想陪同你一起买东西，可是依我现在的身体状态，即使出去了，我看你也买不了东西，就得照顾我了，让你操心我心里可不好受。反正我们也不急于这几天，我看我们改天再去吧！我好好休息一下，你也免得为我担心，怎么样啊？” 2．打开水的时间到了，你由于有急事不得不找同寝室友小徐帮你打水，但小徐平时很不情愿为别人打开水，你如何使小徐心甘情愿的为你打开水，使你们的友谊升温？ 参考答案：“小徐，恐怕得麻烦你帮我打壶水了，原本想和你一起去的，因为我有急事就得请你帮这个忙了，也不知道为什么我第一个想到的帮我打水的人就是你，看来我真把你当成我的好朋友了。想起你平时那么照顾我，你真是一个值得交的人啊！那就麻烦你辛勤的为我服务一次吧！” 3．假如你正在主持一个校园歌手晚会，一个同学正唱到精彩处时，伴奏带断了，他坚持唱到最后。作为主持人，你如何人处理这个场面？ 参考答案：“刚才伴奏带一停，让我们有机会听到这位歌手的清唱，真是此时无声胜有声，他的清唱更显功力、更具魅力啊！” 4．你去图书馆准备学习，可是星期日人满为患，忽然你发现了一个空座位，只见桌子上放着书可能在占座，你已经等了15分钟也不见有人来，而你也发现空座位旁边的同学可能就是占座的同学，为了得到那个位置，你该如何人开口呢？ 参考答案：“同学，你好，打扰了，我可以坐在这个空位上吗？我已经找了很长时间都没有空座，就这里有个座位了，你看能不能让我先坐在这，等一会你的朋友来了我们再商量着办，我再冒昧地说一句，就是在图书馆占座是欠考虑的行为，当然，这是我们的通病，大家都是为了学习嘛！谁都有找不到位置的时候，我也找了半天了，你就让给我吧！呵呵！ 5．电梯的正常负载人数为标准体重13人。而当电梯已进12人时，另有两名同学执意要上电梯，作为电梯礼仪小姐，请你使他们回心转意坐下一次电梯。 参考答案：“这两名同学请留步，莫着急。电梯已够承载人数，您两位要是上去了，恐怕大家谁也别想上楼了，您二位想快些上楼若是为了学习，那么我请二位发扬学习风格的同时也发扬谦让的风格，好的秩序是德行的标志，请您二位稍等下一次电梯，可以吗？ 电梯马上就下来，到时候您二位先上，行吗？” 6.中午食堂人山人海，你非常着急，把一位同学新买的鞋踩脏了，眼看着那个同学就要发 怒了，请你说几句话使他平息怒火，该如何讲呢？ 参考答案：“同学，真是抱歉啊，我实在不是故意的，你这是新买的鞋吧，我就更不应该踩了。你看，中午买饭人挨人，人挤人，你碰我一下，我碰你一下，这些都是难免的，就请你多多担待吧。实在是对不起了。” 7.你和几个好朋友一起坐车去逛街。公交车上十分拥挤，这时上来一位年迈的老奶奶，你 身边坐着的小伙子对此无动于衷，请你说服小伙子给老奶奶让座。 参考答案：“这位大哥，看你坐着挺久了，不如站起来活动活动吧，给这位老奶奶让个座吧，老人家上年纪挤车多不容易啊，谁都有老的一天，你站着就当锻炼身体，老奶奶坐着你我大伙都放心，我想你心里也是这么像的吧，别不好意思站起来，与人方便自己方便嘛！”
口语对话 1 赶时髦(go after fashion) A: Fashion show is around the corner, I’m so excited! 时装表演即将来临，我很兴奋！ B: Are there any good!这有什么好的！ A: I didn't see anything wrong with the clothes; they looked pretty nice to me. I think you don’t like it! Why?我没看出衣服有任何问题；在我看来它们都很不错。我觉得你不喜欢！为什么？ B: It was dumb. I think it's stupid for women to wear clothes like that. 这是愚蠢的。我认为女人们穿成那样是很愚蠢的。 A: The benevolent see benevolence and the wise see wisdom. 仁者见仁，智者见智。 B: Do you really think people can wear that stuff and walk around the streets? 你真的认为人们可以穿那种东西走在街上？ A: Yes, I do. At least, some people certainly can. They wear high-fashion clothes to show off their sense of style and wealth. 是的，我这样认为。至少，有人一定会。他们穿着时尚的衣服展示他们的时尚感和财富。 B: Well. I still think they're dumb. It makes more sense to spend the money on more practical purposes.我仍然认为他们是愚蠢的。把更多的钱花在更有意义的地方比较实际。 A: So you think it's bad if I wear it?所以你认为我穿成这样很不好吗？B: If you wear it I must speak nice! 如果你穿成这样我一定说它很好看！A: I know you will say that.我就知道你会这样说。 B: Only you know me!知我者非你莫属！ 2 食品安全(food security)
Unit 6 This is my family. 词汇：family mum dad sister brother friend 句型：This is... 句子：1.This is my mum. Lesson 2 词汇：my your 缩写：What’s=What is my name’s=my name is 句子：①What’s your name？ My name’s Pop. Unit 7 Happy birthday? 词汇：bike car doll robot train van 缩写：it’s it’s=it is it isn’t it isn’t=it is not 词汇：a an 句子：①It’s a van. ②It isn’t a car. 短语：a train/van/robot/car An apple/insect/egg/umbrella Lesson 2 句型：Is it...? 句子：㈠Is it an apple？ Yes，it is./No,it isn’t. Unit 8 That’s my book. 词汇：book, pen, pencil ,pencil case, rubber, ruler, school bag 缩写：this is this isn’t this isn’t= this is not that is that isn’t that isn’t= that is not 句子：①肯——This is my book. 否——This isn’t my book. ②肯——That’s my book. 否——That’s isn’t my book. Lesson 2 句型：Is this...? Is that...? 句子：①Is this a pencil？ Yes，it is./No,it isn’t. ②Is that a school bag? Yes，it is./No,it isn’t. Unit 9 What’s this,Mum? 词汇:bone, hamburger, salad, sausage, tomato, pizza 缩写：What’s=What is 句型：What’s...?
英语问路情景对话简单 初中英语教学阶段,口语交际能力的培养是教学任务的重要内容之一。整理了简单的英语问路情景对话，欢迎阅读! 简单的英语问路情景对话一A：Could you show me the way to the railway station? 你能告诉我去车站的路吗? B：Yes, of course. 当然。 A：Would you like to be my guide? 给我领领路好吗? B：I'd be very pleased. 我很乐意。 简单的英语问路情景对话二A：Excuse me, but can you tell me the way to the railway station? 请问去火车站怎么走? B：Just go straight along this street, turn left at the first crossing and walk straight ahead about 100 meters. You can’t miss it. 沿着这条街一直往前走，到第一个十字路口向左拐，然后再往前走100米就是了。你一定能看到。
A：About how long will it take me to get there? 去那儿大约要多少时间? B：It’s about 20 minutes’ walk, I think. 步行大概要二十分钟。 A：I see. Is there a bus I can take? 我明白了，可以乘公共汽车去吗? B：Yes, you can take the No. 5 bus over there. Get off at the next stop. 可以，你可以在那儿乘5路车，在下一站下车。 A：Thank you so much for your help. 非常感谢你的帮助。 B：It’s my pleasure. 不客气。 简单的英语问路情景对话三A：Excuse me. Can you tell me where the Wal-Mart supermarket is? 劳驾，请问沃尔玛超市在哪儿? B：Turn right at the second crossing and then go straight for two blocks. 在第二个十字路口往右拐，然后一直往前走，过两个街区。 A：Is it far from here? 离这儿远吗? B：No, it’s only ten-minute's walk.
面试英语情景对话大全 Why are you interested in working for our company? 为什么有兴趣在我们公司工作？ Because your company has a good sales record. 因为你们公司有良好的销售记录。 Because your operations are global, so I feel I can gain the most from working in this kind of environment. 因为你们公司的运作是全球化的，我觉得可以在这样一个环境中工作会有最大的收获。 Because I think my major is suitable for this position. 因为我认为我的专业适合这个职位。
Because I can learn new things in your company, at the same time I can offer my services to you. 因为我可以在贵公司学到新的东西, 同时能为你们提供服务。 Because I'm very interested in your company's training program. 因为我对你们公司的培训计划很感兴趣。 Why did you leave your former company? 为什么离开以前的公司？ Because I'm working in a small company where a further promotion is impossible. 因为我在一家小公司工作，升职的希望很小。 Because I'm capable of more responsibilities, so I decided to change my job.
英语情景对话大全 篇一：英语情景对话大全 美国英语情景对话大全 (1).Introductios and Opening Conversations 介绍和开场白 People in the United States don't always shake hands when they are introduced to one another. However, in a formal or business situation people almost always shake hands. 1.A: Mary, this is Joe's brother David. B; I'm very glad to meet you. C: It's a pleasure to meet you. B: How do you like Texas so far? C: It's really different from what I expected. B: Don't worry. You'll get used to it in no time. 2.A: Mrs. Smith, I'd like to introduce a friend of mine, Pierre Dubois. B: How do you do? C: Hello. B: What's your impression of the United States? C: Well, I can't get over how different the weather is here. B: Oh, you'll get used to it soon! 3.A: Wendy, I'd like you to meet my brother Sam. B: Hi. C: Nice to meet you. B: What do you think of Dallas? C: Well, I'm still feeling a little homesick and so many things seem strange to me. B: You're bound to feel that way at first, I guess. 4.A: Mrs.Hughs, this is Peter Brown. B: Pleased to meet you. C: How do you do? B: I hope you're enjoying your stay here. C: If it weren't for the climate, I'd like it here very much. B: It always takes time to get used to a new place. (2).Special Greetings 节假日的问候 There are eight national holidays celebrated in the United States: New Year's Day(Jan.), Washington's Birthday (Feb.), Memorial Day(May), the Fourth of July, Labor Day(Sep.),Veteran's Day(Nav.),Thanksgiving(Nov.) and Christmas(Dec.), In addition, there are many state and local holidays. 1. A: Merry Christmas! B: The same to you! A: Are you doing anyting special?
1.在中餐馆At a Chinese restaurant A: It's very nice of you to invite me. 你真是太好了，请我来做客。 B: I'm very glad you could come, Mr. Liu. Will you take a seat at the head of the table? It's an informal dinner, please don't stand on ceremony... Mr. Liu, would you like to have some chicken? 刘先生，您能来我很高兴，请上坐。这是一次家常便饭，请大家别客气。刘先生，要吃点鸡肉吗？ A: Thank you. This is my first time to come to a Chinese restaurant. Could you tell me the different features of Chinese food? 谳谢，这是我第一次来中餐馆，请给我讲讲中国菜的不同特色好吗？ B: Generally speaking, Cantonese food is a bit light; Shanghai food is rather oily; and Hunan dishes are very spicy, having a strong and hot taste. 一般来讲，广东菜清淡一些；上海菜比较油腻：湖南菜香味浓，辣味很重。 A: Chinese dishes are exquisitely prepared, delicious, and very palatable. They are very good in colour, flavour, and taste. 中国菜做得很精细，色、香、味俱全。 B: Mr. liu, would you care for another helping? 刘先生，再来一点吧？ A: No more, thank you. I'm quite full. 不啦，谢谢。我已经够饱了。 B: Did you enjoy the meal? 您吃得怎么样？ A: It's the most delicious dinner I've had for a long time. It's such a rich dinner. 好久没有吃过这样美味的饭莱了。这顿饭莱太丰盛了。 B: I'm so glad you like it. 你能喜欢，我不胜荣幸。 A: Thank you very much for your hospitality. 谢谢你的盛情款待。
英语情景对话——常用 英语情景对话一 对春节期间的烟花爆竹燃放问题的讨论 A：Happy new year, LIU, It is nice to enjoy New year holiday, but the noise of the fireworks and firecrackers bothers me recently. B: Happy new year, On the contrary,I think the fireworks and firecrackers is as nice as the Spring festival. A:Oh no, A t New Year’s Eve, I was up all night because there is too much noise outside. I think fireworks and firecrackers should be prohibited. B:I may not agree with you. The noise of them is a symbol of lively festive atmosphere. And I set off some fireworksat New Year’s Eve. It’s so interesting. A: Ok, the noise, is a piece of cake, it is just for me. But fireworks and crackers will cause air pollution. When firework is set off, it will produce lots of sulfur dioxide which is harmful to our environment. What’s more, fireworks and crackers do lots of harm to people. To make the matter worse, children love playing carelessly with firecrackers, which more often cause conflagration. B: Yeah, some of what you have said is true and It should cause the attention of people. But as long as people pay attention to safety when setting off, I think, It'sunderstandable to set off fireworks and firecrackers during the festival and they should not be prohibited. A:There also exist potential hazards in manufacturing, shipping and storing them. B: As a traditional way to celebrate the New Year, quite a few people think firecrackers have become part of our Chinese culture. Lots of fun will be gone with the ban of firecrackers. A: True, to change a custom is not easy, and people are not ready for such drastic action, but it will pay in the long run if the setting off of firecrackers is effectively banned in the cities. B: Maybe, we can celebrate our holiday by other ways instead of setting off firework.
新概念英语青少版入门级A Listening 听力部分30’ 一、听录音，写单词，并写出单词的中文含义(20’)。 二、听录音，选出你所听到的单词。（10’） 1. ( ) A. skirt B. shirt C. sock D. sweater 2. ( ) A. rabbit B. tortoise C. rubber D. parrot 3. ( ) A. twelve B. twenty C. eleven D. seven 4. ( ) A. a duck B. two ducks C. three ducks D. four ducks 5. ( ) A. evening B. morning C. lunch D. breakfast 6. ( ) A. chair B. shelf C. bed D. wall 7. ( ) A. next to B. bedroom C. bathroom D. between 8. ( ) A. dancer B. nurse C. man D.woman 9. ( ) A. aunt B. uncle C. cousin D. sister 10. ( ) A. this B. that C. those D. the
Writing 笔试部分70’ 三．用“a”或“an”填空（10’） 1. apple 2. book 3. rabbit 4.umbrella 5. mouse 6. __ ice cream 7. ___ little monkey 8. __ big egg 9. __ red apple 10．＿banana 四、圈出不同(10’) 1. red blue pink banana 2. frog mouse butterfly big 3. shirt anorak short cap 4. seven eight sister eleven 5. father mother uncle driver 五、请写出缩写（10’） I am___ he is___ you are ___ is not___ are not___ it is___ they are____ she is____ that is ____ 六、选择正确的答案（10’） ( )1.--What’s this? A. That’s a book. B. Yes, it is. C. It’s a bus. ( )2.--What colour is it? A. It’s yellow. B. It’s a dog. C. Thank you. ( )3.-- is your name? --I’m Ann. A.What B. Who C. Where
简单的英语情景对话3篇 学习英语的口语是我们刻不容缓的事情，今天就给大家分享一下英语情景对话，大家一起来学习阅读哦 有关生活的英语口语对话 她总是很乐于助人She is willing to help AMy granny is such a kind-hearted person. She is willing to help. 我奶奶心肠很好，总是乐于助人。 BShe does have a heart of gold. 她确实有个好心肠。 AYou can say that again. 你说得太对了。 简单的英语情景对话参考 他很有远见卓识He has a long head AHe has a long head, I bet he will do well in his business. 他很有远见卓识，我敢肯定他会将生意做得很红火。 BHe does, he started five years ago and now ends up the richest among us. 的确。他五年前开始做生意，现在是我们当中的最富的一个。
AWhen we were students, he was no good in any subject. 学生时代，他没有哪一门功课是非常出色的。 BIt seemed to be useless to study well when we were young if we examine his case. 就他的案例来看，好像年轻时学习出色并没有什么用。 精选情景英语会话 你的项目研究做得很好 AYour search on this project has been excellent. 你对这个项目的研究做得非常好。 BThank you. I couldn’t have done it without the help of a strong team. 谢谢。要是没有一个强大团队的支持，我做不到这些。 AThe final report is exceptionally thorough and well done. I think that you have been come up with some interesting recommendations for the client. 最后的报告非常全面，做得很好。你给客户提出了一些不寻常的建议。 BWell, I hope the client feels the same way! 我希望客户有同样的看法。 AI am sure they will be pleased. 我非常肯定他们会满意的。 简单的英语情景对话3篇
英语生活日常情景口语对话 导读：我根据大家的需要整理了一份关于《英语生活日常情景口语对话》的内容，具体内容：学习好英语在很多地方都是有需要的，下面我就给大家整理了英语的情景口语对话，大家快来看看吧A picnic野餐ChinChin: I love g... 学习好英语在很多地方都是有需要的，下面我就给大家整理了英语的情景口语对话，大家快来看看吧 A picnic 野餐 ChinChin: I love going on a picnic to the mountain! ChinChin: Mimi,have some fruit. ChinChin: They are really fresh. Mimi: Thank you. Mimi: Its a lot of fun! Costa: Fun? Costa: Who is having fun? Costa: Hmmm. Look at that fruit. Costa: I want to have some too. Costa: Hey,red wolf! Costa: Scared them and bring that fruit to me! Samsik: Hey,give me the fruit!
Samsik: My master wants it. ChinChin: Your master? ChinChin: That must be Costa! ChinChin: Hey, Suri Suri,I need your help. Suri: Yes,sir! Costa: Oops,I lost again... 晨晨：我喜欢去山上野餐! 晨晨：咪咪，吃一些水果。 晨晨：它们真的很新鲜。 咪咪：谢谢你。 咪咪：这真有趣! 科斯达：有趣? 科斯达：谁玩得开心了? 科斯达：嗯。看看那些水果。 科斯达：我也想要一些。 科斯达：嘿，红太狼! 科斯达：去吓走他们，把水果给我带过来! 桑姆斯克：嘿，把水果给我! 桑姆斯克：我的主人想要得到它。 晨晨：你的主人? 晨晨：那一定是科斯达! 晨晨：嘿，苏瑞苏瑞，我需要你的帮助。
动物：cat 猫 dog 狗 fish 鱼insect昆虫 monkey 猴子 panda 熊猫 zebra 斑马 pig 猪frog 青蛙 mouse 老鼠 parrot 鹦鹉 rabbit 兔子 tortoise 乌龟 食物：apple 苹果 egg鸡蛋hamburger 汉堡包 salad 沙拉 sausage香肠tomato西红柿 pizza比萨jelly果冻 颜色： green绿色 red 红色 blue 蓝色yellow黄色 orange 橙色 身体部位： leg 腿 mouth 嘴 nose 鼻子 玩具：Ball 球 kite 风筝 violin 小提琴xylophone 木琴doll 木偶 robot 机器人 车：bike 自行车 car 小汽车train 火车 van 货车；面包车 家人亲戚：family 家人 mum 妈妈 dad 爸爸 sister 妹妹 brother 弟弟friend 朋友 文具：book 书 pen 钢笔 pencil铅笔 pencil case 笔袋 rubber 橡皮 ruler 尺子 school bag书包 职业：dancer 跳舞者，舞蹈专家 doctor医生 nurse 护士 policeman男警察policewoman 女警察 postman 邮递员 teacher 老师 数字：one two three four five six seven eight nine ten 形容个人体态：tall 高的short 矮的big大的 little 小的fat 胖的thin瘦的 衣物： anorak 带帽子的夹克衫 cap 鸭舌帽 T-shirt T恤Shirt 衬衣sweater毛衣hat有檐帽 方位介词：behind 在…后面 in 在…里面 in front of 在…前面 on 在…上面under 在…下面 其他词汇： girl女孩 boy 男孩 sun 太阳 table 饭桌 umbrella 雨伞 king 国王 queen王后zoo 动物园 window 窗户 bone 骨头 句子： at my yellow leg! 看我黄色的腿！ is my brother and sister. 这是我的哥哥和姐姐。（用于介绍某人） ’s a robot. 这是一个机器人。 it an apple？ Yes，it is./No，it isn’t.它是一个苹果吗？是的，它是。/不，它不是。 isn’t my schoolbag. 这不是我的书包。 this a schoolbag? Yes,it is. /No,it isn't. 这是一个书包吗？是的，它是。/不，它不是。 ’s this？It’s a hamburger. 这是什么？这是一个汉堡。 What’s that？It’s a salad. 那是什么？那是一盘沙拉。 What is it？It’s an egg. 它是什么？它是一个鸡蛋。 8.How old are you？I’m eleven. 你多大了？我十一岁了。 9.Are you seven？Yes，I am. /No，I’m not.你七岁了吗？是的，我是。/不，我不是。 10.You aren’t five. 你不是五岁。 11.He’s tall.他很高。She isn’t fat. 她不胖。 12.Is she tall？Yes,she is./No，she isn’t.她高吗？是的，她高。/不，她
情景一A：你是北京交通职业技术学院的学生李磊，被派往机场迎接来自哈佛大学的White 教授。你询问他飞行是否愉快，并表示飞机没有晚点就是最好的事情了。B：你是来自哈佛大学的White 教授，你感谢李磊来接机。15个小时的飞行你很累。你希望能够赶快去酒店洗个热水澡，睡一觉。情景二 A：你是学校秘书王小姐，见到了来自哈佛大学的White 教授，你邀请他周日参加你们学校50周年校庆，并在周日下午做一个关于Lifelong Study 的讲座。告诉他全校学生都会参加，并相信学生们会有所收获。B:你是哈佛大学的White 教授，你被邀请周日参加北京交通职业技术学院50周年校庆，并在周日下午做一个关于Lifelong Study 的讲座。你询问了题目、具体时间和地点、参加人员等问题。情景三A: 你是Annie，你的姐姐Jennie下周日结婚，你打电话恭喜她新婚快乐，并祝她早生贵子，并表示一定会按时参加他们的婚礼，期待姐姐穿婚纱的样子。并建议他们巴黎度蜜月。B:你是姐姐Jennie，你下周日结婚，你妹妹打电话祝贺。你表示感谢，并邀请他参加你们的婚礼派对。你询问妹妹对去哪里进行蜜月的建议。情景四A:你是Annie, 你邀请你的搭档Jennie周日一起去看京剧---你们的共同爱好，你们都认为京剧是中国传统文化，体现了中国的文化历史，应该吸引更多青年人喜欢。B: 你是Jennie, 你邀请你周日一起去看京剧,你表示感谢并询问见面的时间地点等内容。你表示现在越来越多的人喜欢京剧了。要赢得更多人喜欢，京剧需要融入新的元素。情景五 A：你是李磊.你的朋友王宇搬入了新房子，你去拜访。你带了一瓶红酒作为礼物。你夸赞了新房子：很大，阳光充足、装修漂亮。 B: 你是王宇。李磊来你新家做客并带了礼物，你表示感谢。你请李磊留下来吃晚餐，并欢迎你经常来做客。情景六A:你是ABC公司总经理秘书Annie, 你接到了来自客户Tommy的电话，他想和总经理谈一下订单发货的事情，但经理在开会。你打算记录留言并转告经理。B:你是客户Tommy，你想想和总经理谈一下订单发货的事情，你希望尽快发货以方便圣诞节前的销售。经理不在，你决定让秘书转告，并期待经理尽快回电话给你。情景七A: 你是Annie, 你最近瘦了10斤，你的方法和健康：每天3千米的长跑，周末游泳一个小时，合理饮食，多吃蔬菜和新鲜水果。B：你是Jennie,你很羡慕Annie最近瘦了10斤，你询问Annie减肥的方法。你觉得自己生活方式不健康：睡得太晚，不爱运动，吃太多肉。你打算改变，从下周开始每天跑步和注意饮食。情景八A: 你是Annie,你来自内蒙古，现在在北京上学，你表示不太喜欢北京的气候，太干燥。而且夏天太热。不过你觉得北京冬天比你们家乡暖和。你觉得内蒙古的空气更好，晚上可以清晰看到很多星星。你邀请你好朋友Jennie 暑假去你们那里玩。B：你是Jennie,你是北京人，没去过内蒙古。你和来自内蒙古的Annie聊天，你询问她对北京天气的看法。你表示北京夏天没有空调不行，北京的空气正在逐渐改善。希望有机会去内蒙古看看。
工作生活英语情景对话 英语情景对话作为真实生活的交际模式，作为语言输出的源头，作为语言练习的最佳途径，作为语言教授的媒介，它对于把英语作为外语来学习的学生，扮演着非常重要的角色。下面为大家带来生活英语情景对话，欢迎大家学习! 工作生活英语情景对话1 Hi, Col, what can I do for you? If you have a few minutes, I would like to talk to you about the future of this company. Sure, have a seat. Thanks. Let me just grab your file. How long have you worked for us just now? I've worked here as a sales representative for about a year now. 1 year already, it's amazing how time flies like that. Are you enjoying your job? Yes, but I'd like to have a chance of job advance. I see. What job did you have in mind? Well, I've noticed that there is a position available as
a sales manager. Do you understand what duties that job would in tell ? Yes, I'll be directly responsible for all the sales representatives in my department. I assume there be more meetings, paper working and other responsibilies too. That's right. Do you have any experience in management? Yes, in fact if you look at my resume you can see that I was a manager before I started this job. Well, I think you'd be the perfect candidate for the position. According to company policy, you still have to go through the formal application procedures, so fill this application form in and I'll call you for an interview next week. Ok, thanks for your support. 工作生活英语情景对话2 Are you ready for the meeting? Yes, come on in. How is your new job going? It's challenging, but I'm enjoying it quite a bit. That's great. I knew you do a good job as a manager. Thanks a lot. How is your assistant manager getting on?