Eukaryotic RNases and their Partners in RNA Degradation and Biogenesis, Part B

227 Pages·2012·5.838 MB·1-229\227

by  Feng Guo and Fuyuhiko Tamanoi (Eds.)

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PREFACE
Study of small RNAs (20–30 nucleotides in length) has made a big impact
onbiomedicalresearchinthepast15–20years.MicroRNAs(miRNAs)are
endogenous non-protein-coding RNAs that are extensively involved in
development and cell physiology. Small interfering RNAs (siRNAs) are
centraltoRNAinterferencethatservesasanRNA-basedimmunesystem.
TheRNAitechnologyhastransformedhowgenefunctionsarestudiedand
is being explored as therapeutics. siRNAs may also be generated from
double-stranded RNAs transcribed from an organism’s own genome, and
these endogenous siRNAs are involved in transposon suppression and
heterochromatin formation. PIWI-interacting RNAs (piRNAs) maintain
genome stability in germ cells by silencing transposon elements.
WhilethesesmallRNAsdifferfromeachotherintheirsequences,bio-
genesis,andfunctions,theyallrequirenucleasesandRNA-bindingproteins
formaturationandforregulatingtheexpressionoftargetgenes.Inthisvol-
ume of The Enzymes, we present most up-to-date reviews of the nucleases
and nucleic acid-binding proteins that play critical roles in processing the
small RNAs from their usually much-longer precursors and in mediating
target gene silencing. Chapter 1 summarizes how the ribonuclease III
enzyme Dicer cleaves both miRNA and siRNA precursors and facilitates
theassemblyoftheRNA-inducedsilencingcomplexes(RICS).Chapter2
describes how three Drosophila RNA-binding proteins, produced from a
singlegeneloquaciousviaalternativesplicing,partnerspecificallywitheither
Dicer-1 or Dicer-2 to direct the cleavage of miRNAs and siRNAs. In
Chapter 3, how a heteromeric complex C3PO, composed of TRAX and
translin,activatestheRISCactivityisdiscussedinthecontextofthecrystal
structureofoctomericC3PO,whichrevealsacuriousfootball-shapedcage
with the RNA-binding residues and ribonuclease active sites located in
the interior. Chapter 4 presents the recent structural studies of the central
component of RISC, the Argonaute proteins.
Whereasthefirstfourchaptersconcernproteinsthatmainlyfunctionin
thecytoplasm,thefollowingtwochaptersdiscussthesmallRNAprocessing
events in the nucleus. Chapter 5 focuses on how another ribonuclease III
enzyme Drosha and its RNA-binding partner DGCR8 work together to
recognize and cleave primary transcripts of miRNAs. Chapter 6 reviews
ix
x Preface
howtheSMADproteins,criticalcomponentsoftheTGFbsignalingpath-
way, regulate the Drosha-mediated processing of a subset of miRNAs.
Chapter7explainshowthePIWIproteinscollaboratewithothernucleases
anduseitsownRNaseH-likesliceractivitytocontributetotheproduction
ofpiRNAsinthenucleusandcytoplasm.Chapter8describeshowthetumor
suppressor p53, often described as “the guardian of the genome,” and the
MCPIP1ribonuclease control miRNA maturation at the Drosha and Dicer
cleavagesteps,respectively.
Chapter9addressesthemostcriticaltechnicalhurdlethatisholdingback
theapplicationofRNAinterferencetechnologyfortherapeuticpurposes—
howtodeliversmallRNAstowheretheyneedtogoincombatingdiseases.
Nanoparticle-based delivery methods and their applications in cancer
treatment are reviewed.
Therearetopicsthatwehavenotbeenabletocoverinthisvolume.We
hopetobeabletodiscussothercriticaltopicsrelatedtosmallRNAsinfuture
volumes. We thank all the authors for their commitment and contribution
to this volume. We also thank Mary Ann Zimmerman for her encourage-
ment and guidance with the preparation of this book.
FENG GUO
FUYUHIKO TAMANOI
CHAPTER ONE
Dicer Proteins and Their Role
in Gene Silencing Pathways
Michael Doyle*,1, Lukasz Jaskiewicz†, Witold Filipowicz‡,1
*CenterforBrainResearch,MedicalUniversityofVienna,Vienna,Austria
†Biozentrum,UniversityofBasel,Basel,Switzerland
‡FriedrichMiescherInstituteforBiomedicalResearch,Basel,Switzerland
1Correspondingauthors:e-mailaddress:[email protected];[email protected]
Contents
1. Introduction 2
2. Dicers:RNaseIIIFamilyMemberswithaRoleinGeneSilencing 3
3. MechanismofProcessingandtheRoleoftheCatalyticRNaseIIIDomains 5
4. RoleofOtherDomainsinDicerFunction 10
4.1 PAZdomain 10
4.2 Helicase/ATPase 11
4.3 DUF283 12
4.4 dsRBD 13
5. DomainArrangementinMetazoanDicers 14
6. Inside-OutProcessingMechanismbytheYeastDicer 16
7. FunctionofDicerintheNucleus 18
8. ProteinsInteractingwithDicerandtheAssemblyofRISCEffectorComplexes 22
8.1 dsRBDproteins 22
8.2 Agoproteins 25
8.3 Otherproteinsinteractingwithdicer 26
9. FuturePerspectives 27
Acknowledgments 28
References 28
Abstract
TheRNaseIIIfamilyendoribonucleaseDiceristhekeyenzymeinvolvedintheRNAin-
terference(RNAi)andmicroRNA(miRNA)pathways.Apartfromitsfunctioninbiogen-
esis of miRNAs and short interfering RNAs (siRNAs), Dicer also plays a role in the
assemblyoftheRNA-inducedsilencingcomplex(RISC),whichfunctionsasaneffector
complexinRNAiandmiRNArepression.Inthischapter,wereviewourcurrentknowl-
edgeofDicerfromvariousorganisms.Wefocusonthefunctionsofindividualdomains
ofDicer,howtheenzymerecognizesandprocessesitssubstrates,andprovideinforma-
tiononDicer-associatedproteinsandtheirroleinsmallRNAbiogenesisandRISCas-
sembly. We also discuss Dicer's role in gene and chromatin regulation processes in
thecellnucleus.
TheEnzymes,Volume32 #2012ElsevierInc. 1
ISSN1874-6047 Allrightsreserved.
http://dx.doi.org/10.1016/B978-0-12-404741-9.00001-5
2 MichaelDoyleetal.
1. INTRODUCTION
In the past 10 years, RNA interference (RNAi) and microRNA
(miRNA)-mediatedgenesilencingpathwayshaveemergedaskeyglobalreg-
ulatorymechanismscontrollinggeneexpressionineukaryotes.Bothpathways
dependuponanenzymaticcascadethatgenerateseither20-to25-ntsmallin-
terferingRNAs(siRNAs)ormiRNAsbothofwhichfunctionbybasepairing
to target RNAs. Together with other proteins, including Argonautes, they
formpartofeffectorribonucleoprotein(RNP)complexesthatultimatelytrig-
ger translational repression and/or degradation of target RNAs resulting in
changesingeneexpression.SeveraldynamicclassesofRNPcomplexesexist
includingsi-ormi-RISCs(RNA-inducedsilencingcomplexes,actingpost-
transcriptionally)orRITS(RNA-inducedtranscriptionalsilencingcomplexes,
actingatthechromatinlevel).RegardlessofthetypeofRNPformed,allcon-
tainsmallRNAsderivedfromlongerdouble-strandedRNA(dsRNA)orhair-
pin precursors that are processed to their shorter lengths by RNase III-type
endonucleases,DroshaandDicer.Thegenome-encodedmiRNAsaretran-
scribedasprimaryprecursormiRNAs(pri-miRNAs)inthenucleusandmost
areinitiallycleavedbytheDrosha/DGCR8complextogenerateprecursor-
miRNAs(pre-miRNAs).ThesearethenexportedviaExportin5tothecyto-
plasmwheretheyareprocessedbyDicertothedouble-strandedsiRNA-like
formofmiRNA.DicerisalsoresponsibleforgenerationofsiRNAs,whichare
processedfromlongerdsRNAsaccumulatinginthecellastheresultofeither
viral infection or antisense transcription, or are experimentally induced to
specificallydownregulategeneexpression.
In1998,Fireetal.[1]werethefirsttodemonstratethatinCaenorhabditis
eleganssequence-specificgenesilencingcanbeinducedbydsRNA.Soonaf-
terward,itbecameapparentthatlongdsRNAisprocessedintosiRNAsthat
actastheeffectorsofRNAsilencing.Afteranintensesearchfortheenzymatic
activity responsible, Dicer was identified in Drosophila and demonstrated to
processdsRNAtosiRNAs[2].Similarenzymesweresoondescribedinother
eukaryotes, including mammals, with a copy number and domain structure
varying among the different organisms [3–5]. For example, four Dicer
proteins are present in Arabidopsis thaliana, two in Drosophila melanogaster,
and one in mammals, C. elegans, and Schizosaccharomyces pombe. Later, more
“primitive” Dicer proteins were characterized from unicellular eukaryotes,
including Trypanosoma brucei, Giardia intestinalis, and some budding yeast
species [6,7].
RoleofDicerinGeneSilencing 3
In this chapter, we review our current knowledge about Dicer and
Dicer-associated proteins. We will focus on the function of Dicer in
RNA silencing and posttranscriptional regulation in various organisms as
well as how structural and mutational studies have provided new insights
in the way Dicer functions.
2. DICERS: RNASE III FAMILY MEMBERS WITH A ROLE
IN GENE SILENCING
DicerisamemberoftheRNaseIIIfamilyofendoribonucleasesthat
specificallycleavedsRNA.Allmembersofthefamilypossesoneortwocop-
iesoftheRNaseIIIdomainwhichisresponsibleforrecognizingandcleav-
ingdsRNA.RNaseIIIwasfirstdescribedinEscherichiacoli,andsincethen,
manyendoribonucleasesrelatedtoithavebeenidentifiedthatareimportant
for RNA processing and posttranscriptional gene regulation in both pro-
karyotes and eukaryotes [8]. One of the most prominent roles of this class
of proteins is their involvement in pre-ribosomal RNA (pre-rRNA)
processing(reviewedbyNicholson[8]).Blaszczyketal. [9]subdivideden-
zymesoftheRNaseIIIfamilyintothreeclasses,accordingtotheirdomain
organization,butin2008,weproposedthatRNaseIIIorthologsbegrouped
into just two classes [3]. In the original classification, Drosha and Dicer
proteinsweregroupedintoseparateclassesduetotheperceivedcomplexity
at the time regarding their respective domain organizations. Since the dis-
covery of primitive Dicers, including those from T. brucei, G. intestinalis,
and some budding yeast species (e.g., Saccharomyces castelli, Kluyveromyces
polysporus, Candida albicans), having a less complex organization, the need
to separate Dicer and Drosha proteins is not necessary and we continue
to advocate that RNase III orthologs be grouped into just two classes.
According to this classification, class I includes all bacterial and fungal pro-
teins that contain a single RNase III domain as well as, generally, a
C-terminal dsRNA-binding domain (dsRBD). This class of RNase III
orthologs,functioningashomodimers,alsocontainssomeprimitiveDicers,
for example, K. polysporus Dicer which has a single RNase III domain and
twodsRBDs.Incontrast,classIIRNaseIIIorthologsfunctionasmonomers
andcontainthemorecomplexDicerandDroshaproteinswithtwotandem
RNaseIIIdomains(termedRNaseIIIaandIIIb)intheC-terminalregion.
In addition, Drosha and Dicer proteins contain multiple other domains in
varying combinations and copies, including helicase/ATPase, PAZ (Piwi/
Ago/Zwille),DUF283(domainofunknownfunction),anddsRBD.Relevant
4 MichaelDoyleetal.
Class I E. coli RNase III
B. subtilis Mini-III
S. cerevisiae Rnt1p
K. polysporus Dicer
Class II
Human Dicer
Drosophila Dicer-1
Drosophila Dicer-2
C. elegans Dicer
Arabidopsis DCL1
Arabidopsis DCL2
Arabidopsis DCL3
Arabidopsis DCL4
Giardia Dicer
Trypanosoma Dicer
Dictyostelium Dicer
Human Drosha
Drosophila Drosha
RNase III domain DUF283
Double-stranded RNA-binding domain PAZ domain
N-terminal domain P-rich domain
Helicase domain Truncated helicase RS-rich domain
Figure1.1 ClassificationofRNaseIIIfamilyenzymesintotwoclasses.ClassIcontains
bacterialandfungalRNaseIIIorthologsaswellasbuddingyeastDicer,allcontaining
asingleRNaseIIIdomain.ClassIIcontainsDicerandDroshaproteinscontainingtwo
RNaseIIIdomains.DomainorganizationoftheselectedproteinsoftheRNaseIIIfamily
isshownschematically.
examplesofproteinsbelongingtobothclassesofRNaseIIIenzymesaresche-
maticallyshowninFig.1.1.
ThecomplexityofDicerproteinscanbeviewedatmultiplelevelsrang-
ing from the various combinations of domains they contain in addition to
the catalytic RNase III domains, the number of different proteins that are
expressed in a single organism, to their localization and site of function in
the cell. In animals, plants, and S. pombe, Dicers are large multidomain
RoleofDicerinGeneSilencing 5
proteins. However, in most of lower eukaryotes, Dicer has a less complex
domain organization. The simplest Dicer proteins studied to date are that
foundinT.bruceithatcontainsjusttwoRNaseIIIdomainsandthoseactive
in some budding yeast species, containing single RNase III domain and
dsRBD (Fig. 1.1). Vertebrates and also some other organisms (e.g.,
C. elegans, S. pombe) express only one Dicer protein. In insects and plants,
which express multiple Dicer proteins, individual Dicers generally have
specialized functions in gene silencing. Two Dicers proteins, termed
Dcr-1 and Dcr-2, are expressed in D. melanogaster. Studies of mutant flies
and biochemical experiments revealed that Dcr-1 is required for the
processing of miRNA precursors, whereas Dcr-2 mainly processes siRNA
precursors [10,11]. Delineating the individual functions of the four
Dicer-like (DCL) proteins expressed in A. thaliana has proved more
challenging. DCL1 functions in miRNA biogenesis at both pri-miRNA
and pre-miRNA-processing steps as well as in siRNA production from
endogenous inverted repeats. The other three DCLs are required for
specific types of siRNA production: DCL2 processes natural cis-acting
antisense transcripts into siRNAs and also functions in viral defense,
DCL3 produces siRNAs which act as guides in chromatin modification,
and DCL4 is the key enzyme generating trans-acting siRNAs (tasiRNA)
which target mRNAs with only partially complementary sequences in a
similar manner as miRNAs [12,13].
3. MECHANISM OF PROCESSING AND THE ROLE OF THE
CATALYTIC RNASE III DOMAINS
Having identified Dicer to be a member of the RNase III family of
endoribonucleases [2], a concerted effort began to understand: (a) how the
enzymecleavesitssubstratestotheproductsofdefinedlength;(b)howsub-
straterecognitionisachieved;and(c)whatistheroleofindividualdomainsin
Dicerfunction.Thesequestionswereaddressedbiochemically,initiallywith
the human Dicer, and were later complemented by structural studies, most
notably those of G. intestinalis, human, and budding yeast Dicers. Human
Dicer is a large protein of approximately 220kDa that comprises, from the
N- to C-terminus, a helicase/ATPase, DUF283 and PAZ domains, two
RNaseIIIdomains,andasingledsRBD(Fig.1.1).Earlybiochemicalstudies
[14,15], performed with the purified human Dicer expressed in insect cells,
revealed that the endonuclease activity of Dicer requires the presence of
Mg2þ ions which can to an extent be substituted by Mn2þ or Co2þ. The
6 MichaelDoyleetal.
enzyme was demonstrated capable of cleaving dsRNA substrates, ranging
in size from 30 to 500bp, to (cid:2)20-bp siRNAs bearing 2-nt 30-overhangs
0 0
and 5-phosphate and 3-OH termini, characteristic features of products
generated by RNase III family enzymes [8]. The Dicer displayed a strong
preference to progressively process dsRNA substrates starting from their
ends. Blocking the ends of the substrate, either by capping them with
stable tetraloops or by DNA–RNA duplexes, resulted in internal cleavages,
but at much reduced levels [15].
The observation that generally Dicers are members of the class II of
RNaseIIIfamilyfocusedattentionon therolethatthetwoRNaseIIIdo-
mains play in the catalysis. The class I RNase III enzymes function as
homodimers andstructural characterization of the bacteriumAquifex aeolicus
(Aa)RNaseIIIandthemutagenicanalysisoftheE.coliRNaseIII(Ec-RNase
III)[9],suggestedamodelproposingthatRNaseIIIdimercontainstwocat-
alytic centers, each cleaving both strands of the dsRNA substrate at nearby
phosphodiesterbonds[9].ThespacingofthecatalyticcentersonthedsRNA
0
predictedgenerationof9-bpproductswith2-nt3-overhangs,consistentwith
the size of products generated by bacterial RNase III in vitro [8,9].
GiventhatmostDicerenzymescontain twotandemlyarrangedRNase
III domains and produce longer products, immediately indicated that the
mechanismofprocessingmustbedifferentfromthatproposedforprokary-
oticenzymes.SequencealignmentsofthehumanDicerRNaseIIIaandIIIb
domains with bacterial enzymes indicated thepresence of conservedacidic
residues likely functioning in dsRNA cleavage and Mg2þ coordination in
thehumanenzyme.Mutationoftheseresiduesincontextofthefull-length
humanprotein(D1320andE1564fromRNaseIIIa;andD1709andE1813
from IIIb; these residues are equivalent to the Ec-RNase III residues D44
and E110) demonstrated that they, but not additional other residues pro-
posed as also catalytic in the bacterial model [9], are essential for dsRNA
cleavage [16]. Analysis of the cleavage products generated by catalytic
residuesmutantsineithertheRNaseIIIaorIIIbdomainrevealedthatDicer
accesses its substrates in a polar fashion. Whereas the RNase IIIa domain
0
always processes the protruding 3-OH-bearing RNA strand, the RNase
0
IIIb cuts the opposite recessed 5-phosphate-containing strand. In
pre-miRNA,thesestandscorrespond tothe descendingarmof thehairpin
cutbydomainIIIaandtheascendingarmcutbyIIIb.Takentogetherwith
the results of gradient sedimentation and modeling of the Dicer RNase III
domainsinacomplexwithdsRNA,thesedataledtoaconclusionthatDicer
functions as an intramolecular pseudo-dimer with the RNase IIIa and IIIb
RoleofDicerinGeneSilencing 7
A B
Connector helix
HDeUliFca2s8e3, D1709, E1813 RIIIb PlatformPAZ RIIIa RIIIb
RIIIa dsRBD Platform
5¢ D1320, E1564 PAZ domain
3¢ PAZ RNase IIIa
C
Connector
PAZ Head helix
RNase III
a/b
Body
RNase III
a/b RNase IIIb
Helicase Helicase
PAZ
Base
Previous model New model
Figure1.2 StructuralorganizationofDicer.(A)Amutagenesis-basedmodelofdsRNA
processing by human Dicer [16]. Individual domains of Dicer are shown in different
colors.TheenzymecontainsasingledsRNAcleavagecenterwithtwoindependentcat-
alyticsites.ThecatalyticcenterisformedbyintramoleculardimerizationoftheRNase
IIIaandRNaseIIIbdomains.Mostimportantcatalyticresiduesareindicated.DsRBDpo-
sitioningisarbitrary.(B)CrystalstructureofG.intestinalisDicer[7].Thisribbonrepresen-
tationofDicershowstheN-terminalplatformdomain(blue),thePAZdomain(orange),
the connector helix (red), the RNase IIIa domain (yellow), and the RNase IIIb domain
(green).Positionsofmetalionsareindicatedbyvioletdots.PredictedlocationofdsRNA
isindicatedbyadashedline.PDB_id2fflwasrenderedusingDeepView[17]andPOV-
Ray3.6(www.povray.org).(C)EMreconstructionoftheshapeofafull-length human
Dicer.Leftpanelshowstheoriginallyproposeddomainarrangementalongwithbound
pre-miRNA [18–20]. Right panel shows the revised domain positioning based on
streptavidin labeling of inserted tags [21]. Positions of individual Dicer domains and
bound pre-miRNA, in relation to the head, body, and base regions of the letter
L-shaped protein, are indicated and see Ref. [21]. Figure (C), Right panel: Adapted
fromRef.[20].
domains forming a single-processing center containing two independent
catalytic“halfsites.”EachsiteiscapableofcuttingonestrandofthedsRNA
0
duplex generating products with a 2-nt 3-overhangs (Fig. 1.2A). Impor-
tantly, mutational analysis of Ec-RNase III and processing of its substrates
indicated that the bacterial enzyme also uses the single dsRNA-processing
center mechanism similar to that described for Dicer [16].
ValidityoftheproposedcatalyticmodelsforbothDicerandprokaryotic
RNase III were soon confirmed by additional biochemical and also