protein synthesis and translational control

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


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.

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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

v


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


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


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.

Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved

Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011528

1


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.)

J.W.B. Hershey et al.

2 Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011528


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).

Principles of Translational Control

Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011528 3


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.)




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




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-




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.




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


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


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


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


start codon recognition role, 39–43

eIF1A, 22, 33, 47–48, 64


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


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



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


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


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


4E1RCAT, 334

4E-BP

cancer

therapeutic targeting, 333–334

translational control, 262–264

4E-BP1, 95

mTORC1 signaling to translational machinery, 129–132


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


Hunchback, 4EHP in translation repression, 202

ICP6, 308–309

IF1, 18–19, 74–75

Index

347


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


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)


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


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

348


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


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


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


overview, 194–195

targeting to posterior pole plasm, 198


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


gurken localization, 198

nanos

targeting to posterior pole plasm, 198


oskar

targeting to posterior pole plasm, 197–198


overview, 194–195

protection from degradation, 199

Vasa as translational activator, 202–203

Oskar, messenger RNA localization in oocyte pattern

specification


overview, 194–195

targeting to posterior pole plasm, 197–198


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



messenger RNA decay

decapping promotion and translation

initiation repression, 179–182

pathways, 177–179


PCBP-2, 94

PDCD4


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

349


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


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


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

350


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


overview, 74–75

messenger RNA imaging in gene expression, 225–227

rationale, 72–74

time scales, 71–72

transfer RNA


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


caprin induction, 288–289


messenger RNA decay

decapping promotion and translation initiation

repression, 179–182

pathways, 177–179


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


oncogenic signaling, 262, 264

TAR RNA-binding protein (TRBP), 312

TDI, 61

Termination, translation

overview, 7


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


conformational changes, 81



ribosome interactions and translocation, 84–85

transit through ribosome, 81–82


Index

351


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


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)


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


protein synthesis and translational control

Documents