Genetic and molecular biology II 课程总结

Technorati Tags: APOBEC,cellular bodies,ChIP-on-chip,CTD,EMSA,FISH,Footprinting,FRAP,gene transcription,Histone chaperon,insulator,LC-MS,RNA world,RNAi,snoRNP,snRNP,TAP,Tiling-array

Genetic and molecular biologyII 课程总结（1）——methods

1、TAP（Tandem affinity purification）

Nature Cell Biology 6, 87 – 89 (2004)
doi:10.1038/ncb0204-87

Tandem affinity purification (TAP) is a two-step approach for the purification of tagged proteins under non-denaturing conditions.

Figure 1. Tagged versions of known and predicted signalling pathway components (blue) are stably expressed at levels similar to those of the endogenous protein.

(a) The TAP tag contains two immunoglobulin-binding domains from Staphyloccus aureus protein A (brown) and a calmodulin-binding domain (red), separated by a tobacco etch virus (TEV) protease cleavage site. After stimulation with TNF-, proteins are purified in two steps.

(b) First, the tagged protein is bound through the high-affinity interaction of protein A with IgG beads, washed of unbound protein, and eluted using the TEV protease.

(c) Second, tagged proteins are captured with calmodulin beads and eluted by the addition of EGTA. Non-interacting proteins are washed from the columns before elution. This two-step approach helps ensure that purified proteins truly associate with the bait protein in vivo.

(d) Eluted proteins are concentrated, separated by denaturing polyacrylamide-gel electrophoresis (PAGE), and sequential gel slices are made and analysed by LC−MS/MS.

Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (aka HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity. Generally its application is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).

2、FISH(Fluorescence In Situ Hybridization)

http://www.mtsinai.on.ca/pdmg/Genetics/cytogenetics.htm

Nature Reviews Genetics 2, 292-301 (April 2001) doi:10.1038/35066075

Chromosome territories：

3、EMSA（Electrophoretic mobility shift assay）

! non-denaturing poly-acrylamide gels !

http://www.promega.com/guides/protein.interactions_guide/chap7.pdf

The Electrophoretic Mobility Shift Assay (EMSA) also referred to as the gel retardation assay or
gel shift assay, is a common technique used to characterize protein:DNA/RNA interactions. Gel
shift assays are often performed concurrently with DNase footprinting, ChIP and primer
extension assays.

Overview of EMSA. The target protein is expressed in mammalian cells and a whole-cell extract is prepared. An alternate method relies on expressing the protein using mammalian-based, cell-free expression systems. In either case an aliquot containing the protein is incubated with the DNA sequence that has been labeled using either radioactive or non-radioactive methods and the DNA:protein complex is allowed to form. In order to maintain the protein:DNA complex, the reaction is run on a non-denaturing polyacrylamide gel. After electrophoresis, the experimental reaction is compared to a control reaction that contains only the labeled DNA to determine whether a protein:DNA interaction has occurred.

4、ChIP-on-chip (also known as ChIP-chip) is a technique that combines chromatin immunoprecipitation (ChIP) with microarray technology (chip)/tiling array.

http://en.wikipedia.org/wiki/ChIP-on-chip

http://en.wikipedia.org/wiki/Tiling_array

Genes & Dev. 2002 16: 245-256

5、FRAP（Fluorescence recovery after photobleaching）

Plant Methods 2006, 2:12

Principle and quantitative assessment of FRET via DFRAP. (a) In case of FRET between the donor CFP and the acceptor YFP due to interaction between two proteins A and B, the photochemical destruction of the acceptor abolishes FRET and leads to an increased emission from the donor, CFP. CFP and YFP are depicted as cyan and yellow ribbon models fused to putative interacting proteins A and B respectively. (b, c). Time-course analysis of fluorescence intensity before and after photobleaching in the presence or absence of a protein-protein interaction. Blue and yellow curves indicate the levels of CFP and YFP fluorescence before and after photobleaching, respectively. In case of FRET, bleaching of the acceptor molecule leads to an increase in donor fluorescence (b). In the absence of interaction between proteins A and B, CFP levels before and after the bleach do not vary considerably (c). BB – Before bleach, AB – After bleach.

6、DNaseI Footprinting

MoGeII课程总结（2）Method——Tiling Array

Human genome: 3 billion bps, i.e. 2m, with about 22000 protein coding genes; several hundred RNA encoding genes(>200bp); correspond to 5% coding sequences; repeat sequences >50%

New array data (using tiling arrays that contain only unique sequences, not repetitive regions) :
at least 15% of the human genome is transcribed (real or false?)

Nature Reviews Genetics 9, 179-191 (March 2008) doi:10.1038/nrg2270

Tiling microarrays： DNA microarrays with densely spaced or overlapping probes that allow for high-resolution genomic mapping.

Trends Genet. 2005 August ; 21(8): 466–475, Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping

Properties of tiling microarrays. (a) The design of a tiling microarray experiment. Each individual probe in the tiling is indicated by a different color and thick overbar. The probes making up the design constitute a ‘tile path’. Nucleotides not incorporated into probes are grayed. Most array designs randomize the position of the adjacent tiles on the array in an attempt to avoid systematic errors. (b) Tiling designs (tile paths) can be overlapping, end-to-end or spaced.

Genome Res. 2006 16: 271-281, Design optimization methods for genomic DNA tiling arrays

(Left) Evolution of genomic tiling arrays. Representing large spans of genomic DNA with bacterial artificial chromosome (BAC) clones facilitates global experimentation using relatively few array features, at the expense of low-tiling resolution. Higher-resolution designs using PCR products or oligonucleotides allow precise mapping of transcripts and regulatory elements, but require labor-intensive or technologically sophisticated approaches to implement. (Upper right) Linear feature tiling with gapped and end-to-end oligonucleotide placement. (Lower right) Overlapping tiles using fractional offset (e.g., one 25-mer probe placed every 5 nt) and single-base offset placement. The latter strategy provides a finer-resolution tiling of the genomic sequence, and can give a more precise indication of where hybridizing sequences are located on the chromosome.

The problem of sequence similarity in tiling genomic DNA.
(A) The level of similarity of oligonucleotide sequences to the remainder of the genome is represented by descending bars, where longer bars indicate more redundant sequences. If the redundancy exceeds a given threshold, indicated by the dashed line, the sequence is omitted from the tile path (B). Avoiding redundant or repetitive sequences inhibits adequate tiling of the sequence (C). Here, the level of non-repetitive sequence coverage decreases as the minimum tile size increases. At this point it also becomes necessary to use approximations that identify instances of known DNA transposons, retroelements, satellites, and other repetitive sequences, rather than calculating an explicit measure of sequence similarity. (D) In order to recover a higher percentage of nonrepetitive DNA, tiling algorithms can be devised that incorporate some redundant sequences (gray) in an optimal fashion, which balances the cost of inclusion against the gain in sequence coverage.

MoGeII课程总结（3）questions for transcription

Alberts et al., Molecular Biology of the Cell 4th ed

I、transcription, how nucleosome positioned on the DNA, how to open this structure and opose DNA sequence to the TFs and PolII? Interaction between DNA and TFs? DNA accessibility ? How the PolII moved? How will nascent mRNA be capped, spliced, polyA tail added? How about exportation of mRNA and the quality control?

The situation:75-90% DNA are packed in nucleosomes. Nucleosome: 2 DNA loops(~150bp)+ core histione octamer 2*(H2A, H2B, H3, H4),linker DNA(~10-50bp),11nm； -> 30nm dense packing of nucleosomes, histone H1

H3.3 is enriched of promotors with high Pol density

Euchromatin and Heterochromatin: interchromatin compartments of euchromatin, less condensed chromatin expands into these areas.

Histone chaperons: Dissolubility of histones, depositon(e.g. in replication and transcription) or eviction(e.g. after the passing by of PolII)

Deposition of histones and assembly of chromatin requires transient acetylation and several chaperon and chromatin assembly factors translocase.

Insulator: Not only insulate genes but also insulate heterochromatin from euchromatin

Gaszner and Felsenfeld, 2006, Nat Rev Gen 7, 703

core promoter: Promoter decide the start site and direction

e.g. CpG island promoter. CpG dinucleotides repetitive, gene associated, 0.5-2kb long, regulation by me(methylation), typically lack TATA box and DPE(downstream promoter element), may have INR(initiator), multiple weak TSS(transcription start site), half of the gene habe CpG island.

J. Biol. Chem., Vol. 282, Issue 20, 14685-14689, May 18, 2007

The textbook description states that the initiation of mRNA synthesis requires the recruitment and binding of the TATA-binding protein (TBP) as a component of the TFIID complex to a consensus sequence that is found in core promoters and is known as the TATA box. This view is now seriously challenged by a series of observations. (i) The TATA box is not a general component of all Pol II core promoters; (ii) not only TBP but different types of TBP-related factors can mediate Pol II transcription initiation; (iii) TBP- or TBP-related factor-independent Pol II transcription has been described; and (iv) TBP binding is not necessarily a prerequisite or even an indicator of promoter activation in vivo.

A variety of hypothetical core promoters and a schematic representation of protein complexes that may bind to them. Sequence specificity of the distinct promoter recognition factors (on the right) results in a variety of choices of transcription initiation sites (blue arrowheads) on the different core promoters (on the left). The number of blue arrowheads represents the strength of the given initiation site on the promoter. The different consensus sequence elements (TATA box, INR, DPE, CpG island, XCPE1 (X)) provide binding sites for subunits of promoter recognition factors . None of these elements are present in all promoters, and the interactions described may not fully represent all the possible protein-DNA interactions on the specified elements or may only be hypothetical.

TFs(transcriptional factors), coactivators: TFIID(TBP,TAF),TFIIB,TFIIA(not essential),PolII,TFIIF, TFIIH(helicase, ATPase, kinase(cyclin/CDK),interacte with CTD,phosphorylation), TFIIE(regulates helicase, kinase and ATPase activities)

*TBP is essential for transcription, but when knock down, the mouse zygote can still divede into several cells, why? Because the egg cell cytoplasm can supply the cell with TBP, when used up, die.*

Possible interaction forms between DNA and proteins:hydrogen bonds, electrostatic interactions, hydrophobic surface, major groove, minor groove

Modifications on the N-terminal histion tails(NTD,N-terminal domain): Acetylation(typicaly positive), Methylation(lysines,K), Methylation(arginines,R), Phosphorylation, Ubiquitylation(positive), Sumoylation(negatibe), ADP ribosylation, Biotinylation, Citrullination, cis/trans Isomerization

H3 me: K4me3(by Mll/WDR5/ASH2 methylated) and R2me2a(by PRMT6, Protein arginine methyltransferase6) are mutually exclusive.

Histone H3K4me3(trimethylation) stimulates splicing by enhancing the recruitment of U2 snRNP(small nuclear ribonucleoprotein particle)

HAT(histione acetyltransferase)(positive for transcription)<->HDAC(histione deacetylase),SIRT(negative)

HMT(histione methyltransferase)(H3 K4me, positive; H3 K9me negative)<->histone demethyllases(LSD1, JmjCs, others)

Ubi(activation)<->SUMO(repression)

In differentiation: RA(retionic acid receptor) is a molecular switch. RA replace HDAC -> opening of chromatin -> access of factors -> induction of differentiation genes

me(methylation) of DNA:->bingding site for specific proteins(the recruited corepressors HDACs)-> deacetylation of chromatin -> condensation of corresponding chromosome sections -> reduced accessibility of DNA for TFs

CTD(C-terminal domain) of PolII:52 tandem 7 a.a. repeats, Ser2 and Ser 5 phosphorylation are decisive for the processing of transcription(Enzyme: e.g. TFIIH,p-TEFb). the change ratio of Ser 2 and Ser 5: Ser5 -> Ser 2

When not Ph -> bing with basal factors -> PolII retard

Ph of Ser 5 -> initial actiation of PolII, release, promoter clearance, also capping proteins get to work(capping: 5′-5′ bond mGpppG/ApNpNp… protect mRNA from exonuclease)(Enzymes: guanylyl transferase, phosphohydrolase, guanine-7-methyltransferase for cap0, 2-prime-O-methyl-transferase for cap1 and cap2, cap1 is dominant)

Ph of Ser 5 and Ser 2 -> splicing(for spliceosome-mediated splicing：U1、U2、U4、U5、U6 participated in the process, U2 and U6 catalysed；SR(splicing regulator),U1,U2 with other factors are responsible for intron-exon definition, i.e. alternative splicing；EJC,exon-exon junction complex, bingding for afterwards checking by ribosome in cytoplasm) proeins get to work

*For splicing, there are two types: spliceosome-mediated splicing(GT/AG introns, GC/AG introns, AT/AC introns), Autocatalytic splicing(GroupI, GroupII)*

Ph of Ser 2 -> elongation of the reaction, PolyA tail adding(AAUAAA signal, A/U rich downstream region；CPSF,CstF,CFI,CFII,PAP, PolII CTD；after about 10ntA AAUAAA is not essential any more, and afterwards the tail will be binded with PABP for protection, in order to add long A)(abbr.:Cleavage and Polyadenylation Specificity Factor,Cleavage stimulation factor, Cleavage Factors, Poly A polymerase)

J. Biochem. 141, 601–608 (2007) doi:10.1093/jb/mvm090 :

Regulation and recognition of the phosphorylated CTD. Rpb1, the largest subunit of Pol II, has a unique C-terminal domain consisting of heptapeptide (YSPTSPS) repeats. The repeat number varies among different organisms, ranging from 26 in yeast to 52 in human. The CTD is mostly phosphorylated at Ser2 and Ser5 within the heptapeptide repeat during transcription. The kinases and phosphatases with specificity for Ser2 and Ser5 and the factors that bind to the CTD phosphorylated at Ser2, Ser5 or both, are indicated. Those factors include pre-mRNA processing factors, histone methyltransferases (HMT), nuclear peptidylprolyl cis/trans isomerase Pin1 and a novel WW domain containing protein PCIF1.

Dynamic changes in the CTD phosphorylation profile coordinate the Pol II transcription cycle with pre-mRNA processing and histone modification. (A) The general transcription factors (GTFs) form a complex with initiation-competent hypo-phosphorylated Pol II (Pol IIA) at the promoter. Transcription starts at the same time as Ser5 phosphorylation of the CTD (thick black line) by TFIIH. (B) Shortly after transcription initiation, capping enzyme (CE) is recruited to the phosphorylated Pol II (Pol IIO) through its direct binding to Ser5-phosphorylated CTD. The histone methyltransferase Set1-containing complex is also recruited and tri-methylates histone H3 Lysine 4 (H3K4). Transcription pausing induced by DSIF/NELF is relieved by P-TEFb-mediated CTD phosphorylation. (C) Elongating Pol IIO is increasingly phosphorylated at Ser2 by P-TEFb and associated with histone methyltransferase Set2, which tri-methylates histone H3 Lysine 36 (H3K36). Pol IIO also helps the recruitment of the splicing machinery (SP), which splices sites in the pre-mRNA (red line). This step is mediated by an unknown phosphorylated CTD-binding factor (X) that facilitates the efficient excision of introns (red broken line). (D) Near the 30 end of the gene, 30 end processing factors (PA) are increasingly recruited to Pol IIO through direct interaction between Pcf11 and the Ser2-phosphorylated CTD. After transcribing the poly(A) signal (AATAAA), 30 end processing factors possibly transfer to RNA to catalyse endonucleolytic cleavage (black arrow) and induce subsequent transcription termination, which is presumably helped by the 50–30 exonucleases Xrn2 and Pcf11. (E) After dissociating from the DNA template, Pol IIO is possibly dephosphorylated by the action of the CTD phosphatases, FCP1 and Ssu72, before recycling or reinitiation.

RNA transcriptional editing:ADAR(adenosine deaminases acting on RNA) recognises duplex RNA that is formed between the editing site and the ECS(Editing Site complementary Sequence) that is often located in a downstream intron, A to I(functioned as G) editing, base leve editing(there are also nucleotide level editing by gRNA, guid RNA, insertion or deletion of uridines).

*Another base level editing: APOBEC(apolioprotein B editing complex),C to U editing. antivirus (e.g. anti HIV) by APOBEC 3G, but Vif, HIV encode, a small protein mediates APOBEC degradation, they compete each other?*

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 13, pp. 8309–8312, March 31, 2006

Nature Reviews Immunology 4, 868-877 (November 2004) doi:10.1038/nri1489

mRNA export: mostly Ran-GTPase-independent.

Other nuclear RNA(mostly non-coding RNAs) exports:

Journal of Cell Science 116, 587-597 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00268

Karyopherin-mediated nuclear RNA export pathways:

Nucleocytoplasmic transport is mediated by the RanGTPase, karyopherin family of proteins

RanGAP: Ran-specific GTPase-activating protein -> cytoplasm

RanGEF: Ran-specific guanine-nucleotide-exchange factor -> nucleus

Cytoplasmic Tan -> inactive, GDP-bound form; nuclear-Ran -> bound to GTP

Import: cargo +Importin -> cargo, Imp+RanGTP；Export: Cargo+Exp+RanGTP -> RanGAP Cargo RanGDP Exportin(back to the nucleus)

more about RNA export: Nature Reviews Molecular Cell Biology 8, 761-773 (October 2007) doi:10.1038/nrm2255

mRNA possible structure: 5’cap –5′-UTR– AUG ——(ORF)—PTC?—-UAG/UGA/UAA 3′-UTR(ARE?)–PolyA tail (abbr.: PTC, premature termination codon；ARE: A/U rich element)

Checking/RNA stability: three major control mechaisms——NMD(Nonsense-mediated mRNA decay), NSD(non-stop decay), NGD(no-go decay)

*NMD(DNA intron!):premature termination codon, ribosome, EJC, decapping, 5′ to 3′ exonulease；NSD(in S. cerevisiae): “no stop of ribosome”, no stop codon mRNA degradation, 5′ to 3′ decay by Ski7 exosome, 3′ to 5′ decay by Xrn1；NGD(in S. cerevisiae):ribosome cannot go on moving because of e.g. a hairpin structure in the intron. Endonucleolytic decay, 3′ to 5′ decay by Ski7 exosome, 5′ to 3′ decay by Xrn1*

Possible models:

TRENDS in Biochemical Sciences Vol.30 No.3 March 2005

Hypothetical model for the coactivator complexes that contribute sequentially to the multiple subreactions of transcription for steroid-regulated genes. The order of coactivator association with the promoter might vary between different nuclear receptors (NR) or promoters from this simplified diagram. (1) The target-gene promoter is in its initial basal state with histone octamers, assuming a repressive configuration. (2) Ligand-bound NR – a steroid-receptor homodimer in this example – binds to a sequence specific hormone-response element on the promoter. The chromatin ATPase-dependent remodeling complex (3), SRC–CBP/p300 histone acetyltransferase (HAT) complex(4), histone methyltransferase (HMT) activity from CARM1 or PRMT1 (5) all bind to the NR in a sequential manner and alter the position of the nucleosome on the DNA and modify the N-terminal histone tails of nucleosome histones by acetylation (Ac) or methylation (Me). (6) The mediator complex facilitates interaction of the receptor with the pol II basal transcription apparatus. Splicing-related coactivators (7), such as PGC-1, p72, CoAA or CAPER, intervene to control the alternative splicing composition of the mRNA transcript, and ubiquitin-conjugating enzymes (Ubc), ubiquitin ligases (Ubl) and the ATPases of the proteasome cap (8) function to disrupt the initiation complex, enabling transcription elongation and cessation.

II、What does nucleus looks like? How dose nuclear structure affect gene transcription?

i、Active polymerases are immobilized and clustered(nature genetics • volume 32 • november 2002)

i.e. Transcription occurs in discrete foci within the nucleus

ii、Gene Order and Dynamic Domains(Science 306, 644, 2004)

Interphase SKY:

Given the evidence for chromatin mobility and the looping of gene loci from chromosome territories, it is an attractive possibility to consider that coregulated gene clusters from different genomic regions may also be proximal in the nuclear volume, as depicted in the theoretical magnification at right. The regulation of these localized gene clusters may take advantage of protein
concentrations (or may be the basis for them), as exemplified by the various types of nuclear bodies
found in the nucleus.

III、mRNA transportation in the cytoplasm and localization?

Possible roles of localized RNAs

Localisation by protection from degradation: maternal RNAs in egg cels are protected in a position-dependent manner.Elements in the 3′-UTR protect transcripts from degradation in the pole cells.

Localisation by transport: active transport and diffusion, active:

Vol. 13 March 1999 The FASEB Journal JANSEN:

Messenger RNA can be transported in the form of RNP particles (‘granules’) via microtubules or microfilaments. Transported RNA is subsequently anchored via cytoskeletal-associated proteins to either intersecting microfilaments (‘vertices’) or loosely bundled microtubules. Both transport and anchoring seem to rely mRNA signals in the 3’UTR.

The 3`UTR zipcode is the general control element of localisation, if the RNA will be sent to P bodies(processing bodies) for degradation.

more for reading:

Cap-tabolism,TRENDS in Biochemical Sciences Vol.29 No.8 August 2004

APOBEC3 Cytidine Deaminases: Distinct Antiviral Actions along the Retroviral Life Cycle,JBC Papers in Press, December 30, 2005, DOI 10.1074/jbc.R500021200

THE MANY ROLES OF AN RNA EDITOR, NATURE REVIEWS GENETICS VOLUME 2 NOVEMBER 2001 869

HOW DID ALTERNATIVE SPLICING EVOLVE?, NATURE REVIEWS GENETICS VOLUME 5 OCTOBER 2004 773

Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function, GENES & DEVELOPMENT 21:1833–3856 2007

Human housekeeping genes are compact, TRENDS in Genetics Vol.19 No.7 July 2003

MoGeII（4）Biogenesis of sn RNPs & cellular bodies

In vertebrates, the intron regions of protein-coding genes frequently encode snoRNAs.

Sm proteins + other proteins -> particles in nucleus -> snRNPs

scaRNA-> snRNA(U1 U2 U4 U5,PolII; U6,PolIII) & snoRNA

snRNA -> mRNA; snoRNA(C/D box-> Cajal bodies, H/ACA box) -> rRNA(PolI,28S,18S,5.8S; PolIII, 5S)

Telomerase and SRP(signal recognition particle) in CB(Cajal bodies) and in nucleus modification.

*RNAse P -> tRNA 5′ end processing; RNAse D -> tRNA 3′ end processing, add for typeII tRNAs CCA is added by terminal nucleotidyltransferase; PolII: miRNA*

J Cell Sci. 2004 Dec 1;117(Pt 25):5949-51

Current Opinion in Cell Biology, Volume 14, Issue 3, 1 June 2002, Pages 319-327

Structure and expression of snoRNAs:

EMBO reports 7, 6, 590–592 (2006) doi:10.1038/sj.embor.7400715

SnapShot: Cellular Bodies

Cell 127, December 1, 2006 ©2006 Elsevier Inc. DOI 10.1016/j.cell.2006.11.026

MoGeII（5）catalytic RNAs, RNA world, RNAi

catalytic RNAs:

GroupI、II introns; RNAse P; Viroids and virosoids

NON-CODING RNA GENES AND THE MODERN RNA WORLD, NATURE REVIEWS GENETICS VOLUME 2 DECEMBER 2001

The antiquity of RNA-based evolution, NATURE VOL 418 11 JULY 2002

“All life that is known to exist on Earth today and all life for which there is evidence in the geological record seems to be of the same form — one based on DNA genomes and protein enzymes. Yet there are strong reasons to conclude that DNA- and protein-based life was preceded by a simpler life form based primarily on RNA. This earlier era is referred to as the ‘RNA world’, during which the genetic information resided in the sequence of RNA molecules and the phenotype derived from the catalytic properties of RNA.”

Small RNAs: Classification, Biogenesis, and Function， Mol. Cells, Vol. 19, No. 1, pp. 1-15， Argonaute; MicroRNA; RNA Interference; RNA Silencing; RNase III; siRNA; Small RNA.

RNAi: RNA inerference, PTGS: post-transcriptional silencing, VIGS: cirus induced gene silencing, Homology-dependent silencing, Quelling, Cosuppression

Drosha/Pasha: RNAseIII, dsRNA binding activity

Dicer complex: RNAseIII, dsRNA bingding activity, RNA helicase, Ago proteins

RISC(RNA-induced silencing complex): Argonaute proteins(slicer nuclease, RNAse H)

Secondary siRNA production: RdRP(RNA-dependent RNA polymerase)

RITS(RNA-induced initiation of transcriptional silencing)

Argonaut: Ago; PAZ (Piwi, Argonaut, Zwille). Family, under family

Specialization and evolution of endogenous small RNA pathways, Nature Reviews Genetics 8, 884-896 (November 2007) doi:10.1038/nrg2179

Summary

· Eukaryotes have evolved small-RNA-guided regulatory systems for the control of RNA transcripts, chromatin, genome content and invasive agents.

· Specialized silencing systems evolved in eukaryotic lineages through proliferation and specialization of small-RNA biogenesis and effector factors.

· Genomes spawn new types of RNA silencing triggers through sequence duplications, bidirectional transcription and evolution of self-complementary foldbacks

· Amplification of endogenous silencing signals occurs by distinct secondary small interfering RNA (siRNA)-biogenesis mechanisms that involve RNA-dependent RNA polymerases in various lineages.

· Plant genomes can spawn new microRNA (miRNA)-generating loci de novo by inverted duplication of protein-coding sequences followed by accommodation by the specialized miRNA-biogenesis apparatus through sequence drift

· miRNA families expand through gene duplication, yielding sets of miRNAs with redundant, overlapping and specific functions. miRNA specialization within families can result from miRNA sequence differences and differential regulation of family members.

Model for Piwi-interacting RNA (piRNA) biogenesis. piRNAs are found in association with members of the Piwi subfamily of Argonaute proteins. The proposed piRNA-biogenesis model involves initial targeting of transcripts from transposons and retroelements by a Piwi-like protein that is programmed with a small RNA. Cleavage of the transcript generates the 5′ end of a new piRNA. Further 3′-end processing might require a distinct Piwi-like protein, such as Drosophila melanogaster AGO3, generating a new piRNA with a 3′ end that is offset by 10 nucleotides from the initial small RNA.

The chromatin-associated small interfering RNA (siRNA) pathway in Arabidopsis thaliana. RNA-directed DNA methylation (for example, at histone H3 lysines 4 and 9 (H3K4me and H3K9me, respectively) and chromatin remodelling in A. thaliana involves 24-nucleotide siRNAs formed through an RNADEPENDENT RNA POLYMERASE 2 (RDR2)–DICER-LIKE 3 (DCL3)–POLYMERASE IVA (PolIVa)-dependent pathway. Effector complexes containing siRNAs, ARGONAUTE 4 (AGO4) and PolIVb direct DNA and chromatin modifications through the activities of many factors, including DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1 and DRM2), CHROMOMETHYLASE 3 (CMT3), DEFECTIVE IN RNADIRECTED DNA METHYLATION 1 (DRD1) and SU(VAR)3-9 HOMOLOGUE 4 (SUVH4) (also known as KYP).

Table 1 Classes of small RNA identified in eukaryotes.

www.agrikola.org

AGRIKOLA project: Arabidopsis Genomic RNAi Knock-out Line Analysis

Viral suppressors can inactivate RNA interference

There are analogues of RNAi in prokaryots

more about gene expression and regulation:

SURVEY AND SUMMARY Alu elements as regulators of gene expression, Nucleic Acids Research, 2006, Vol. 34, No. 19 5491–5497

！Editor meets silencer: crosstalk between RNA editing and RNA interference,NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 7 DECEMBER 2006 919

Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? february 2008 volumume 9 http://www.nature.com/reviews/genetics