Protein Synthesis andTranslational Control
A subject collection from Cold Spring Harbor Perspectives in Biology
Copyright 2012 Cold Spring Harbor Laboratory Press.
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Protein Synthesis andTranslational Control
A subject collection from Cold Spring Harbor Perspectives in Biology
EDITED BY
COLD SPRING HARBOR LABORATORY PRESS
Cold Spring Harbor, New York † www.cshlpress.org
John W.B. Hershey Nahum Sonenberg
University of California, Davis McGill University
Michael B. Mathews
UMDNJ–New Jersey Medical School
Copyright 2012 Cold Spring Harbor Laboratory Press.
Protein Synthesis and Translational ControlA Subject Collection from Cold Spring Harbor Perspectives in BiologyArticles online at www.cshperspectives.org
All rights reserved# 2012 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkPrinted in the United States of America
Executive Editor Richard SeverManaging Editor Maria SmitProject Manager Barbara AcostaPermissions Administrator Carol BrownProduction Editor Diane SchubachProduction Manager/Cover Designer Denise Weiss
Publisher John Inglis
Front cover artwork: The cover art depicts the structure of the eukaryotic ribosome from the yeastSaccharomyces cerevisiae. The ribosome consists of four RNA chains (gray ribbons) and 79 dif-ferent proteins (colored ribbons). With a total mass of 3.3 MDa, it is more intricate and �40%larger than its bacterial counterpart. The previous edition of the translational control series,Translational Control in Biology and Medicine (2007), showed the structure of the bacterial ribo-some on its cover. The display of the structure of the eukaryotic ribosome on the cover of thisvolume is a demonstration of the recent remarkable advances made in the protein synthesisfield. The image was kindly provided by Marat Yusupov, Directeur de Recherche du CNRS.
Library of Congress Cataloging-in-Publication Data
Protein synthesis and translational control : a subjectcollection from Cold Spring Harbor perspectives in biology/
edited by John W.B. Hershey, Nahum Sonenberg, Michael B. Mathews.p. cm. -- (Cold Spring Harbor perspectives in biology)
Includes bibliographical references and index.ISBN 978-1-936113-46-0 (hardcover : alk. paper)
1. Proteins--Synthesis. 2. Genetic translation. 3. Geneticregulation. I. Hershey, John W. B. II. Sonenberg, Nahum. III.Mathews, Michael.
QH450.5.P77 2012572’.6--dc23
2012016831
10 9 8 7 6 5 4 3 2 1
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Contents
Preface, vii
Principles of Translational Control: An Overview, 1
John W.B. Hershey, Nahum Sonenberg, and Michael B. Mathews
The Structure and Function of the Eukaryotic Ribosome, 11
Daniel N. Wilson and Jamie H. Doudna Cate
The Mechanism of Eukaryotic Translation Initiation: New Insights and Challenges, 29
Alan G. Hinnebusch and Jon R. Lorsch
The Elongation, Termination, and Recyling Phases of Translation in Eukaryotes, 55
Thomas E. Dever and Rachel Green
Single-Molecule Analysis of Translational Dynamics, 71
Alexey Petrov, Jin Chen, Sean O’Leary, Albert Tsai, and Joseph D. Puglisi
The Current Status of Vertebrate Cellular mRNA IRESs, 89
Richard J. Jackson
From Cis-Regulatory Elements to Complex RNPs and Back, 109
Fatima Gebauer, Thomas Preiss, and Matthias W. Hentze
Regulation of mRNATranslation by Signaling Pathways, 123
Philippe P. Roux and Ivan Topisirovic
Protein Secretion and the Endoplasmic Reticulum, 147
Adam M. Benham
New Insights into Translational Regulation in the Endoplasmic Reticulum
Unfolded Protein Response, 163
Graham D. Pavitt and David Ron
P-Bodies and Stress Granules: Possible Roles in the Control of Translation and
mRNA Degradation, 177
Carolyn J. Decker and Roy Parker
mRNA Localization and Translational Control in Drosophila Oogenesis, 193
Paul Lasko
Toward a Genome-Wide Landscape of Translational Control, 209
Ola Larsson, Bin Tian, and Nahum Sonenberg
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Copyright 2012 Cold Spring Harbor Laboratory Press.
Imaging Translation in Single Cells Using Fluorescent Microscopy, 225
Jeffrey A. Chao, Young J. Yoon, and Robert H. Singer
A Molecular Link between miRISCs and Deadenylases Provides New Insight
into the Mechanism of Gene Silencing by MicroRNAs, 237
Joerg E. Braun, Eric Huntzinger, and Elisa Izaurralde
Translational Control in Cancer Etiology, 253
Davide Ruggero
Cytoplasmic RNA-Binding Proteins and the Control of Complex Brain Function, 281
Jennifer C. Darnell and Joel D. Richter
Tinkering with Translation: Protein Synthesis in Virus-Infected Cells, 299
Derek Walsh, Michael B. Mathews, and Ian Mohr
Emerging Therapeutics Targeting mRNATranslation, 327
Abba Malina, John R. Mills, and Jerry Pelletier
Index, 345
Contents
vi
Copyright 2012 Cold Spring Harbor Laboratory Press.
Preface
THE MECHANISM OF PROTEIN SYNTHESIS and its regulation have been studied intensively for more thana half-century, yet much remains to be learned. This is a particularly exciting time for such
studies, as the role of translational control in regulating gene expression is broadly recognized asmore important than previously thought. In the past, many studies focused on defining the transla-tional machinery and how it functions. The translation of specific mRNAs suspected of being regu-lated was also studied, establishing a variety of mechanisms for controlling the translational efficiencyof mRNAs. During the past few years, high-throughput methods have been applied to studies oftranslational control, resulting in the realization that such regulation is applied to the majority ofmRNAs. Situated at the nexus between nucleic acids and proteins, the importance of translationalcontrol, now appreciated for its role in establishing the cell’s proteome, is comparable to that of tran-scriptional control—a realization that makes studies of translational control even more compellingand essential.
The fact that protein synthesis is regulated broadly means that we need to understand a vast rangeof translational controls that operate on most mRNAs. This is an enormous challenge, as mRNAsdiffer in structure, in their modes of initiation, and in the assortment of cis-acting sequences thatcoordinate different regulating elements. Many mRNAs are themselves a collection of different struc-tures due to alternative promoters, splicing, or processing. In addition, multiple regulatory mechan-isms may operate on individual mRNAs, complicating their identification. To address this problemeffectively, a precise knowledge of the mechanism of protein synthesis is required. Recent advances inribosome structure, single-molecule studies, and reaction kinetics should provide the depth ofunderstanding required to explain regulation.
While we were contemplating editing a fourth edition of Translational Control, John Inglissuggested that we consider creating a book for the Perspectives series for the Cold Spring HarborLaboratory Press. Our previous editions, namely Translational Control (1996), TranslationalControl of Gene Expression (2000), and Translational Control in Biology and Medicine (2007), providedcomprehensive reviews of the process and regulation of protein synthesis. For the Perspectives series,we have attempted to focus on the current status of the field, with emphasis on aspects that needfurther elucidation and development. We have chosen a limited number of specialized areas thatwe feel are particularly important for future developments in the field. The volume begins with anumber of chapters that examine fundamental mechanisms of protein synthesis and continueswith chapters that describe approaches or mechanisms that apply broadly to many mRNAs. Anumber of chapters address a specific aspect of cell metabolism where translational control plays aprominent role. The volume ends with an examination of how insights into translational controlcan be used to develop therapeutic agents.
We thank all of the authors for their superb efforts in generating thoughtful and exciting chap-ters. The quality of the book rests on their efforts. We also thank John Inglis and Richard Sever fortheir encouragement, project manager Barbara Acosta for her competent and tireless attention to oursubmissions, and the production staff of the Press.
JOHN W.B. HERSHEY
NAHUM SONENBERG
MICHAEL B. MATHEWS
vii
Copyright 2012 Cold Spring Harbor Laboratory Press.
Principles of Translational Control: An Overview
John W.B. Hershey1, Nahum Sonenberg2, and Michael B. Mathews3
1Department of Biochemistry and Molecular Medicine, School of Medicine, University of California,Davis, California 95616
2Department of Biochemistry and Goodman Cancer Research Center, 1160 Pine Avenue West,Montreal, Quebec H3A 1A3, Canada
3Department of Biochemistry and Molecular Biology, UMDNJ—New Jersey Medical School,Newark, New Jersey 07103-2714
Correspondence: [email protected]; [email protected]; [email protected]
Translational control plays an essential role in the regulation of gene expression. It is espe-cially important in defining the proteome, maintaining homeostasis, and controlling cellproliferation, growth, and development. Numerous disease states result from aberrant regu-lation of protein synthesis, so understanding the molecular basis and mechanisms of trans-lational control is critical. Here we outline the pathway of protein synthesis, with specialemphasis on the initiation phase, and identify areas needing further clarification. Features oftranslational control are described together with numerous specific examples, and wediscuss prospects for future conceptual advances.
Protein synthesis is an indispensable processin the pathwayof gene expression, and is a key
component in its control. Regulation of trans-lation plays a prominent role in most processesin the cell and is critical for maintaining homeo-stasis in the cell and the organism. The synthesisrate of a protein in general is proportional to theconcentration and translational efficiency of itsmRNA. Translational control governs the effi-ciency of mRNAs and thus plays an importantrole in modulating the expression of many genesthat respond to endogenous or exogenous sig-nals such as nutrient supply, hormones, or stress.Because the vast majority of eukaryotic mRNAshave quite long half-lives (.2 h) (Raghavan etal. 2002), rapid regulation of the cellular levels ofthe proteins they encode must be achieved by
controlling their mRNA translational efficien-cies and protein degradation rates. During earlystages of viral infection (Walsh et al. 2012) and incells lacking active transcription such as oocytesand reticulocytes, translational control is oftenthe only mechanism to regulate the synthesisof proteins. Furthermore, protein synthesis ac-counts for a large proportion of the energy bud-get of a cell, especially one that is rapidly growingor biosynthetically active, and therefore requirestight regulation. Because protein synthesis is in-timately integrated with cell metabolism, aber-rations in its regulation contribute to a numberof disease states. It is therefore apparent that adetailed knowledge of the mechanisms that con-tribute to translational control is essential in un-derstanding cell homeostasis and disease.
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PROTEIN SYNTHESIS PATHWAY
Protein synthesis is a highly conserved processthat links amino acids together on ribosomesbased on the sequence of an mRNA template.To appreciate the complex translation pathwayin human cells, it is useful first to consider pro-tein synthesis in bacteria. The bacterial pathwayis coupled to transcription of the DNA intomRNA, made possible because no nuclear mem-braneseparatestheseprocesses. Itcomprises fourphases: initiation, elongation, termination, andrecycling (Fig. 1). The initiation phase involvesthe binding of the small ribosomal subunit (30S)to an unstructured region in the mRNA (theShine-Dalgarno region) that is complementaryto a portion of the 16S rRNA, and is stabilizedthrough an interaction between the ribosome-bound initiator formyl-methionyl-tRNA and
the initiation codon, usually AUG. Althoughformation of the 30S initiation complex is pro-moted by three initiation factors, identificationof the initiation site in the mRNA is based pri-marily on RNA–RNA interactions. The largeribosomal subunit (50S) then binds to form a70S initiation complex, which contains the for-myl-methionyl-tRNA in the ribosomal P-siteand which is prepared to enter the elongationphase. Elongation involves three steps: the bind-ing of an aminoacyl-tRNA whose anticodon iscomplementary to the mRNA codon in the ri-bosomal A-site; formation of a peptide bond bytransfer of the amino acid or peptide attached tothe tRNA in the P-site to the aminoacyl-tRNA inthe A-site; and translocation of the newly formedpeptidyl-tRNA from the A-site to the P-site, to-gether with the mRNA. These reactions are pro-moted by a number of elongation factors and by
fMet
30S AUG
50S
Initiation
GDP + Pi
GDP + Pi
Binding
fMet
AUG AUG
EPA
fMet aaGTP
GDP + Pi GTP
aaRecycling
mRNA GDP + Pi
Elongationcycle
Termination Translocation
Protein EPAEPA EPAAUGAUG
Peptidyltransferase
GTP
GTP
mRNA
fMet
Figure 1. Pathway of protein synthesis in bacteria. The simplified cartoon shows the four phases of proteinsynthesis and how the ribosomes, tRNAs, mRNA, and GTP interact. Not shown are the initiation, elongation,and termination factors that promote the reactions. Following initiation, each turn of the elongation cycle resultsin the addition of another amino acid (gray pentagon) to the growing peptide chain (not shown). Terminationoccurs when a termination codon (UAA, UAG, UGA) appears in the ribosomal A-site and involves hydrolysis ofthe peptidy-tRNA in the P-site. (Figure constructed by Nancy Villa, University of California, Davis.)
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the ribosome itself, with rRNA playing a partic-ularly notable part. When a termination codon(UAA, UAG, UGA) enters the A-site, termina-tion factors bind to the ribosome and promotethe hydrolysis of the peptidyl-tRNA. Ribosomesare then recycled through interactions with anumber of protein factors to generate ribosomalsubunits capable of undergoing another roundof protein synthesis. Detailed descriptions ofthe bacterial pathway are found in a number ofreviews (Laursen et al. 2005; Noller 2007).
Protein synthesis in higher cells shares manysimilarities with that in bacteria. The geneticcode is identical and the aminoacyl-tRNAs andtheir synthetases are very similar, but eukaryoticribosomal subunits, named 40S and 60S, arelarger and richer in protein, as illustrated byrecent high-resolution structures (see Wilsonand Cate 2012). Whereas the elongation phaseis strongly conserved, the initiation and termi-nation/recycling phases differ substantially. Aconspicuous feature of eukaryotic protein syn-thesis is the fact that mRNAs are translated inthe cytoplasm, making translation uncoupledfrom transcription. Mature eukaryotic mRNAspossess a m7G-cap at their 50-terminus and, inmost cases, a poly (A) tail at their 30-terminus.They are generally monocistronic, unlike mostbacterial mRNAs, and the pathway and mecha-nism for the formation of 40S and 80S initiationcomplexes differ substantially from those in bac-teria. For example, a large number of initiationfactors (at least 12) promote the binding of themRNA and initiator methionyl-tRNAi (Met-tRNAi)—which is not formylated—to the 40Sribosomal subunit. Therefore 40S initiationcomplex formation involves numerous pro-tein–RNA and protein–protein interactions, incontrast to what occurs in bacteria. Given thepreeminence of the initiation phase in the reg-ulation of protein synthesis, we develop themechanism of eukaryotic initiation in consid-erable detail in the following section. Termina-tion and recycling resemble the reactions in pro-karyotes, except that different sets of proteinspromote these phases. Eukaryotic initiationpathways are outlined in Figure 2; detailed de-scriptions of the molecular mechanisms arefound in Hinnebusch and Lorsch (2012).
MECHANISM OF EUKARYOTIC INITIATION
To elucidate translational control mechanisms,it is essential to define the detailed molecularmechanism of protein synthesis. The major ini-tiation pathway, scanning, involves binding of a40S–Met-tRNAi complex to the 50-terminusof an m7G-capped mRNA, followed by down-stream scanning along the mRNA until anAUG (or near-cognate) initiation codon is rec-ognized. The 60S ribosomal subunit then joinsthe 40S initiation complex to form an 80S ini-tiation complex capable of entry into the elon-gation phase. These reactions are promoted bytwelve or more initiation factors comprisingover 25 proteins (see Hinnebusch and Lorsch2012). Although much has been learned abouthow mammalian cells initiate protein synthesis,a number of gaps and uncertainties remain.For example, identification of initiation factorshas been based on their stimulation of in vitroinitiation assays constructed with purified com-ponents, and verified by genetic methods. How-ever, the recent discoveries of new proteins ap-parently involved in the pathway (e.g., DHX29[Parsyan et al. 2009] and DDX3 [Lai et al.2008]) suggest that all relevant initiation factorsmay not have been identified. In addition, therelevance of some identified factors is uncer-tain. For example, eIF2A promotes the bindingof Met-tRNAi to 40S ribosomal subunits, but itsrole in translation initiation is not well estab-lished (Komar et al. 2005; Ventoso et al. 2006). Anewly identified factor, eIF2D, promotes tRNAbinding into the ribosomal P-site in the absenceof GTP, but its mechanism of action and role ininitiation have not been defined (Dmitriev et al.2010). eIF5A promotes protein synthesis, butwhether it is involved in the initiation or elon-gation phase (Saini et al. 2009), or possibly justin formation of the first peptide bond, is con-troversial (reviewed in Henderson and Hershey2011). eIF6 was first identified as an antiribo-some association factor but its role in initiationwas then questioned (Si and Maitra 1999); how-ever, recent structural studies show clearly howbinding to the nascent premature 60S subunitprevents junction with the 40S initiation com-plex (Klinge et al. 2011).
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Despite its centrality, aspects of the scan-ning mechanism are not yet well elucidated.eIF4A is a well-established RNA helicase thatfunctions while tethered to the cap-bindingcomplex. However, does it continue to unwindRNA following 40S ribosomal subunit recruit-ment to the mRNA, or do other identified hel-icases such as DHX29 (Parsyan et al. 2009) andDDX3 (Lai et al. 2008) provide the helicasefunction during scanning? Are there mechanis-tic clues in the unusual bidirectionality of thehelicase activity of eIF4A, and the departures
from stoichiometry in the levels of some ofthe factors? Ribosome profiling methods detectinitiation at numerous sites in a large numberof mRNAs, some at non-AUG codons (Ingoliaet al. 2011), but how such initiation events areregulated is unclear. Because rigorous kineticanalyses of many of the reactions in initiationhave not been performed, we do not have a fulldescription of their reaction rates, yet such in-formation is essential for detecting and under-standing regulation during initiation. In con-trast, great progress has been made recently in
Cap dependent
HelicaseATP
ADP + Pi
IRES dependent
AUG 40S
GTPMet
EPA
Scanning
Initiation codon recognition
Subunit joining
ATP
ADP + Pi
GDP + Pi
60S
GDP + Pi
GTP
AUG
AUG
AUG
AUG
AUG
AUG
AUG
Figure 2. Pathway of eukaryotic initiation. The simplified cartoon shows two types of initiation mechanisms(m7G-cap-dependent scanning and IRES-dependent internal), and how the ribosomes, methionyl-tRNAi,mRNA, and ATP/GTP interact. Not shown are the initiation factors or the possibility that scanning followsIRES-directed binding of the 40S ribosomal subunit during internal initiation. (Figure constructed by NancyVilla, University of California, Davis.)
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elucidating the structure of eukaryotic ribo-somes (see Ben-Shem et al. 2011; Klinge et al.2011; Rabl et al. 2011; Wilson and Cate 2012),although atomic resolution structures of initia-tion complexes are still lacking.
Besides the scanning pathway, a sizablenumber of cellular mRNAs—generally estimat-ed as 5%–10%—may use a different way to re-cruit the ribosome. Direct binding of the 40Sribosomal subunit to an internal region of themRNA, called the internal ribosome entry site(IRES), bypasses the necessary recognition ofthe m7G cap (Fig. 2). IRES-mediated initiationis often used for the translation of viral mRNAs,and is reported for some cellular mRNAs as well(see Jackson 2012 for a critical analysis of cellularmRNAs containing IRESs). Still other initiationpathways have been described, involving shunt-ing (Yueh and Schneider 2000; Pooggin et al.2006), tethering (Martin et al. 2011), translationenhancers (Vivinus et al. 2001), the TISU ele-ment that frequently functions with mRNAspossessing very short 50-UTRs (Elfakess et al.2011), and a poly-adenylate leader in the 50-UTR that appears to function in the absence ofa number of the canonically required initiationfactors (Shirokikh and Spirin 2008). Althoughwe already possess much sophisticated knowl-edge of how these initiation pathways proceed,there remains much to be learned that is essentialfor a full understanding of translational control.
FEATURES OF TRANSLATIONAL CONTROL
Regulation of protein synthesis may occur atdifferent steps of the pathway, with the initia-tion phase being the most common target.Which phase of protein synthesis is affected isoften identified by determining polysome pro-files (Merrick and Hensold 2000) and ribosometransit times (Fan and Penman 1970; Palmiter1972). One of the salient features of translation-al control involves the number of mRNAs affect-ed. A given mechanism might affect the trans-lation of a single mRNA, a subset of mRNAs, ormost mRNAs. Global regulation often is basedon the activation or inhibition of one or morecomponents of the translational machinery,whereas specific regulation frequently occurs
through the action of trans-acting proteins (seeGebauer et al. 2012) or microRNAs (see Rouxand Topisirovic 2012) binding to cis-elements inthe mRNA. Some mRNAs are capable of escap-ing the effects of global activation or inhibition.Therefore, the response caused by a given mech-anism may be complex in terms of the mRNAsaffected. Methods that address this latter issueare ribosome profiling (Ingolia et al. 2009) in-volving high-throughput deep sequencing ofribosome-protected mRNA sequences, or DNAmicroarray technology, involving identificationof mRNAs in size-fractionated polysomes (seeLarsson et al. 2012). Such analyses of changes inribosome profiles caused by a difference inphysiological state enable identification of themRNAs that are most affected. The polysomeprofiling method is particularly powerful, as itdetermines where ribosomes are positioned onessentially all cellular mRNAs at a specific pointin time, thereby shedding light on the phase ofprotein synthesis that changes.
Another important feature of translationalcontrol is that a change in physiological state canactivate multiple regulatory mechanisms thataffect the rate of protein synthesis. Such redun-dancy complicates mechanistic studies, becauseinterfering with one mechanism does not nec-essarily alter the overall extent of inhibitionor activation. A further complication is that agiven mechanism may itself cause only a minorchange in protein synthesis rate. However, whenmultiple weak mechanisms act on the systemtogether, significant translational control canresult. Mechanisms that are modest in their ac-tion are especially difficult to elucidate, as theireffects sometimes only slightly alter a specificreaction rate. To detect and assess the impor-tance of such mechanisms, sophisticated andhighly accurate kinetic analyses are required,and are increasingly being pursued.
REGULATORY MECHANISMS
Recruitment of the mRNA to the 40S ribosomalsubunit is thought to be the rate-limiting step ofinitiation, and is often modulated. The bindingof methionyl-tRNAi also is frequently regulated,and subsequent steps such as scanning and
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initiator codon recognition may be affected aswell. How are these reactions regulated? Thecellular levels of the canonical initiation factorsdiffer in various cell or tissue types, thereby af-fecting initiation rates. Modulating the activi-ties of the initiation factors by phosphorylationis often used to regulate global rates of proteinsynthesis. The best-studied examples are phos-phorylation of eIF2a, which results in an inhi-bition of Met-tRNAi binding to ribosomes (seeHinnebusch and Lorsch 2012; Pavitt and Ron2012), and phosphorylation of 4E-BPs (seques-tors of the cap-binding protein, eIF4E), whichrelieves translational repression caused by de-creased mRNA recognition and binding to ri-bosomes (see Hinnebusch and Lorsch 2012;Roux and Topisirovic 2012). Numerous otherinitiation factors are phosphorylated, often astargets of signal transduction pathways, as areribosomes and the elongation factor eEF2, buthow such events regulate protein synthesis is notyet well established. Besides phosphorylation,posttranslational modifications such as methyl-ation, ubiquitination, and glycosylation, mayaffect protein synthesis, but these have not beenstudied extensively. One can anticipate thatmass spectrometric methods will identify newmodifications of importance in the near future.
mRNA levels appear not to be rate-limitingfor global protein synthesis in many cells, as asubstantial number of mRNAs are found as un-translated, freemRNPsrather thaninactivepoly-somes. However, mRNAs also can be seques-tered in stress granules or P-bodies (see Deckerand Parker 2012) or localized in specific regionsof a cell’s cytoplasm (see Chao et al. 2012; Lasko2012), indicating that mRNA accessibility caninfluence the efficiency of translation.
Regulation of translation through the actionof microRNAs is an exciting new area of study.MicroRNAs can stimulate the degradation ofmRNAs or affect protein synthesis directly (seeBraun et al. 2012). The mode of regulation ismRNA-specific, although a single microRNAmay affect a number of different mRNAs. Pre-cise mechanisms whereby the microRNAs affectprotein synthesis are yet to be elucidated. Recentin vitro experiments detecting early microRNA-based inhibition of protein synthesis prior to
mRNA deadenylation may resolve the contro-versy of which effect is dominant (Fabian et al.2009).
Trans-acting proteins affect initiation ratesby binding to specific mRNAs and interferingwith various steps of the pathway (see Gebaueret al. 2012). Such proteins frequently recognizea binding site in the 30-UTR, yet affect eventsoccurring near the 50-m7G cap. These regulatorymechanisms often function during early devel-opment (see Gebauer et al 2012; Lasko 2012) viaprotein-mediated crosstalk between the twoends of the mRNAs. Indeed, active mRNAs arethought to be circularized through an interac-tion between the poly (A)-binding protein(PABP) and eIF4G, which is a component of them7G-cap-binding complex (Wells et al. 1998).Some mRNAs, for example the histone mRNAsthat lack a poly (A) tail, are circularized throughspecialized proteins that bind near the 30-termi-nus of the mRNA and react with the cap-bind-ing protein complex (Cakmakci et al. 2008).However, mutant yeast lacking the PABP-eIF4Ginteraction show normal translation rates (Parket al. 2011), which suggests that circularizationdoes not invariably promote initiation, at leastnot in yeast. Another possibility, not yet estab-lished, is that circularization enhances the abil-ity of a terminating ribosome to reinitiate on thesame mRNA, perhaps by a mechanism that dif-fers from the canonical scanning mechanism.During analyses of the generation of polysomesin vitro, the rate of addition of a new ribosometo a polysome was slower than the ribosometransit time, yet polysome size increased, sug-gesting that ribosomes already present on apolysome reinitiate more efficiently on thesame mRNA than new ribosomes initiate (Nel-son and Winkler 1987).
Although less commonly reported, the elon-gation and termination phases (see Dever andGreen 2012) also are targets of translational con-trol. The rate of elongation is thought to be max-imal under most conditions (Ingolia et al. 2011),but can be inhibited by specific mechanisms.Whether such inhibition affects the elongationrates of all mRNAs equally is not well estab-lished. Only when elongation is slowed suffi-ciently, such that initiation is no longer rate-
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limiting, is the rate of protein synthesis affected.The occurrence of rare codons or strong second-ary structure in the coding region of a mRNA isthought to slow the rate of elongation. SomemRNAs can thereby be induced to undergo ashift in reading frame at a specific region, gen-erating a protein whose sequence and length dif-fer from the unshifted one. For proteins that areto be inserted into the endoplasmic reticulum,their elongation is paused by the signal recogni-tion particle, enabling the amino terminus ofthe nascent protein to dock onto the endoplas-mic reticulum, after which elongation resumes(see Benham 2012). The rate of elongation af-fects the folding of proteins (see Cabrita et al.2010); if elongation is too fast, e.g., when recom-binant eukaryotic proteins are synthesized inbacteria, many proteins fail to fold properly un-less the overall rate is reduced (Siller et al. 2010).Alternatively, slowing the elongation rate at spe-cific regions of the mRNA may enhance properfolding (Zhang et al. 2009), further demonstrat-ing the link between rates or elongation and pro-tein folding. Furthermore, the folding of thenascent protein as it emerges from the large ri-bosomal subunit can affect the elongation rate,either positively or negatively (see Cabrita et al.2010).
The termination phase also may be regulated(reviewed in Dinman and Berry 2007). Undermost circumstances, termination is not rate-limiting for protein synthesis, because ribo-somes are not found stacked up at the endsof mRNAs. However, termination can be sup-pressed, enabling either frame-shifting or read-through to occur, thereby extending the nascentprotein at its carboxyl terminus. The UGA stopcodon can be reprogrammed to enable the in-sertion of a seleno-cysteine residue rather thanto terminate synthesis. Incorporated into pro-teins by the translational process itself, seleno-cysteine has been called the “21st amino acid,”and it is now followed by the 22nd—pyrroly-sine, encoded by a UAG codon in some meth-anogenic archaea and bacteria (Atkins and Ges-teland 2002). Such “amendments” to theelongation and termination steps are influencedby the sequence context of the codon, or byother features of the mRNA.
FUTURE PROSPECTS
New discoveries of the involvement of transla-tional control in cell metabolism, proliferationand disease are being reported constantly. Theribosome profiling method has already identi-fied unexpected changes in the translation ofnumerous specific mRNAs and can be expectedto generate a vast amount of new data. Handlingthe plethora of information requires new andsophisticated bioinformatic methods that arerapidly being developed and refined (see Larssonet al. 2012). A challenge is presented by the pro-liferation of gene products, including alterna-tively processed mRNAs and protein isoforms,produced in higher cells. This diversification in-troduces additional levels of complexity thatneed to be accommodated in these analyses.Such high-throughput approaches do not gen-erally elucidate details of the molecular mecha-nisms involved, however. To understand the ob-served changes in mRNA translation, many ofthem rather modest in extent, it is necessary tohave a precise knowledge of the mechanism ofprotein synthesis.
What are the major challenges for under-standing translational control mechanisms? Wealready have a fairly detailed description of theprocess of protein synthesis during the initia-tion, elongation, termination, and recyclingphases. With the recent ability to obtain crystalsof eukaryotic ribosomes (Ben-Shem et al. 2011;Klinge et al. 2011; Rabl et al. 2011), we can an-ticipate atomic level structures of ribosomecomplexes that are essential for describing howpeptide bonds are formed and how the variousfactors interact on the surface of the ribosome topromote initiation, elongation, and termina-tion. However, crystals of 40S and 80S initiationcomplexes have eluded researchers, and evencryo-electron microscopic approaches have notyet yielded sufficiently precise structures of theseimportant intermediates. Another area lackingstructural information at high resolution per-tains to mRNAs. Although computer programscan predict structural motifs in RNA (Cruz andWesthof 2011), the actual 3-dimensional struc-tures, especially those of the 50-UTR, are onlynow beginning to be determined (Steen et al.
Principles of Translational Control
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Copyright 2012 Cold Spring Harbor Laboratory Press.
2011). Such detailed structures of mRNAs andtheir native mRNP complexes are eagerly await-ed, as they surely are important for mRNA bind-ing, scanning and initiator codon recognitionduring initiation. mRNA structures also affectthe elongation and termination rates, therebyaffecting protein folding and the regulation offrameshifting. So high-resolution mRNA struc-tural information, especially pertaining to theinitiation phase, is needed.
Another area in which our knowledge islimited pertains to the kinetics of the variousreactions, interactions and conformationalchanges involved in protein synthesis. The elon-gation phase is relatively well characterized, es-pecially for prokaryotes, but there are numerousinitiation steps that are yet to be studied in de-tail. Insights into the kinetics of initiation com-plex formation have been gained from studiesprimarily performed with yeast components(reviewed in Lorsch and Dever 2010 and Hin-nebusch and Lorsch 2012), yet much is yet to belearned. Do initiation factors form subcom-plexes off the ribosome, or do they first bindto the 40S subunits, and if so, in what order?Which proteins mediate the binding of Met-tRNAi to 40S ribosomes, and how is that rateaffected by other initiation factors? What is therate of ribosome scanning of the 50-UTR, and isthis rate affected by changes in the activities ofassociated initiation factors, e.g., those involvedin RNA helicase activity? Why do ribosomesappear to idle at the initiator AUG codon? Thatis, what limits their rapid progression into theelongation phase? Most kinetic analyses averagethe effects of many molecules over time. Theapplication of single molecule studies to thekinetics of protein synthesis (see Petrov et al.2012) likely will generate new views of howsuch reactions proceed. Additional work em-ploying both single molecule and standard ki-netic methods are needed to properly recognizeand evaluate many of the translational controlmechanisms that operate at the initiation phase,especially those mechanisms that only margin-ally affect reaction rates.
Complementing our constantly increasingunderstanding of the molecular mechanismsof translational control is the expectation that
more and more examples of regulation at thelevel of protein synthesis will be discovered. Anumber of promising areas of research are fea-tured in this volume. We anticipate that regula-tion by microRNAs will prove to be importantfor the translation for most mRNAs (Braunet al. 2012). How does the secretion or cotrans-lational folding of nascent proteins affect theirsynthesis (Cabrita et al. 2010; Benham 2012)?Other areas in which translational control al-ready is firmly established are described in lit-erature dealing with cell development (Lasko2012), cancer (Ruggero 2012), synaptic plastic-ity and memory (Darnell and Richter 2012),and viruses (Walsh et al. 2012). As translationalcontrol mechanisms are better understood andas high throughput methods identify when suchregulation occurs, we can confidently anticipatethat we will learn of additional areas of cellularmetabolism that are modulated through pro-tein synthesis. Indeed, it is becoming clear thattranslational control and transcriptional con-trol are comparably important in regulatinggene expression.
The relevance and importance of proteinsynthesis in disease and medicine is increasinglyrecognized and appreciated. The dysregulationof protein synthesis in a specific disease pro-vides a target for therapeutic intervention (seeMalina et al. 2012). As our knowledge of thestructures and detailed mechanisms of proteinsynthesis improve, this information can be ap-plied to enable more rational drug design.Therefore, research in the area of translationalcontrol will contribute to a better understand-ing of many disease states and to the develop-ment of novel therapeutic agents.
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Index
ABCE1, 22, 62–66
aIF2, 31–33, 44
AKT
mTORC1 modulation, 126–127
oncogenic signaling, 262, 267
AMP-dependent protein kinase (AMPK), 128
AMPK. See AMP-dependent protein kinase
Antisense inhibition. See eIF4E
Argonaute, microRNA-induced silencing complex role,
238–239, 245
A-site, 2, 56–57, 81, 83
ATF4
transcript in upstream open reading frame-dependent
translation initiation, 166–167
unfolded protein response, 166–167
ATF5
transcript in upstream open reading frame-dependent
translation initiation, 167
unfolded protein response, 167
BACCE501, 152
BDNF. See Brain-derived neurotrophic factor
BEZ235, 270
BHQ. See Black hole quencher
Biarsenical fluorescent dyes, fluorescence imaging in single
cells, 232–233
Bicaudal-C, 200
Bicaudal-D, 195
Bicoid, messenger RNA localization in oocyte pattern
specification
anterior–posterior protein gradient formation,
196–197
anterior targeting, anchoring, and translational
regulation, 196
cis-acting elements, 195
overview, 194–195
BiP, 152
Black hole quencher (BHQ), 81
BNIP3, 128
Brain-derived neurotrophic factor (BDNF), 228, 230, 289
CAF1, 244–245, 248
Calnexin cycle, protein quality control, 152–153
Calreticulin
calcium binding, 153
protein quality control, 152–153
structure, 153
Cancer
evolutionary considerations, 265–266
gene defects in translational machinery
initiation factors, 255, 257–258
ribosome protein mutations, 258–261
table, 256–257
oncogenic signaling and translation perturbation, 261
therapeutic targeting of translation components
eIF4E
antisense oligonucleotides, 334–335
cap interaction blockers, 333
eIF4G interaction uncoupling, 334
helicase inhibitors, 335–336
phosphorylation inhibitors, 336–337
eIF4F
phosphatidylinositol 3-kinase inhibitors, 332
rapamycin analogs, 329
target of rapamycin kinase inhibitors,
330–332
tumorigenesis role, 328–329
overview, 270–271, 328
prospects, 338
ternary complex formation inhibitors, 337–338
translational control
defects by cancer stage
progression and metastasis, 268–270
transformation and tumor initiation,
266–268
degradation, 253–255
Cap-independent translation enhancer
(CITE), 94, 307
Caprin, neuron function, 289–290
Cartilage–hair hypoplasia syndrome (CHH), 261
Caudal, 4EHP in translation repression, 202
CBP20, 333
CBP80, 333
CCR4, 117, 177, 179, 182, 199–201, 241–242,
244–245, 249
CDK11, 268
Cercosporamide, 337
CHH. See Cartilage–hair hypoplasia syndrome
CHOP
transcript in upstream open reading frame-dependent
translation initiation, 167–168
unfolded protein response, 167
CITE. See Cap-independent translation enhancer
COPII vesicle, 156, 158
CPEB. See Cytoplasmic polyadenylation element-binding
protein
cTAGE5, 156
Cup, 116, 203
345
Copyright 2012 Cold Spring Harbor Laboratory Press.
Cytoplasmic polyadenylation element-binding protein
(CPEB), 268
cognitive function, 287–288
CPEB4, 266
functional overview, 286
isoforms, 287
translation repression, 287
DAP5, 38
DAPK, 119
DBA. See Diamond-Blackfan anemia
DC. See Dyskeratosis congenita
DCP1, 181, 245
DCP2, 181, 183, 188, 245
Ddx3, 3–4, 35, 46
Ded1, 35, 38, 46
DENR, 64
Dhh1, 179, 181, 183
Dhx9, 3–4, 46
Diamond-Blackfan anemia (DBA), 259–261
Disulfide bond, formation, 154–156
DOM34, 64–65
Dyskeratosis congenita (DC), X-linked, 258–259, 261
Edc3, 183–184
EDC4, 245, 247
EDD, 241–242, 246
EDEM1, 152–154
eEF1, 20
recycling, 57
eEF1A, 55–57, 137
eEF2, 20, 32, 56–57, 132, 308
diphthamide modification, 57–58
phosphorylation sites, 137
eEF2K, phosphorylation sites, 137
eEF3, 58–59, 65
EF-G, 23, 57, 60–61, 63, 81, 83–85
EF-P, 59–60
EF-Tu, 22–23, 32, 55–56, 60–61, 64, 81–83
Egalitarian, 195
eIF1, 64
binding site on ribosome, 19–20
phosphorylation sites, 137
start codon recognition role, 39–43
eIF1A, 22, 33, 47–48, 64
start codon recognition role, 39–43
eIF2, 31, 33–34, 36, 47, 93
innate immunity
overview, 310
phosphorylation inhibition by viruses
bypassing, 312–313
combinatorial strategies, 312
inhibitors, 312
Met-tRNAi recruitment to small ribosomal subunit
eIF2.GDP recycling, 33–34, 47
overview, 31–32
ternary complex binding promotion factors, 32–33
start codon recognition role, 43
eIF2a, 136, 164, 172, 312–313, 337
eIF2B, 33, 165–166
eIF2Be, phosphorylation sites, 137
eIF2b, 32
eIF2D, 64
eIF2g, 32, 34, 43
eIF3, 31–33, 38, 63–64, 90, 93, 117, 183, 255, 257–258,
300, 302, 314
messenger RNA recruitment, 37–38
phosphorylation sites, 137
eIF3c, start codon recognition role, 43–44
eIF3e, 36
eIF4A, 4, 31, 34–38, 46, 93, 306, 309, 335–336
eIF4B, 36–37, 131–132, 266, 310
eIF4E, 31, 34–35, 38, 93, 129, 134, 199, 202, 257, 262–265,
268–270, 300, 302, 306, 309–310, 315,
328–330
cancer therapeutic targeting
antisense oligonucleotides, 270, 334–335
cap interaction blockers, 333
eIF4G interaction uncoupling, 334
helicase inhibitors, 335–336
phosphatidylinositol 3-kinase inhibitors, 332
phosphorylation inhibitors, 336–337
rapamycin analogs, 329
target of rapamycin kinase inhibitors, 330–332
tumorigenesis role, 328–329
eIF4F, 37, 46, 94–95, 266, 304, 306, 309, 315, 329
phosphorylation
sites, 136
viral DNA replication promotion, 309–310
eIF4G, 6, 31, 34–36, 38, 46, 62, 92–94, 117, 199, 257, 300,
302, 306, 309–310, 315
eIF4GI, 38–39, 96, 136, 181, 269
eIF4GII, 38–39
eIF4H, 37, 46, 131–132, 136
eIF5, 32–33, 36, 47, 90
eIF2-mediated translational control response
role, 165–166
phosphorylation sites, 137
start codon recognition role, 39–44
eIF5A, 59–60, 65, 257, 268
eIF5B, 47–48, 90, 137, 202
eIF5G, 47
eIF6, 3, 137, 265, 267
Elongation, translation
eEF1 recycling, 57
eEF2 diphthamide modification, 57–58
eEF3 function, 58–59
EF-P, 59–60
eIF5A, 59–60
overview in eukaryotes, 55–57
prospects for study, 65–66
single-molecule studies in bacteria
initiation transition to elongation, 76–77
ribosome
conformational changes, 79–81
tracking, 78
Shine-Dalgarno sequence clearing, 78
Index
346
Copyright 2012 Cold Spring Harbor Laboratory Press.
EMT. See Epithelial-to-mesenchymal transition
Encephalomyocarditis virus. See Picornavirus internal
ribosome entry sites
Endoplasmic reticulum (ER)
calnexin cycle in protein quality control, 152–153
disulfide bond formation, 154–156
glycosylation of proteins, 150–152, 154
inositol-requiring enzyme-1 ribonuclease activity and
protein-folding homeostasis, 172
protein exit and secretion regulation, 156–158
protein targeting, 147–150
unfolded protein response. See Unfolded protein
response
Endoplasmic reticulum oxidoreductase, 154–155
Epithelial-to-mesenchymal transition (EMT), cancer, 269
EPRS, 117–118
ER. See Endoplasmic reticulum
ERdJ5, 152–153
eRF1, 22, 60–61, 63–66, 314
eRF3, 22, 60–61, 63–65
ERGIC53, 157–158
ES. See Expansion segment
E-site, 57, 81–82
Expansion segment (ES), ribosomal RNA, 14, 16–17
FKBP12, 125, 330–331
FlAsH, 232
FLuc, small interfering RNA screening for internal ribosome
entry site, 100–101
Fluorescence microscopy. See Single-cell imaging;
Single-molecule studies
Fluorescence resonance energy transfer (FRET)
principles, 73–74
single-molecule studies in bacteria
ribosome conformational changes during initiation
and elongation, 79–81
transfer RNA
conformational changes, 81
dynamics in ribosome, 82–84
ribosome interactions, 84–85
Fluorescent noncanonical amino acid tagging (FUNCAT),
global measurement of translation in single
cells, 228
FMR1. See Fragile X syndrome
FMRP. See Fragile X syndrome
Foot and mouth disease virus. See Picornavirus internal
ribosome entry sites
4E1RCAT, 334
4E-BP
cancer
therapeutic targeting, 333–334
translational control, 262–264
4E-BP1, 95
mTORC1 signaling to translational machinery, 129–132
phosphorylation sites, 136
4EGI-1, 334
4EHP, translation repression of Caudal and Hunchback
messenger RNAs, 202
Fragile X syndrome, FMRP
function and defects, 282–285
messenger RNA target identification, 283–284
therapeutic targeting, 291
FRET. See Fluorescence resonance energy transfer
FUNCAT. See Fluorescent noncanonical amino acid tagging
GADD34, 167–169, 315
GAIT complex, temporal control of translation, 117–118
GCN2, 337
GCN4, 37, 47, 109, 168
Genome-wide analysis, posttranscriptional gene expression
cis and trans factor identification, 216–219
data analysis, 215–216
dynamic regulation, 212
techniques for study, 209–211
translational activity analysis, 213–215
Gld2, 286
Glucosyl transferase (GT), 152
Glyceraldehyde 3-phosphate dehydrogenase (GPDH),
117–118
Glycosylation
endoplasmic reticulum proteins, 150–152
protein secretion effects, 154
GPDH. See Glyceraldehyde 3-phosphate dehydrogenase
GT. See Glucosyl transferase
Gtr1, 127
Gtr2, 127
GW182
domain organization, 240–241
microRNA-induced silencing complex
plant studies, 246–247
protein interactions
deadenylase complex, 242
plasticity, 242–243
poly(A)-binding protein interactions and
function, 240, 243–244
redundant and combinatorial interactions, 245
recruitment, 239
proline-rich motif, 242
Hac1p, 165
HBS1, 64–65
HHT. See Homoharringtonine
Hippuristanol, 335–336
HITS-CLIP, messenger RNA-binding protein target
identification, 265, 283–285
Homoharringtonine (HHT), 328
HRI, 337–338
Hrp48, 200
Hu, neuron function, 290
Human rhinovirus. See Picornavirus internal
ribosome entry sites
Hunchback, 4EHP in translation repression, 202
ICP6, 308–309
IF1, 18–19, 74–75
Index
347
Copyright 2012 Cold Spring Harbor Laboratory Press.
IF2, 18–19, 74–75, 79
order of IF2 and transfer RNA arrival in
bacteria, 75–76
IF3, 18–19, 63, 75
Initiation, translation
bacteria overview, 2–3
cancer defects in initiation factors, 255, 257–258
eukaryote overview, 3–5, 29–31
initiation factor binding sites on ribosome, 18–20
initiator transfer RNA recruitment, 34
internal ribosome entry site. See Internal
ribosome entry site
messenger RNA recruitment to ribosome. See
Messenger RNA
prospects for study, 48
ribosomal subunit joining, 47–48
RNA helicases, 45–46
single-molecule studies in bacteria
elongation transition, 76–77
order of IF2 and transfer RNA arrival, 75–76
overview, 74–75
ribosome conformational changes, 79–81
start codon recognition
eIF1, 39–43
eIF1A, 39–43
eIF2, 43
eIF3c, 43–44
eIF5, 39–44
messenger RNA sequence context, 44
ribosomal RNA role, 44–45
transfer RNA role, 44–45
transfer RNA recruitment to ribosome.
See Transfer RNA
INK128, 270
Inositol-requiring enzyme 1 (IRE1)
functional overview, 165
ribonuclease activity and protein folding
homeostasis, 172
translational pausing and colocalization of XBP1
messenger RNA with IRE1 effector domain,
170–172
Internal ribosome entry site (IRES)
cap-independent mechanisms of initiation,
94–95
ITAFs, 93–94
messenger RNA in cells
bicistronic plasmid test, 96–97
controls for screening from cryptic promoters or
splicing, 98–100
evidence, 95–96
mapping, 103
prospects for study, 103–105
RNA polymerase II transcription dependence,
97–98
small interfering RNA screening for FLuc
expression, 100–101
transfection and in vitro translation
assay, 101–102
overview, 89–90
picornavirus internal ribosome entry sites
class III and class IV site mediation, 306–307
classification, 90–93
initiation factor requirements, 93–94
overview, 306
trans-acting factor requirements, 94–95
virus distribution, 307
IRE1. See Inositol-requiring enzyme 1
IRES. See Internal ribosome entry site
ITAFs. See Internal ribosome entry site
K10, 198
L13a, GAIT complex, 117–119
L30e, 14
L41e, 22
La, 94
Long-term depression (LTD), translational regulation
in neurons, 282
Long-term potentiation (LTP), translational regulation in
neurons, 282
LTD. See Long-term depression
LTP. See Long-term potentiation
Mammalian target of rapamycin. See Target of rapamycin
MAPKs. See Mitogen-activated protein kinases
Mass spectrometry, interactome capture, 113
MCFD2, 157
MCT-1, 64
MDM2, 266
Messenger RNA (mRNA)
decay
decapping promotion and translation initiation
repression, 179–182
messenger ribonucleoprotein granules
aggregation, 186
assembly in cytoplasm, 183–184
dynamics in cytoplasm, 185–186
mRNA cycle model, 186–187
nontranslating messenger RNA assembly into
RNA–protein granules, 182–183
pathways, 177–179
decoding, 22–23
internal ribosome entry site. See Internal ribosome
entry site
oogenesis studies in Drosophila. See Oogenesis,
Drosophila
recruitment to ribosome
eIF3 role, 37–38
eIF4B role, 36–37
eIF4F role, 34–36
initiation factor knockout studies in yeast, 38–39
overview, 5–6
single-molecule studies in bacteria, 78
start codon recognition
eIF1, 39–43
eIF1A, 39–43
eIF2, 43
Index
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Copyright 2012 Cold Spring Harbor Laboratory Press.
eIF3c, 43–44
eIF5, 39–44
messenger RNA sequence context, 44
ribosomal RNA role, 44–45
transfer RNA role, 44–45
MFC. See Multifactor complex
MicroRNA
Drosophila ovary messenger RNA protection
from degradation, 199
functional overview, 237–238
translation regulation, 5–6
MicroRNA-induced silencing complex (miRISC)
Argonaute role, 238–239, 245
cytoplasmic deadenylase complexes, 244–245
deadenylation interaction with translational repression,
247–248
decapping enzymes, 245
GW182
domain organization, 240–241
plant studies, 246–247
proline-rich motif, 242
protein interactions
deadenylase complex, 242
plasticity, 242–243
poly(A)-binding protein interactions and
function, 240, 243–244
redundant and combinatorial interactions, 245
recruitment, 239
mechanism, 238–240, 247
prospects for study, 248–249
miRISC. See MicroRNA-induced silencing complex
Mitogen-activated protein kinases (MAPKs)
interacting kinase inhibitor therapy in cancer, 336–337
mTORC1 modulation, 127–128
signaling to translational machinery
interacting kinases, 132–134
overview, 132–133
prospects for study, 135–137
ribosomal S6 kinase, 134–135
mRNA. See Messenger RNA
MSL2, translational repression of messenger RNA, 114–115
mTORC. See Target of rapamycin
Multifactor complex (MFC), 29, 33, 65
Myc, 267
Nanos
messenger RNA localization in oocyte pattern
specification
cis-acting elements, 195
overview, 194–195
targeting to posterior pole plasm, 198
translational control, 200–201
temporal and spatial control of translation, 115–117
Neuroligin, 233
NOT, 177, 179, 182, 241–242, 244–246, 249
NSAP1, 117–118
OAS. See Oligoadenylate synthase
Oligoadenylate synthase (OAS), 303
Oligosaccharide transferase (OST), 151–152
Oogenesis, Drosophila
advantages as model system, 193
4EHP in translation repression of Caudal and
Hunchback messenger RNAs, 202
messenger RNA localization in pattern specification
bicoid
anterior–posterior protein gradient formation,
196–197
anterior targeting, anchoring, and translational
regulation, 196
cis-acting elements, 195
gurken localization, 198
nanos
targeting to posterior pole plasm, 198
translational control, 200–201
oskar
targeting to posterior pole plasm, 197–198
translational control, 199–200
overview, 194–195
protection from degradation, 199
Vasa as translational activator, 202–203
Oskar, messenger RNA localization in oocyte pattern
specification
cis-acting elements, 195
overview, 194–195
targeting to posterior pole plasm, 197–198
translational control, 199–200
OST. See Oligosaccharide transferase
p27, 259
p53, 259–260, 268
Pab1, 179
PABP. See Poly(A)-binding protein
PAN2, 241–242, 244–245
PAN3, 241–242, 244–246
PAR-CLIP, 111–113, 218
PARN, 286
Pat, 245
Pat1, 179, 181–182, 185
Pateamine A, 335
P-body
aggregation, 186
assembly in cytoplasm, 183–184
dynamics in cytoplasm, 185–18
messenger RNA decay
decapping promotion and translation
initiation repression, 179–182
pathways, 177–179
mRNA cycle model, 186–187
PCBP-2, 94
PDCD4
phosphorylation sites, 136
translational regulation, 131
PDI. See Protein disulfide isomerase
PDK1, 131
PDX1, 155
Peptidyl transfer center (PTC), 56, 61
PERK, 164, 168–169, 172, 265–265, 310, 312, 337
Index
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Copyright 2012 Cold Spring Harbor Laboratory Press.
Peroxiredoxin IV, 156
Phosphatidylinositol 3-kinase (PI3K)
inhibitors for cancer treatments, 332
mTORC1 modulation, 126, 135
oncogenic signaling, 262
PI3K. See Phosphatidylinositol 3-kinase
PIC. See Preinitiation complex
Picornavirus internal ribosome entry sites
class III and class IV site mediation, 306–307
classification, 90–93
initiation factor requirements, 93–94
overview, 306
trans-acting factor requirements, 94–95
PIKK, 329, 332
PIM2, 336
PKR. See RNA-dependent protein kinase
Poglut, 154
Poly(A)-binding protein (PABP), 6, 31, 34, 62, 66, 117,
240–244, 286, 302, 309, 314
Polypyrimidine tract-binding protein
(PTB), 92, 94, 199
POP2, 244–245
Pop2, 177, 179, 182
PP242, 270
PPIR15A, 169–170
PPIR15B, 169–170
PRAS40, 127
Preinitiation complex (PIC), 29–31, 34–42, 77
PRF. See Programmed ribosomal frameshifting
Programmed ribosomal frameshifting (PRF), 259
Protein disulfide isomerase (PDI), 152, 154–156
PRTE. See Pyrimidine-rich translation element
PSD95, 233
P-site, 2, 18, 39, 44, 56–57, 78, 81, 83–85
PTB. See Polypyrimidine tract-binding protein
PTC. See Peptidyl transfer center
PTEN, 329, 336
Pumilio
mechanism of action, 288
neuron function, 288
Puromycin, fluorescent analogs for global measurement
of translation, 228–229
Pyrimidine-rich translation element (PRTE), 270
RACK1, 14, 188, 265
Rapamycin, analogs for cancer treatment, 329
Ras, 135
RCK, 179, 245–246
ReAsH, 232
REDD1, 128
RF1, 22, 61, 63
RF2, 22, 61, 63
RF3, 60, 63
Rft1, 150
Rheb, 127–128
Ribonucleoprotein particles (RNPs)
cis/trans interactions, 113–114
cross-linking studies, 111–113
interactome capture, 113
messenger particles as templates for translation control,
110–111
messenger ribonucleoprotein granules. See P-body;
Stress granule
prospects for study, 119
RNA affinity chromatography, 113
Ribosomal recycling factor (RRF), 63, 65
Ribosomal RNA (rRNA)
expansion segments, 14, 16–17
features in eukaryotes, 14–16
start codon recognition role, 44–45
Ribosomal S6 kinase (RSK), mitogen-activated protein
kinase signaling to translational machinery,
132, 134–135
Ribosome
binding sites
initiation factors, 18–20
transfer RNA, 17–18
cancer and protein mutations, 258–261
messenger RNA recruitment. See Messenger RNA
proteins of eukaryotes, 16–17
recycling, 22–23, 62–63–65
single-molecule studies in bacteria
conformational changes, 79–81
tracking during elongation, 78
transfer RNA
dynamics, 82–84
interactions, 84–85
transit, 81–82
structure
large subunit, 13
overview, 11, 13
small subunit, 12
subunit interactions, 21–22
ternary complex binding to small subunit, 32–33
transfer RNA recruitment. See Transfer RNA
tunnel in eukaryotes, 20–21
RIDD, 172
RISP, 314
RLI1, 64–66
RNA2, 34
RNA3, 35
RNA affinity chromatography, RNA-binding protein
identification, 113
RNA-dependent protein kinase (PKR), 264, 312, 337
RNA helicase, translation initiation, 45–46
RNA-induced silencing complex. See MicroRNA-induced
silencing complex
RNA polymerase II, transcription dependence for
messenger RNA internal ribosome
entry site, 97–98
RNPs. See Ribonucleoprotein particles
RPL38, 260
RPS25, 307
RRF. See Ribosomal recycling factor
rRNA. See Ribosomal RNA
RSK. See Ribosomal S6 kinase
Rumi, 154
Rumpelstiltskin, 198
Index
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Copyright 2012 Cold Spring Harbor Laboratory Press.
S6 kinase. See also Ribosomal S6 kinase
mTORC1 signaling to translational machinery, 132
substrates, 131–132
target of rapamycin activation, 129, 131
therapeutic targeting, 330–332
Scd6, 181–183
SDS. See Shwachman-Diamond syndrome
Sec12p, 156
Sex-lethal (SXL), 114–115
Shine-Dalgarno sequence, ribosome clearing studies, 78
Shwachman-Diamond syndrome (SDS), 261
Signal recognition particle (SRP), 148–149
Silvestrol, 335–336
Single-cell imaging
global measurement of translation
fluorescent noncanonical amino acid
tagging, 228
overview, 227–228
puromycin fluorescent analogs, 228–229
prospects for translation studies, 233–234
transcript-specific translation imaging
biarsenical fluorescent dyes, 232–233
overview, 229–230
reporter proteins, 230–232
TimeSTAMP, 233
transfer RNA fluorescent derivatives, 229
Single-molecule studies, translation dynamics
elongation studies in bacteria
ribosome tracking, 78
Shine-Dalgarno sequence clearing, 78
eukaryote study prospects, 85–86
fluorescence resonance energy transfer
principles, 73–74
ribosome conformational changes during initiation
and elongation, 79–81
transfer RNA conformational changes, 81
initiation studies in bacteria
elongation transition, 76–77
order of IF2 and transfer RNA arrival, 75–76
overview, 74–75
messenger RNA imaging in gene expression, 225–227
rationale, 72–74
time scales, 71–72
transfer RNA
dynamics in ribosome, 82–84
ribosome interactions and translocation, 84–85
transit through ribosome, 81–82
siRNA. See Small interfering RNA
SKI2, 172
Small interfering RNA (siRNA), screening for internal
ribosome entry sites, 100–101
Smaug, 116–117
SOX, 303
Squid, 198
SRP. See Signal recognition particle
Stm1, 181
Stress granule
aggregation, 186
assembly in cytoplasm, 183–184
caprin induction, 288–289
dynamics in cytoplasm, 185–18
messenger RNA decay
decapping promotion and translation initiation
repression, 179–182
pathways, 177–179
mRNA cycle model, 186–187
SUO, 246–248
SXL. See Sex-lethal
TANGO1, 156
Target of rapamycin (TOR)
complexes and functions, 124–126
kinase inhibitors for cancer treatment, 330–332
mTORC1 signaling to translational machinery
4E-BPs, 129–132
overview, 126–126
S6 kinase, 132
upstream factors
growth factors and hormones, 126–127
nutrients, oxygen, and energy status, 127–128
prospects for study, 128–129
oncogenic signaling, 262, 264
TAR RNA-binding protein (TRBP), 312
TDI, 61
Termination, translation
overview, 7
prospects for study, 65–66
release factors, 60–62
structural insights, 65
virus regulation, 313–314
Ternary complex (TC), 29, 31–33, 39, 44–45
inhibitors for cancer treatment, 337–338
TIA-1, 185
TIA-R, 185
TimeSTAMP, fluorescence imaging in single cells, 233
TISU element, 5
TOR. See Target of rapamycin
Tpa1, 66
TPI. See Triose phosphate isomerase
TPL. See Tripartite leader
TRAM, 150
Transfer RNA (tRNA)
fluorescent derivatives for global measurement of
translation, 229
Met-tRNAi recruitment to small ribosomal subunit
eIF2-GDP recycling, 33–34
eiF2-independent recruitment, 34
eIF2 role, 31–32
ternary complex binding promotion
factors, 32–33
ribosome binding sites in eukaryotes, 17–18
single-molecule studies in bacteria
conformational changes, 81
dynamics in ribosome, 82–84
order of IF2 and transfer RNA arrival, 75–76
ribosome interactions and translocation, 84–85
transit through ribosome, 81–82
start codon recognition role, 44–45
Index
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Copyright 2012 Cold Spring Harbor Laboratory Press.
TRAP, 150
TRBP. See TAR RNA-binding protein
Triose phosphate isomerase (TPI), 96
Tripartite leader (TPL), 308
tRNA. See Transfer RNA
TSC, 305, 308, 329
Unfolded protein response (UPR)
eIF2-mediated translational control response
eIF5 role, 165–166
phosphorylation relationship to fitness of stressed
cells, 169–170
transcripts in upstream open reading frame-depen-
dent translation initiation, 166–168
overview, 164
UNR, 115
Unr, 94
Upf1, 66
UPR. See Unfolded protein response
Vanishing white matter disease (VWM), 166
Vasa, translational activation in Drosophila oogenesis,
202–203
Vascular endothelial growth factor (VEGF), 269
VEGF. See Vascular endothelial growth factor
Virus translational control
balancing translation, replication, and
encapsidation, 314
cap-dependent initiation
adenoviruses, 307–308
asfarviruses, 309
eIF4E phosphorylation and DNA replication
promotion, 309–310
herpesviruses, 308
megaviruses, 309
mimiviruses, 309
papillomaviruses, 307–308
polyomaviruses, 307–308
poxviruses, 309
RNA viruses, 310
cap-independent translation. See also Picornavirus
internal ribosome entry sites
internal ribosome entry site virus distribution, 307
overview, 305–306
protein-linked 50 ends, 306
eIF2 in innate immunity
overview, 310
phosphorylation inhibition by viruses
bypassing, 312–313
combinatorial strategies, 312
inhibitors, 312
host translation impairment
cell translation factors
direct effects, 300–302
indirect effects, 302–303
overview, 304–305
RNA manipulation, 303–305
prospects for study, 314–315
replication strategies, 300
termination and reinitiation regulation, 313–314
VP1, 313
VP2, 313
VWM. See Vanishing white matter disease
Wispy, 200
X-box-binding protein 1 (XBP1)
functional overview, 165
translational pausing and colocalization of messenger
RNA with IRE1 effector domain, 170–172
XBP1. See X-box-binding protein 1
XRN1, 172, 187
YB-1, 35
ZBP1. See Zip code binding protein 1
Zip code binding protein 1 (ZBP1), neuron function,
288–289
ZIPK, 119
Index
352
Copyright 2012 Cold Spring Harbor Laboratory Press.