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The RNA Exosome Targets the AIDCytidine Deaminase to Both Strandsof Transcribed Duplex DNA SubstratesUttiya Basu,1,2,7,* Fei-Long Meng,1,7 Celia Keim,2,7 Veronika Grinstein,2 Evangelos Pefanis,2 Jennifer Eccleston,1
Tingting Zhang,1 Darienne Myers,1 Caitlyn R. Wasserman,1 Duane R. Wesemann,1 Kurt Januszyk,5 Richard I. Gregory,4
Haiteng Deng,3,6 Christopher D. Lima,5 and Frederick W. Alt1,*1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine and Immune Disease Institute, Children’s Hospital Boston,
Department of Genetics, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA2Department of Microbiology and Immunology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA3Rockefeller University, Proteomic Research Center, New York, NY 10065, USA4Children’s Hospital Boston, Harvard Stem Cell Institute, Department of Biological Chemistry andMolecular Pharmacology, Harvard MedicalSchool, 300 Longwood Avenue, Boston, MA 02115, USA5Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA6School of Life Sciences, Tsinghua University, Beijing 100084, China7These authors contributed equally to this work*Correspondence: ub2121@columbia.edu (U.B.), alt@enders.tch.harvard.edu (F.W.A.)
DOI 10.1016/j.cell.2011.01.001
SUMMARY
Activation-induced cytidine deaminase (AID) initiatesimmunoglobulin (Ig) heavy-chain (IgH) class switchrecombination (CSR) and Ig variable region somatichypermutation (SHM) in B lymphocytes by deami-nating cytidines on template and nontemplatestrands of transcribed DNA substrates. However,the mechanism of AID access to the template DNAstrand, particularly when hybridized to a nascentRNA transcript, has been an enigma. We now impli-cate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNAstrands. In B lineage cells activated for CSR, theRNA exosome associates with AID, accumulates onIgH switch regions in an AID-dependent fashion,and is required for optimal CSR. Moreover, boththe cellular RNA exosome complex and a recombi-nant RNA exosome core complex impart robustAID- and transcription-dependent DNA deaminationof both strands of transcribed SHM substratesin vitro. Our findings reveal a role for noncodingRNA surveillance machinery in generating antibodydiversity.
INTRODUCTION
Antigen-activated B lymphocytes undergo two distinct immuno-
globulin (Ig) gene diversification processes, namely somatic
hypermutation (SHM) and Ig heavy-chain (IgH) class switch
recombination (CSR). SHM diversifies IgH and Ig light-chain
(IgL) variable region exons to allow generation of B cells with
the potential to secrete higher-affinity antibodies (reviewed by
Odegard and Schatz, 2006; Di Noia and Neuberger, 2007;
Maul andGearhart, 2010). IgH class switch recombination allows
B cells to express different classes of antibodies with different
IgH constant regions (CHs) and, as a result, different antibody
effector functions (reviewed by Chaudhuri et al., 2007; Honjo
et al., 2002). CSR involves joining DNA double-strand breaks
(DSBs) in the large repetitive switch (S) region (Sm) that lies
upstream of the Cm constant region exons to DSBs within
a downstream S region (e.g., Sg1), which replaces Cm exons
with a set of downstream CH exons to complete CSR (e.g.,
switching from IgM to IgG1). Activation-induced cytidine deam-
inase (AID) initiates both SHM and CSR (Muramatsu et al., 2000;
Revy et al., 2000) by deaminating cytidines on, respectively, tran-
scribed IgH or IgL variable region exons or transcribed IgH
S regions (Petersen-Mahrt et al., 2002). The deaminated cyti-
dines become targets of co-opted DNA repair pathways that
lead to mutations associated with variable region exon SHM or
to S region DSBs that initiate CSR (reviewed by Di Noia and Neu-
berger, 2007; Neuberger et al., 2003). To initiate both SHM and
CSR, AID equally deaminates both template and nontemplate
strands of transcribed target DNA sequences (Milstein et al.,
1998; Shen et al., 2006; Xue et al., 2006).
AID is a single-stranded (ss) DNA-specific cytidine deaminase
that lacks activity on double-stranded (ds) DNA (Chaudhuri et al.,
2003; Dickerson et al., 2003; Ramiro et al., 2003; Sohail et al.,
2003). Correspondingly, SHM and CSR require transcription
through duplex substrate V(D)J exons or S regions to target
AID activity, consistent with transcription generating a ssDNA
AID substrate (reviewed by Chaudhuri et al., 2007; Yang and
Schatz, 2007; Di Noia and Neuberger, 2007; Maul and Gearhart,
2010). Transcription through mammalian S regions generates
R loops in which the template strand is hybridized to the nascent
transcript and the nontemplate strand is looped out as ssDNA
Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc. 353
(Daniels and Lieber, 1995; Shinkura et al., 2003; Tian and Alt,
2000; Yu et al., 2003a). In biochemical studies, transcription-
generated R loops within duplex substrates allow AID deamina-
tion butmainly on the looped-out nontemplate strand (Chaudhuri
et al., 2003). AID that is phosphorylated on serine 38 by protein
kinase A can access in vitro transcribed dsDNA SHM substrates
(e.g., V(D)J exons) that lack R loop-forming ability—again,
however, mainly on the nontemplate strand (Basu et al., 2005,
2008; Chaudhuri et al., 2004; Zarrin et al., 2004; Vuong et al.,
2009). Ectopically expressed AID mutates transcribed
substrates in bacteria and yeast but, once again, predominantly
on the nontemplate strand (Gomez-Gonzalez and Aguilera,
2007; Ramiro et al., 2003). Thus, the mechanism by which AID
accesses the template DNA strand of substrates has been
a major question (e.g., Chaudhuri et al., 2007; Chelico et al.,
2009; Di Noia and Neuberger, 2007; Liu and Schatz, 2009;
Longerich et al., 2006; Maul and Gearhart, 2010; Pavri et al.,
2010; Peled et al., 2008).
Biochemical studies suggested that AID may gain access to
both DNA strands of certain types of transcribed plasmids via
formation of RNA polymerase-generated supercoils (Besmer
et al., 2006; Shen et al., 2005; Shen and Storb, 2004). However,
such a mechanism does not readily explain how AID accesses
the template strand of a transcribed substrate if it is blocked
by a nascent RNA transcript (Maul and Gearhart, 2010). Indeed,
AID has no known activity on RNA/DNA hybrids, which form over
wide regions of mammalian S regions in the form of R loops
(Huang et al., 2006, 2007; Roy et al., 2008; Yu et al., 2003a)
and which may also form in the context of transcription bubbles
(Gomez-Gonzalez and Aguilera, 2007; Li and Manley, 2005).
RNaseH has been proposed as a candidate for exposing regions
of R-looped S region template strands to AID based on biochem-
ical ability to degrade RNA in the context of R loops (Lieber,
2010; Yu and Lieber, 2003b); however, to date, there has been
no direct evidence that implicated either RNase H or other
cellular RNA degradation factors in CSR.
The RNA exosome is an evolutionarily conserved RNA-pro-
cessing/degradation complex (reviewed by Houseley et al.,
2006; Lykke-Andersen et al., 2009; Schmid and Jensen, 2008;
Shen and Kiledjian, 2006). The nine-subunit core of the eukaryotic
RNA exosome, which includes the CsL4, Rrp4, Rrp40, Rrp41,
Rrp46,Mtr3, Rrp42, Rrp43, andRrp45 subunits, hasRNA-binding
activitybut lacks ribonucleaseactivity (Andersonetal., 2006;Grei-
mann and Lima, 2008; Oddone et al., 2007; Figure S1 available
online). RNA exosome ribonuclease activity is provided by non-
core subunits, including Rrp6 and Rrp44, that have RNA 30-50
exonuclease or endonuclease activity (Greimann and Lima,
2008; Houseley et al., 2006; Lebreton et al., 2008; Schaeffer
et al., 2009; Figure S1). The mammalian RNA exosome complex
interacts with cofactors, such as the TRAMP complex, that target
it toparticularsubstratesbasedonsequenceorstructural features
(reviewed by Houseley et al., 2006; Houseley and Tollervey, 2008;
LaCava et al., 2005). In Drosophila, the RNA exosome complex
interacts with elongating RNA polymerase II (Pol II) complexes
via the Spt5/6 transcription elongation cofactors (Andrulis et al.,
2002), and in yeast, it binds to and removes nascent RNP
complexes from template DNA (El Hage et al., 2010; Houseley
et al., 2006; Houseley and Tollervey, 2008). We now describe
354 Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc.
studies that implicate the RNA exosome as a long-speculated
cofactor that targets AID deamination activity to both template
and nontemplate strands of transcribed dsDNA substrates.
RESULTS
AID Associates with the RNA Exosome ComplexTo elucidate factors that might promote AID access to the
template strand of transcribed substrates in the context
of RNA/DNA hybrid structures, we in vitro transcribed an
RGYW-rich dsDNA SHM substrate with T7 RNA polymerase in
the presence of Ramos human B cell lymphoma line extracts.
Transcribed DNA-protein complexes were purified via
chromatographic steps that would enrich for DNA binding
(CM-sepharose, DEAE cellulose) and macromolecular complex
formation (gel-filtration chromatography), followed by heparin
sepharose chromatography and anti-AID antibody-mediated
affinity purification to enrich complexes containing AID (Figures
1A and 1B, Figure S1B, Table S1, and Extended Experimental
Procedures). At each step, fractions enriched for AID deamina-
tion stimulatory activity were identified via a 3H-release assay
(e.g., Figure S1B and Extended Experimental Procedures).
Among proteins identified by mass spectrometric analysis of
purified complexes were multiple subunits of the RNA exosome
complex, including Mtr3, Csl4, Rrp43, Rrp40, and Rrp42 (Fig-
ure 1B and Table S1). To further elucidate potential functions,
we assayed the ability of the AID-associated, transcribed DNA
complex to enhance deamination activity of purified AID in a tran-
scribed dsDNA SHM substrate assay (Chaudhuri et al., 2003)
and found that it markedly stimulated AID activity (Figure 1C).
Of note, AID association with the RNA exosome complex and
purification of an AID stimulatory activity was not observed if
the purification was performed from a reaction without T7
polymerase, indicating that complex formation is enhanced by
transcription (Figure S1B and data not shown).
To assay for AID/RNA exosome association in vivo, we immu-
noprecipitated AID from mouse primary splenic B cells stimu-
lated with anti-CD40 plus IL-4 to induce AID and CSR to IgG1
and then assayed immunoprecipitates for RNA exosome
subunits by western blotting. These analyses revealed that AID
associated with Rrp40, Rrp46, and Mtr3 RNA exosome subunits
(Figure 2A). Likewise, AID immunoprecipitated from mouse
CH12F3 B lymphoma cells stimulated with anti-CD40, IL-4,
and TGF-b to undergo CSR to IgA, as well as AID immunoprecip-
itated from the humanRamosB lymphoma cells, associatedwith
core Rrp40 andRrp46 subunits, as well as with the Rrp6 catalytic
exosome subunit (Figure 2B). By individually expressing FLAG-
tagged versions of RNA exosome subunits along with AID in
HEK293T cells, we observed that immunoprecipitation of any
of the 11 exosome core and catalytic subunits via anti-FLAG
antibodies also pulled down AID (Figure 2C). Together, our find-
ings indicate that AID either directly or indirectly associates with
the RNA exosome complex in cells.
Exosome Core Subunit Rrp40 Is Requiredfor Optimal CSRPrevious work indicates that the absence of a given core exo-
some subunit leads to a severe defect in overall RNA exosome
A B
C
Figure 2. AID Complexes with RNA Exosome Subunits In Vivo
(A) AID immunoprecipitates from extracts of CSR-activated AID-deficient and
wild-type B cells were assayed for Rrp40, Rrp46, Mtr3, and AID (indicated on
the right) via western blotting. The left two lanes show western blotting of total
extract, and the right two lanes show western blotting of immunoprecipitated
products.
(B) AID immunoprecipitates (‘‘Anti-AIDIP’’ lanes) or control reactions without
anti-AID antibody (‘‘�AbIP’’ lanes) from Ramos (‘‘Ramos’’) and CSR-activated
CH12F3 cells (‘‘CH12F3 Sti’’) were assayed for Rrp46, Rrp40, Mtr3, and AID
(indicated on right) by western blotting. Unstimulated CH12F3 cells (‘‘CH12F3
unsti’’) were used as a negative control.
(C) AID was coexpressed with individual FLAG epitope-tagged RNA exosome
subunits in HEK293T cells. Lanes from left to right represent cells transfected
with empty vector (‘‘vector’’) as a control or the individual FLAG epitope-tag-
ged subunit indicated at the top. The top two panels show western blotting of
total extract (‘‘input’’), and the bottom three panels show western blotting with
indicated antibodies (anti-AID, anti-Rrp40, and anti-Rrp6) following immuno-
precipitation with anti-Flag antibodies. Asterisks indicate bands correspond-
ing to Flag-tagged exosome subunits. A background band corresponding to
the anti-Flag Ig light chain also is indicated (‘‘Background’’).
A B
C
Figure 1. AID Forms a Transcription-Dependent Complex with RNA
Exosome
(A) Schematic outlining steps for enrichment of transcription-dependent
AID/RNA exosome/SHM substrate complex. Details are in text and Extended
Experimental Procedures.
(B) Proteins enriched by purification scheme in (A) were analyzed by
SDS-PAGE followed by staining with Coomassie blue. Identity of proteins from
selected bands was determined by mass spectrometry; bands that contain
RNA exosome subunits are indicted on the right withmolecular weight markers
on the left.
(C) AID purified following ectopic expression in HEK293 cells was assayed in
a 3H-release in vitro transcription-dependent SHM substrate assay (Chaudhuri
et al., 2003; see also Figure 5) in the presence or absence of complex enriched
by purification scheme in (A). Percent of total transcribed DNA substrate
deaminated is presented for three separate assays.
See also Figure S1 and Table S1.
function (Jensen andMoore, 2005). Therefore, to evaluate poten-
tial roles of the RNA exosome complex in CSR, we used a knock-
down approach to reduceRrp40 in theCH12F3B lymphoma line.
For this purpose, we lentivirally introduced two different shRNAs
that targeted Rrp40, respectively, into CH12F3 cells to generate
three different knockdown lines that had Rrp40 levels ranging
from about 50% to less than 10% those of controls, including
aWT line anda line harboring a nonspecific (‘‘scrambled’’) shRNA
(Figure3BandFigureS2C). Following stimulationwith anti-CD40,
TGFb, and IL4 for 48 hr to induce CSR to IgA, Rrp40 knockdown
lines consistently displayed reduced CSR, with levels ranging
from 30%–50% of those of controls (Figures 3A and 3C and Fig-
ure S2B). However, the various Rrp40 knockdown lines prolifer-
ated similarly to controls after stimulation (Figure 3E and Figures
S2A and S2E). In addition, the knockdown and control lines ex-
pressed similar levels of Im and Ia transcripts and similar levels
of AID; whereas there were variations in transcript levels from
clone to clone, there was no correlation with Im and Ia transcripts
and Rrp40 levels (Figure 3D and Figure S2F). Finally, similar
knockdowns of the Mtr3 RNA exosome core subunit also led to
decreased CSR without markedly affecting cell proliferation,
AID levels, or Im and Ia transcription (Figure S3). Together, these
findings demonstrate that physiological levels of RNA exosome
core subunits are required for efficient CSR.
Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc. 355
A
B C
D E
Figure 3. RNA Exosome Subunit Rrp40 Is Required for Normal CSR
(A) CH12F3 cells lentivirally infected with a scrambled short-hairpin plasmid (NS) or with shRNA against Rrp40 (shRrp40) were either not stimulated (‘‘Unsti’’) or
stimulated (‘‘Sti’’) for 2 days with anti-CD40, IL4, and TGFb and analyzed for IgA CSR by flow cytometry. ShRrp40-1 and shRrp40-2 are independent shRrp40-
expressing CH12F3 isolates. Results are representative of eight experiments; additional experiments are shown in Figures S2A and S2B.
(B) NS, shRrp40-1, and shRrp40-2 expressing stimulated and unstimulated CH12F3 isolates (shown in A) were assayed for Rrp40 and AID by western blotting.
Results are representative of four experiments; additional experiment is shown in Figure S2.
(C) Average levels and standard deviation from the mean of CSR to IgA from three independent experiments (one shown in A) performed simultaneously with
unstimulated (‘‘unsti’’) NS and stimulated (‘‘Sti’’) NS, shRrp40-1 (‘‘#1’’), and ShRrp40-2 (‘‘#2’’) CH12F3 isolates. Five additional experiments gave similar results
(Figures S2A and S2B).
(D) Total cellular RNA from three independently stimulated samples of indicated CH12F3 isolates (the ones used for C) was assayed for Im transcripts (left) and Ia
transcripts (right) via quantitative RT-PCR. Average and standard deviation from the mean is shown for the three separate experiments. An additional experiment
based on northern or RT-PCR is shown in Figure S2F.
(E) Growth curves of stimulated (NS) shRrp40-1 and shRrp40-2 CH12F3 cells calculated from three independent sets of three experiments (one used for C and
others shown Figure S2A) with a fourth set of three experiments indicated in Figure S2E. Values represent average and standard deviation from the mean. See
also Figure S2 and Figure S3.
Association of Rrp40 with S Regions in B CellsActivated for CSRIf the RNA exosome functions to target AID activity, it should
be found in association with transcribed S regions in B cells
356 Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc.
activated for CSR. To evaluate this possibility, we performed
chromatin immunoprecipitation (ChIP) assays to test whether
Rrp40 associates with transcribed Sm sequences in activated
CH12F3 cells before and after stimulation for CSR to IgA.We first
A B
C D
Figure 4. RNA Exosome Subunit Rrp40 Is Recruited to S Regions
(A) Ch12F3 cells were either stimulated with TGFb, IL4, and CD40 or kept
unstimulated for 48 hr. Subsequently, Rrp40 was immunoprecipitated from
cell extracts under chromatin immunoprecipitation (ChIP) conditions and
immunoprecipitates analyzed for Rrp40 by western blotting with anti-Rrp40.
(B) The ‘‘ChIPed’’ Rrp40-DNA complex from Ch12F3 cells was processed to
isolate bound DNA. ChIPed DNA was tested for Sm and Sa sequences via
q-PCR. The average and standard deviations from themean for three separate
ChIP experiments are shown (see also Figure S4). Numbers indicate average
fold changes comparing stimulated and unstimulated samples. Unstimulated
samples were arbitrarily normalized as 1 (Experimental Procedures).
(C) Rrp40 ChIPs were performed on extracts from primary splenic B cells
stimulated for 2 days with anti-CD40 plus IL4. Sm and Sg1 were tested via
semiquantitative PCR; results are shown for two independent ChIP samples
for each genotype. A 5-fold serial dilution of inputs is shown with the highest
input concentration corresponding to 1/20 of total input.
(D) ChIPed DNA from activated splenic B cells was tested for Sm and Sg1 via
q-PCR. Numbers indicate average fold changes comparing WT and AID�/�
samples. AID�/� samples were arbitrarily normalized as 1 (Experimental
Procedures). Values represent the average and standard deviation from the
mean for three experiments.
See Figure S4 for more details.
employed western blotting to confirm that Rrp40 is specifically
precipitated with an anti-Rrp40 antibody, but not control IgG,
under ChIP conditions (Figure 4A). After processing immunopre-
cipitates for isolation of bound DNA, we utilized quantitative PCR
(q-PCR) to determine levels of Rrp40 bound to Sm and Sa. These
analyses demonstrated enrichment of Sm and, to a lesser extent,
Sa in the anti-Rrp40 ChIPs from stimulated versus unstimulated
CH12F3 cells (Figure 4B and Figure S4). As there is some Sm
transcription in nonactivated B cells (Muramatsu et al., 2000),
the question arises as to whether transcription per se is sufficient
to recruit the RNA exosome to S regions. To explore this ques-
tion, we assayed for Rrp40 recruitment to Sm and Sg1 in WT
and AID-deficient primary B cells activated with anti-CD40 and
IL-4, which induce germline Sg1 transcription in both cell types
(Muramatsu et al., 2000). Consistent with targeting dependent
on germline transcription, Rrp40 was recruited to Sm and Sg1
in the activated WT B cells (Figures 4C and 4D and Figure S4).
Of note, however, Rrp40 was not measurably recruited to Sm
and Sg1 in AID-deficient B cells. Together, our results indicate
that the RNA exosome complex is recruited to transcribed S
regions in B cells activated for CSR in an AID-dependent fashion.
RNA Exosome Stimulates AID Activity on Templateand Nontemplate StrandsTo further evaluate the potential ability of the cellular RNA
exosome complex to act as an AID cofactor, we substantially
purified this complex from cell-free nuclear extracts prepared
from HEK293T cells that expressed a FLAG epitope-tagged
Rrp6 exosome subunit. In this purification, we maintained rela-
tively low salt concentrations to prevent disaggregation of
protein complexes (Figure S5). To test activity, we added varying
amounts of the exosome-enriched extract to a 3H-uracil-release
in vitro transcription-dependent AID deamination assay, which
measures overall SHM substrate deamination (Figure 5A). In
this assay, T7 polymerase transcription of the SHM substrate
leads to little or no AID deamination activity, and addition of
partially purified RNA exosome extract in the absence of AID
also gives no deamination activity on the T7 transcribed
substrate (Figure 5B). However, addition of both AID and partially
purified RNA exosome led to substantial deamination of the
transcribed substrate (Figure 5B), with activity appearing to be
roughly within a range similar to that observed with phosphory-
lated AID and RPA (Basu et al., 2005, 2008; Chaudhuri et al.,
2004; see below). The AID deamination stimulatory activity
observed in these extracts is likely mediated by the exosome
complex, as we found that deamination activity cofractionated
with the RNA exosome during purification (Figure S5). Likewise,
we found similar results when we purified the exosome complex
from HEK293T cells via an approach in which affinity purification
of the complex was performed with antibodies against endoge-
nous Rrp40 (Figure S5D). Finally, we found that the RNA
exosome also stimulated AID deamination of a transcribed
dsDNA core Sm substrate and a synthetic R loop-forming
substrate (Figure 5B).
Because the RNA exosome can associate with Pol II transcrip-
tion complexes and remove nascent transcripts from transcribed
DNA (El Hage et al., 2010), we considered it as a candidate AID
cofactor for template DNA strand deamination. To test this
possibility, we performed in vitro transcription-dependent AID
dsDNA SHM substrate deamination assays in which the
Southern blotting readout reveals deamination of either template
or nontemplate strands, respectively (Figure 5C) (Chaudhuri
et al., 2004). In this assay, no deamination of either strand was
observed when only AID was added in the presence or absence
of T7 polymerase (Figure 5D and Figure S6A). As observed previ-
ously, addition of PKA (to phosphorylate AID on S38) and RPA
along with T7 polymerase and AID led to deamination of the
nontemplate strand, but not the template strand (Figure 5D
and Figure S6A). Strikingly, addition of both AID and partially
purified RNA exosome (in the absence of RPA or PKA) to the
T7 transcription reaction led to deamination of both strands of
the SHM substrate (Figure 5D and Figures S6A and S6E). In
most assays, activity on both template and nontemplate strands,
respectively, was robust, as evidenced by greatly diminished
Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc. 357
A B
C D
Figure 5. Cellular RNA Exosome Augments
Transcription-Dependent AID Deamination
Activity on Template and Nontemplate
DNA Strands
(A) Schematic representation of 3H release assay
for AID deamination of transcribed dsDNA SHM
substrate.
(B) (Top) Results of 3H release assay in which
a SHM substrate was transcribed by T7 poly-
merase (T) in the presence of purified AID (AID),
purified HEK293 RNA exosome (Exo293), or both.
(Middle) Results of 3H release assay in which
a synthetic R loop-forming substrate was tran-
scribed by T7 polymerase (T) in the presence of
purified AID (AID), recombinant RNA exosome
(rExo), or both. (Bottom) Results of 3H release
assay in core Sm substrate were transcribed by T7
polymerase (T) in the presence of purified AID
(AID), purified HEK293 RNA exosome (Exo293), or
both. In all three panels, values represent average
and standard deviation from the mean from three
independent experiments.
(C) A schematic representation of an assay for
measuring strand-specific AID deamination of
a transcribed dsDNA SHM. The location of
template (T) and nontemplate (NT) strand probes is
indicated.
(D) The strand specificity of RPA-dependent or
RNA exosome-dependent DNA deamination was
analyzed by the assay in (C) using either non-
template (left) or template (right) strand-specific
probes. Reactions contained AID, T7 polymerase
(T), RPA, and PKA or purified HEK293 exosome
(‘‘Exosome’’) as indicated.
See also Figure S5 and Figure S6.
levels of full-length substrate strands (Figure 5D and Figure S6A).
The RNA exosome also enhances AID template strand deamina-
tion activity on transcribed core Sm substrate and synthetic R
loop-forming substrates (Figure 5B and Figures S6B and S6C).
Together, our findings indicate that the endogenous RNA exo-
some complex can function as a stimulatory cofactor for AID
deamination activity on both strands of transcribed duplex
SHM substrates.
Recombinant Core RNA Exosome StimulatesAID-Dependent Deamination on Templateand Nontemplate StrandsThe partially purified endogenous RNA exosome preparation
should contain the core structural complex as well as catalytic
subunits (e.g., Rrp6, which was used to pull down the complex)
and potentially other cofactors or contaminating proteins.
To unequivocally evaluate the ability of theRNAexosome to stim-
ulate AID activity on transcribed SHM substrates and to also
358 Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc.
obtain insight into potential mechanisms,
we assayed a nine-subunit recombinant
human RNA exosome core complex
reconstituted from subunits that were
individually purified subsequent to
expression in bacteria (Figure 6A) (Grei-
mann and Lima, 2008; Liu et al., 2006). We found that, when
titrated for optimal activity (Figure S6E), the recombinant RNA
exosome core complex robustly stimulated AID-dependent
deamination of transcribed SHM substrates, as measured by
the H3-uracil release assay (Figure 6B and Figure S6F). In fact,
maximum deamination levels stimulated by the recombinant
core complexwere roughly similar to thoseobtainedwith partially
purified RNA exosome complex (Figure 6B and Figure S6F).
Moreover, the recombinant RNA exosome core complex also
stimulated AID-dependent deamination of both template and
nontemplate strands of the SHM substrate (Figure 6C and Fig-
ure S6G) and synthetic R loop-forming substrates (Figure S6C).
Thus, the core RNA exosome complex alone, in the absence of
the RNAase catalytic subunits, promotes AID- and transcrip-
tion-dependent SHM substrate deamination.
To further characterize potential functions of the core RNA
exosome, we assayed individual core subunits (Figure 6D and
Figure S7). Individual subunits were assayed at approximately
A B
C D
Figure 6. Recombinant Core RNA Exosome
and Individual Subunits Stimulate AID
Activity
(A) Coomassie blue stained polyacrylamide gel
electrophoresis analysis of the nine-subunit
recombinant RNA exosome complex generated
from individual subunits.
(B) Comparative analysis of the ability of RNA
exosome complex from 293T cells (Exo293) and
recombinant RNA core exosome complex (rExo)
to stimulate AID deamination activity as measured
by 3H release/SHM substrate assay in Figure 5A.
See Extended Experimental Procedures and
Figure S6 for details. Optimal amounts of RNA
exosome293T and recombinant RNA exosome
core complex were used (Figure S6 and Extended
Experimental Procedures).
(C) Assay of recombinant exosome complex to
stimulate strand-specific AID deamination of
a transcribed SHM substrate via assay in Fig-
ure 5C. Nontemplate and template strand deami-
nation are shown on left and right panels,
respectively. Reactions contained either AID,
recombinant core RNA exosome (rExo9wt), or both
(AID + rExo9wt) as indicated.
(D) Individual recombinant RNA exosome subunits
were assayed for ability to promote AID deami-
nation of a T7-transcribed SHM substrate as out-
lined in Figure 5A. Added exosome components
are indicated. Control reactions with AID alone or
AID plus T7 polymerase are on the left. A positive
control with complete recombinant core RNA
exosome (rExo9) plus T7 and AID is on the right.
For (B) and (D), values represent the average
and standard deviation from the mean for three
independent experiments. See also Figure S7 and
Figure S8.
the same molarity as when assayed in the context of the core
complex. Some, but not all, recombinant core RNA exosome
subunits stimulated AID- and transcription-dependent SHM
substrate deamination activity; however, the level of stimulation
was low compared to that of the full core complex (Figure 6D and
Figure S7A). The RNA-binding subunits Rrp40, Rrp46, Rrp41,
and Mtr3 showed low levels of activity (Figure 6D and Figures
S7A and S7C). On the other hand, other core subunits with
demonstrated RNA-binding activity and/or RNA-binding
domains, along with the Rrp6 ribonuclease subunit, lacked
detectable activity, indicating that the observed activity is not
a property of all such proteins.
DISCUSSION
Enigma of AID Targeting of the TemplateSubstrate StrandRecent studies revealed a mechanism by which AID can be
brought to transcribed substrates in the context of RNA poly-
merase II pausing (Pavri et al., 2010). Once recruited to the
substrate, transcription-basedmechanismsalso provide a poten-
tial means of providing AID with a ssDNA substrate in the context
of a duplex substrate via looping out of the nontemplate strand
(Chaudhuri et al., 2003, 2004; Gomez-Gonzalez and Aguilera,
2007; Ramiro et al., 2003; Yu and Lieber, 2003). However, AID
equally deaminates both substrate DNA strands during CSR and
SHM (Milstein et al., 1998; Shen et al., 2006; Xue et al., 2006). In
the latter context, the mechanism by which AID generates DSB
intermediates that are substrates for CSR (Petersen et al., 2001;
Schrader et al., 2005; Wuerffel et al., 1997) and for chromosomal
translocations (Ramiro et al., 2004; Robbiani et al., 2008) requires
targeting of both DNA strands (e.g., Xue et al., 2006; reviewed by
DiNoia andNeuberger, 2007). Thus, themechanismbywhichAID
accesses the templateDNAstrandhasbeenamajorquestion (e.g.
Chaudhuri et al., 2007; Chelico et al., 2009; Di Noia and Neu-
berger, 2007; Liu and Schatz, 2009; Longerich et al., 2006; Maul
and Gearhart, 2010; Pavri et al., 2010; Peled et al., 2008). We
now show that the core RNA exosome complex promotes AID
deamination of both template and nontemplate strands of
in vitro transcribedSHMsubstrates.Moreover, inBcells activated
for CSR, the RNA exosome complex associates with AID and
accumulates on S regions in an AID-dependent manner. Finally,
integrity of the RNA exosome complex is required for optimal
CSR. Thus, the RNA exosome is a long-sought cofactor that can
target AID activity to both template and nontemplate strands of
transcribed SHM and CSR targets.
Implications of AID/RNA Exosome BiochemistryOur in vivo knockdown, ChIP, and physical association studies
provide strong biological evidence that the RNA exosome
Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc. 359
functions in CSR. However, such studies often provide limited
mechanistic insight. In addition, as elimination of RNA exosome
core subunits is cell lethal in yeast, it may be difficult to achieve
complete knockdowns or knockouts to fully test the extent of
exosome function in promoting AID activity in vivo. Classical
biochemistry has been extremely useful for elucidating potential
functions of essential replication, repair, and recombination
factors (e.g., Chaudhuri et al., 2004; Dzantiev et al., 2004; Gen-
schel and Modrich, 2003; Maldonado et al., 1996; Stillman and
Gluzman, 1985). In this context, our biochemical studies reveal
that the core RNA exosome robustly targets AID to both strands
of T7 polymerase-transcribed SHM substrates in vitro. Clearly,
T7 polymerase is structurally and mechanistically distinct from
mammalian RNA PoI II, and the in vitro system that we have
employed does not operate in the chromatin context in which
SHM and CSR occur in vivo. Also, the concentration of the
RNA exosome and of other factors in our assays may be greater
than their cellular levels. But again, it is just these aspects of the
biochemical assay that allow us to definewhat the RNA exosome
has the potential of achieving. Additional mechanisms and
factors (e.g., AID) likely will work to specifically recruit the RNA
exosome to transcribed SHM and CSR targets in vivo (see
below), potentially increasing local concentration to levels suffi-
cient to achieve activities observed in vitro.
The evolutionarily conserved RNA exosome complex is
required for degradation and/or processing of various RNAs,
including noncoding RNAs generated in cells due to error-prone
transcription or from transcription initiated by cryptic promoters
(Houseley et al., 2006; Jensen and Moore, 2005; Lykke-Ander-
sen et al., 2009; Preker et al., 2008). In bacteria, the exosome
core complex contains ribonuclease activity; however, in yeast
and mammalian cells, the core subunits lack such activity and
generally have been considered structural scaffolds (Greimann
and Lima, 2008; Houseley et al., 2006; Jensen and Moore,
2005). In eukaryotes, noncore exosome subunits or cofactors
provide ribonuclease activity (Callahan and Butler, 2010; Grei-
mann and Lima, 2008; Houseley et al., 2006; Lebreton et al.,
2008; Schaeffer et al., 2009; Staals et al., 2010; Tomecki et al.,
2010). Our biochemical data demonstrate that the recombinant
nine-subunit exosome core, in the absence of catalytic subunits,
robustly targets AID to both strands of T7 transcribed substrates.
The precise molecular mechanism by which the RNA exosome
core achieves this activity remains to be determined. In theory,
it may compete with duplex DNA for binding the nascent tran-
script, thereby displacing it from the template strand, while
also forming a scaffold that stabilizes the transcriptionally
opened template to expose both DNA strands. Our finding that
certain core exosome subunits have low but significant ability
to enhance AID deamination of in vitro transcribed substrates
is intriguing. Currently, however, there is no clear correlation
between any particular known subunit activity and which
subunits promote low-level AID access.
We previously have shown that RPA, a ssDNA-binding protein
involved in replication and repair, binds AID phosphorylated on
S38 and, thereby, targets AID to transcribed SHM substrates
in vitro, albeit mostly to the nontemplate strand (Basu et al.,
2005; Chaudhuri et al., 2004). A substantial amount of evidence
supports the significance of the AID/RPA interaction for AID
360 Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc.
function during CSRand SHM, both in augmenting AID deamina-
tion activity and also potentially in recruiting downstream repair
pathways involved in further processing AID-initiated lesions
(Basu et al., 2005, 2008; Cheng et al., 2009; McBride et al.,
2006, 2008; Pasqualucci et al., 2006). Of note, RNA exosome-
mediated AID-targeting activity in our in vitro SHM assay does
not require AID phosphorylation on S38 or RPA, demonstrating
that the RNA exosome function in AID targeting is distinct.
However, these two AID-targeting mechanisms still might be
complementary in vivo (see below).
Potential Mechanism by Which the RNA ExosomePromotes AID Activity In VivoBoth CSR and SHM in activated B cells require transcription
through target S regions or variable region exons. Such tran-
scriptional targeting was proposed to result from association
of a mutator factor with stalled Pol II within the target sequence
(Peters and Storb, 1996). Over the years, this model has been
supported by numerous findings, including the following: AID,
now known to be the hypothetical mutator, is targeted by tran-
scription (reviewed by Chaudhuri et al., 2007; Yang and Schatz,
2007; Di Noia and Neuberger, 2007); AID associates with Pol II
(Nambu et al., 2003); and Pol II indeed stalls during transcription
through S regions (Rajagopal et al., 2009; Wang et al., 2009),
potentially augmented by transcribed S regions forming
secondary structures such as R loops (reviewed by Maul and
Gearhart, 2010). Recent studies have implicated the Spt5 tran-
scriptional elongation cofactor in recruiting AID to stalled Pol II
and, thereby, bringing AID to transcribed genomic targets (Pavri
et al., 2010). However, the AID/Spt5/Pol II interaction did not
explain how AID actually accesses its target sequences once
there and, in particular, how AID accesses the template DNA
strand (Pavri et al., 2010), a process in which we now have impli-
cated the RNA exosome. The recruitment of the RNA exosome
to S regions in B cells activated for CSR, in theory, might involve
a putative Spt5/RNA exosome interaction (Andrulis et al., 2002)
or generation of the noncoding S region transcripts (Lennon
and Perry, 1985 Lutzker and Alt, 1988), the latter of which might
attract the RNA exosome complex (reviewed by Houseley et al.,
2006; Jensen and Moore, 2005; Schmid and Jensen, 2008).
However, the marked AID dependence of exosome recruitment
to targeted S regions in activated B cells rules out Spt5 interac-
tion or germline transcription alone for RNA exosome recruit-
ment. Though AID might directly recruit the RNA exosome
complex, we thus far have not found interaction with individual
purified subunits, raising the additional possibility of indirect
recruitment, for example, via an AID/transcription-related
complex.
We propose a working model for how the RNA exosome may
enhance targeting of AID to both DNA strands of its in vivo
substrates (Figure S8). This model is based on our current
biochemical and cellular findings regarding relationships
between AID and the RNA exosome, known aspects of AID
function and recruitment to transcribed target sequences, and
known RNA exosome properties. To enhance AID activity on
the template strand, the RNA exosome must in some way
remove the template RNA. As the RNA exosome is not known
to engage RNA substrates that lack a free single-stranded 30
end, cotranscriptional RNA exosome activity in which an RNA
in the RNA/DNA hybrid is still attached to the RNA polymerase
would seem unlikely. Therefore, we suggest that the RNA
exosome would have its relevant activity on stalled Pol II units
that backtrack to reveal a free 30 end (Adelman et al., 2005) (Fig-
ure S8A). In this context, stalled RNA polymerase recruits AID
via the Spt5 transcription factor (Pavri et al., 2010). Thus, Pol II
stalling would indirectly recruit the RNA exosome via AID (Fig-
ure S8B). Likewise, Pol II stalling would also provide a suitable
substrate to engage the RNA exosome and allow it to enhance
AID substrate activity (Figure S8B), potentially via mechanisms
mentioned in the preceding section of the Discussion or below.
We further suggest that RPA might bind to and stabilize ssDNA
targets opened up by the RNA exosome and facilitate S38-phos-
phorylated AID accumulation via direct interaction (Basu et al.,
2005, 2008; Vuong et al., 2009) (Figures S8C and S8D). In addi-
tion, bound RPA may facilitate engagement of downstream
repair pathways (Chaudhuri, Khuong, and Alt, 2004; Vuong
et al., 2009). We must note that it remains possible, in vivo,
that the catalytic subunit of the RNA exosome could provide
ribonuclease activity that would degrade the nascent transcript
from the DNA/RNA hybrids on transcribed AID substrates and,
thereby, further contribute to exposing the template strand to
AID activity (Figure S8E). Finally, we do not rule out the possibility
that RNA exosome, in the context of an AID complex, acquires
unique properties as compared to properties already described
for the RNA exosome and that these unique properties are
relevant to enhancing AID activity.
EXPERIMENTAL PROCEDURES
Recombinant Plasmids, Antibodies, and Proteins
RNA exosome subunits, AID, and RPA were expressed as described (Grei-
mann and Lima, 2008; Chaudhuri et al., 2003, 2004; Stillman and Gluzman,
1985). AID antibodies were generated as described (Chaudhuri et al., 2003),
and anti-exosome subunit antibodies were purchased from Genway or
AbCam. Exosome subunit and complex purifications were as described
(Greimann and Lima, 2008). Details are in Extended Experimental Procedures.
AID Activity Assays
The 3H release assay was performed essentially as described (Chaudhuri
et al., 2003; Basu et al., 2005). The strand-specific AID and transcription-
dependent dsDNA deamination assays were performed as described (Chaud-
huri et al., 2003, 2004). Details are in Extended Experimental Procedures.
AID Complex Purification from Ramos Cells
We used a combination of DNA affinity, size chromatography, and affinity
purification for isolating AID complexes. We analyzed components of the
complex via mass spectrometry. For details, see Extended Experimental
Procedures.
RNA Exosome Purification from 293T Cells
We generated protein extracts from HEK293 cells expressing FLAG epitope-
tagged Rrp6. We used a combination of size chromatography, glycerol
gradient sedimentation, and FLAG affinity purification to isolate a protein
complex enriched with RNA exosome complex. We also isolated RNA
exosome from HEK293 cells using the same approach except that the affinity
purification step employed anti-Rrp40 antibodies. For details, see Extended
Experimental Procedures.
Immunoprecipitation of AID/RNA Exosome Complex
from B Cells and HEK293 Cells
We utilized previously published protocols to immunoprecipitate the AID
complex from B cells (Chaudhuri et al., 2004; Basu et al., 2005) or used
similarly adapted protocols for immunoprecipitating FLAG-exosome subunits
from 293 cells. For details, see Extended Experimental Procedures.
CSR Analysis of Rrp40-Deficient CH12F3 Cells
We performed shRNA-mediated knockdowns in Ch12F3 according to TRC
shRNA library protocols. Knockdown efficiencies were measured by detecting
levels of Rrp40 in these cells by western blotting. We determined levels of
germline S region transcripts in Rrp40-deficient cells via previously published
protocols (Muramatsu et al., 2000). For details, see Extended Experimental
Procedures.
Chromatin Immunoprecipitation Assays
We utilized adaptations of previously published protocols (Chaudhuri et al.,
2004) for isolating chromatin immunoprecipitates of Rrp40. See Extended
Experimental Procedures for details.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, eight
figures, and one table and can be found with this article online at doi:10.1016/j.
cell.2011.01.001.
ACKNOWLEDGMENTS
We thank Bjoern Schwer, Jason Rodriguez, Stephen Goff, Bruce Stillman, and
Tasuku Honjo for providing reagents and/or protocols. We also thank Bjoern
Schwer and Cosmas Giallourakis for critically reading the manuscript. This
work was supported by National Institute of Health grants AI31541 (to
F.W.A.) and GM079196 (to K.J. and C.D.L.); by Trustees of Columbia Univer-
sity Faculty startup funds (to U.B.); and by funds from Regeneron Pharmaceu-
ticals (in support of E.P.). F.-L.M. is a fellow of Cancer Research Institute of
New York. C.K. was supported by a Cancer Biology training grant
CA09503-23. K.J. is a fellow of the American Cancer Society (PF-10-236-
01-RMC). U.B. is a Special Fellow of the Leukemia and Lymphoma Society
of America and the recipient of a New Investigator Award from the Leukemia
Research Foundation. F.W.A. is an Investigator of the Howard Hughes
Medical Institute.
Received: September 30, 2010
Revised: December 27, 2010
Accepted: December 31, 2010
Published online: January 20, 2011
REFERENCES
Adelman, K., Marr, M.T., Werner, J., Saunders, A., Ni, Z., Andrulis, E.D., and
Lis, J.T. (2005). Efficient release from promoter-proximal stall sites requires
transcript cleavage factor TFIIS. Mol. Cell 17, 103–112.
Anderson, J.R., Mukherjee, D., Muthukumaraswamy, K., Moraes, K.C.,Wilusz,
C.J., and Wilusz, J. (2006). Sequence-specific RNA binding mediated by the
RNase PH domain of components of the exosome. RNA 12, 1810–1816.
Andrulis, E.D., Werner, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P.,
and Lis, J.T. (2002). The RNA processing exosome is linked to elongating
RNA polymerase II in Drosophila. Nature 420, 837–841.
Basu, U., Chaudhuri, J., Alpert, C., Dutt, S., Ranganath, S., Li, G., Schrum, J.P.,
Manis, J.P., and Alt, F.W. (2005). The AID antibody diversification enzyme is
regulated by protein kinase A phosphorylation. Nature 438, 508–511.
Basu, U., Wang, Y., and Alt, F.W. (2008). Evolution of phosphorylation-
dependent regulation of activation-induced cytidine deaminase. Mol. Cell
32, 285–291.
Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc. 361
Besmer, E., Market, E., and Papavasiliou, F.N. (2006). The transcription
elongation complex directs activation-induced cytidine deaminase-mediated
DNA deamination. Mol. Cell. Biol. 26, 4378–4385.
Callahan, K.P., and Butler, J.S. (2010). TRAMP complex enhances RNA
degradation by the nuclear exosome component Rrp6. J. Biol. Chem. 285,
3540–3547.
Chaudhuri, J., Basu, U., Zarrin, A., Yan, C., Franco, S., Perlot, T., Vuong, B.,
Wang, J., Phan, R.T., Datta, A., et al. (2007). Evolution of the immunoglob-
ulin heavy chain class switch recombination mechanism. Adv. Immunol. 94,
157–214.
Chaudhuri, J., Khuong, C., and Alt, F.W. (2004). Replication protein A interacts
with AID to promote deamination of somatic hypermutation targets. Nature
430, 992–998.
Chaudhuri, J., Tian, M., Khuong, C., Chua, K., Pinaud, E., and Alt, F.W. (2003).
Transcription-targeted DNA deamination by the AID antibody diversification
enzyme. Nature 422, 726–730.
Chelico, L., Pham, P., Petruska, J., and Goodman, M.F. (2009). Biochemical
basis of immunological and retroviral responses to DNA-targeted cytosine
deamination by activation-induced cytidine deaminase and APOBEC3G. J.
Biol. Chem. 284, 27761–27765.
Cheng, H.L., Vuong, B.Q., Basu, U., Franklin, A., Schwer, B., Astarita, J., Phan,
R.T., Datta, A., Alt, F.W., andChaudhuri, J. (2009). Integrity of the AID serine-38
phosphorylation site is critical for class switch recombination and somatic
hypermutation in mice. Proc. Natl. Acad. Sci. USA 106, 2717–2722.
Daniels, G.A., and Lieber, M.R. (1995). RNA:DNA complex formation upon
transcription of immunoglobulin switch regions: implications for the mecha-
nism and regulation of class switch recombination. Nucleic Acids Res. 23,
5006–5011.
Di Noia, J.M., and Neuberger, M.S. (2007). Molecular mechanisms of antibody
somatic hypermutation. Annu. Rev. Biochem. 76, 1–22.
Dickerson, S.K., Market, E., Besmer, E., and Papavasiliou, F.N. (2003). AID
mediates hypermutation by deaminating single stranded DNA. J. Exp. Med.
197, 1291–1296.
Dzantiev, L., Constantin, N., Genschel, J., Iyer, R.R., Burgers, P.M., and Mod-
rich, P. (2004). A defined human system that supports bidirectional mismatch-
provoked excision. Mol. Cell 15, 31–41.
El Hage, A., French, S.L., Beyer, A.L., and Tollervey, D. (2010). Loss of Topoi-
somerase I leads to R-loop-mediated transcriptional blocks during ribosomal
RNA synthesis. Genes Dev. 24, 1546–1558.
Genschel, J., and Modrich, P. (2003). Mechanism of 50-directed excision in
human mismatch repair. Mol. Cell 12, 1077–1086.
Gomez-Gonzalez, B., and Aguilera, A. (2007). Activation-induced cytidine
deaminase action is strongly stimulated by mutations of the THO complex.
Proc. Natl. Acad. Sci. USA 104, 8409–8414.
Greimann, J.C., and Lima, C.D. (2008). Reconstitution of RNA exosomes from
human and Saccharomyces cerevisiae cloning, expression, purification, and
activity assays. Methods Enzymol. 448, 185–210.
Honjo, T., Kinoshita, K., and Muramatsu, M. (2002). Molecular mechanism of
class switch recombination: linkage with somatic hypermutation. Annu. Rev.
Immunol. 20, 165–196.
Houseley, J., LaCava, J., and Tollervey, D. (2006). RNA-quality control by the
exosome. Nat. Rev. Mol. Cell Biol. 7, 529–539.
Houseley, J., and Tollervey, D. (2008). The nuclear RNA surveillance
machinery: the link between ncRNAs and genome structure in budding yeast?
Biochim. Biophys. Acta 1779, 239–246.
Huang, F.T., Yu, K., Balter, B.B., Selsing, E., Oruc, Z., Khamlichi, A.A., Hsieh,
C.L., and Lieber, M.R. (2007). Sequence dependence of chromosomal R-loops
at the immunoglobulin heavy-chain Smu class switch region. Mol. Cell. Biol.
27, 5921–5932.
Huang, F.T., Yu, K., Hsieh, C.L., and Lieber, M.R. (2006). Downstream
boundary of chromosomal R-loops at murine switch regions: implications for
the mechanism of class switch recombination. Proc. Natl. Acad. Sci. USA
103, 5030–5035.
362 Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc.
Jensen, T.H., and Moore, C. (2005). Reviving the exosome. Cell 121, 660–662.
LaCava, J., Houseley, J., Saveanu, C., Petfalski, E., Thompson, E., Jacquier,
A., and Tollervey, D. (2005). RNA degradation by the exosome is promoted
by a nuclear polyadenylation complex. Cell 121, 713–724.
Lebreton, A., Tomecki, R., Dziembowski, A., and Seraphin, B. (2008). Endonu-
cleolytic RNA cleavage by a eukaryotic exosome. Nature 456, 993–996.
Lennon, G.G., and Perry, R.P. (1985). C mu-containing transcripts initiate
heterogeneously within the IgH enhancer region and contain a novel 50-non-translatable exon. Nature 318, 475–478.
Li, X., and Manley, J.L. (2005). Inactivation of the SR protein splicing factor
ASF/SF2 results in genomic instability. Cell 122, 365–378.
Lieber, M.R. (2010). The mechanism of double-strand DNA break repair by the
nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211.
Liu, M., and Schatz, D.G. (2009). Balancing AID and DNA repair during somatic
hypermutation. Trends Immunol. 30, 173–181.
Liu, Q., Greimann, J.C., and Lima, C.D. (2006). Reconstitution, activities, and
structure of the eukaryotic RNA exosome. Cell 127, 1223–1237.
Longerich, S., Basu, U., Alt, F., and Storb, U. (2006). AID in somatic hypermu-
tation and class switch recombination. Curr. Opin. Immunol. 18, 164–174.
Lykke-Andersen, S., Brodersen, D.E., and Jensen, T.H. (2009). Origins and
activities of the eukaryotic exosome. J. Cell Sci. 122, 1487–1494.
Lutzker, S., and Alt, F.W. (1988). Structure and expression of germ line immu-
noglobulin gamma 2b transcripts. Mol. Cell. Biol. 8, 1849–1852.
Maldonado, E., Shiekhattar, R., Sheldon, M., Cho, H., Drapkin, R., Rickert, P.,
Lees, E., Anderson, C.W., Linn, S., and Reinberg, D. (1996). A human RNA
polymerase II complex associated with SRB and DNA-repair proteins. Nature
381, 86–89.
Maul, R.W., and Gearhart, P.J. (2010). AID and somatic hypermutation. Adv.
Immunol. 105, 159–191.
McBride, K.M., Gazumyan, A., Woo, E.M., Barreto, V.M., Robbiani, D.F., Chait,
B.T., and Nussenzweig, M.C. (2006). Regulation of hypermutation by activa-
tion-induced cytidine deaminase phosphorylation. Proc. Natl. Acad. Sci.
USA 103, 8798–8803.
McBride, K.M., Gazumyan, A., Woo, E.M., Schwickert, T.A., Chait, B.T., and
Nussenzweig, M.C. (2008). Regulation of class switch recombination and
somatic mutation by AID phosphorylation. J. Exp. Med. 205, 2585–2594.
Milstein, C., Neuberger, M.S., and Staden, R. (1998). Both DNA strands of
antibody genes are hypermutation targets. Proc. Natl. Acad. Sci. USA 95,
8791–8794.
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and
Honjo, T. (2000). Class switch recombination and hypermutation require acti-
vation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell
102, 553–563.
Nambu, Y., Sugai, M., Gonda, H., Lee, C.G., Katakai, T., Agata, Y., Yokota, Y.,
and Shimizu, A. (2003). Transcription-coupled events associating with immu-
noglobulin switch region chromatin. Science 302, 2137–2140.
Neuberger, M.S., Harris, R.S., Di Noia, J., and Petersen-Mahrt, S.K. (2003).
Immunity through DNA deamination. Trends Biochem. Sci. 28, 305–312.
Oddone, A., Lorentzen, E., Basquin, J., Gasch, A., Rybin, V., Conti, E., and Sat-
tler, M. (2007). Structural and biochemical characterization of the yeast exo-
some component Rrp40. EMBO Rep. 8, 63–69.
Odegard, V.H., and Schatz, D.G. (2006). Targeting of somatic hypermutation.
Nat. Rev. Immunol. 6, 573–583.
Pasqualucci, L., Kitaura, Y., Gu, H., and Dalla-Favera, R. (2006). PKA-medi-
ated phosphorylation regulates the function of activation-induced deaminase
(AID) in B cells. Proc. Natl. Acad. Sci. USA 103, 395–400.
Pavri, R., Gazumyan, A., Jankovic, M., Di Virgolio, M., Kein, I., Ansarah-
Sobrinho, C., Resch, W., Yamane, A., Reina San-Martin, B., Barreto, V.,
et al. (2010). Activation-induced cytidine deaminase targets DNA at sites of
RNA polymerase II stalling by interaction with Spt5. Cell 143, 122–133.
Peled, J.U., Kuang, F.L., Iglesias-Ussel, M.D., Roa, S., Kalis, S.L., Goodman,
M.F., and Scharff, M.D. (2008). The biochemistry of somatic hypermutation.
Annu. Rev. Immunol. 26, 481–511.
Peters, A., and Storb, U. (1996). Somatic hypermutation of immunoglobulin
genes is linked to transcription initiation. Immunity 4, 57–65.
Petersen, S., Casellas, R., Reina-San-Martin, B., Chen, H.T., Difilippantonio,
M.J., Wilson, P.C., Hanitsch, L., Celeste, A., Muramatsu, M., Pilch, D.R.,
et al. (2001). AID is required to initiate Nbs1/gamma-H2AX focus formation
and mutations at sites of class switching. Nature 414, 660–665.
Petersen-Mahrt, S.K., Harris, R.S., and Neuberger, M.S. (2002). AID mutates
E. coli suggesting a DNA deamination mechanism for antibody diversification.
Nature 418, 99–103.
Preker, P., Nielsen, J., Kammler, S., Lykke-Andersen, S., Christensen, M.S.,
Mapendano, C.K., Schierup, M.H., and Jensen, T.H. (2008). RNA exosome
depletion reveals transcription upstream of active human promoters. Science
322, 1851–1854.
Rajagopal, D., Maul, R.W., Ghosh, A., Chakraborty, T., Khamlichi, A.A., Sen,
R., and Gearhart, P.J. (2009). Immunoglobulin switch mu sequence causes
RNA polymerase II accumulation and reduces dA hypermutation. J. Exp.
Med. 206, 1237–1244.
Ramiro, A.R., Jankovic, M., Eisenreich, T., Difilippantonio, S., Chen-Kiang, S.,
Muramatsu, M., Honjo, T., Nussenzweig, A., and Nussenzweig, M.C. (2004).
AID is required for c-myc/IgH chromosome translocations in vivo. Cell 118,
431–438.
Ramiro, A.R., Stavropoulos, P., Jankovic, M., and Nussenzweig, M.C. (2003).
Transcription enhances AID-mediated cytidine deamination by exposing
single-stranded DNA on the nontemplate strand. Nat. Immunol. 4, 452–456.
Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N.,
Forveille, M., Dufourcq-Labelouse, R., Gennery, A., et al. (2000). Activation-
induced cytidine deaminase (AID) deficiency causes the autosomal recessive
form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575.
Robbiani, D.F., Bothmer, A., Callen, E., Reina-San-Martin, B., Dorsett, Y., Di-
filippantonio, S., Bolland, D.J., Chen, H.T., Corcoran, A.E., Nussenzweig, A.,
and Nussenzweig, M.C. (2008). AID is required for the chromosomal breaks
in c-myc that lead to c-myc/IgH translocations. Cell 135, 1028–1038.
Roy, D., Yu, K., and Lieber, M.R. (2008). Mechanism of R-loop formation at
immunoglobulin class switch sequences. Mol. Cell. Biol. 28, 50–60.
Schaeffer, D., Tsanova, B., Barbas, A., Reis, F.P., Dastidar, E.G., Sanchez-
Rotunno, M., Arraiano, C.M., and van Hoof, A. (2009). The exosome contains
domains with specific endoribonuclease, exoribonuclease and cytoplasmic
mRNA decay activities. Nat. Struct. Mol. Biol. 16, 56–62.
Schmid, M., and Jensen, T.H. (2008). The exosome: a multipurpose RNA-
decay machine. Trends Biochem. Sci. 33, 501–510.
Schrader, C.E., Linehan, E.K., Mochegova, S.N., Woodland, R.T., and Stav-
nezer, J. (2005). Inducible DNA breaks in Ig S regions are dependent on AID
and UNG. J. Exp. Med. 202, 561–568.
Shen, H.M., Ratnam, S., and Storb, U. (2005). Targeting of the activation-
induced cytosine deaminase is strongly influenced by the sequence and struc-
ture of the targeted DNA. Mol. Cell. Biol. 25, 10815–10821.
Shen, H.M., and Storb, U. (2004). Activation-induced cytidine deaminase (AID)
can target both DNA strands when the DNA is supercoiled. Proc. Natl. Acad.
Sci. USA 101, 12997–13002.
Shen, H.M., Tanaka, A., Bozek, G., Nicolae, D., and Storb, U. (2006). Somatic
hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double-
knockout mice. J. Immunol. 177, 5386–5392.
Shen, V., and Kiledjian, M. (2006). A view to a kill: structure of the RNA
exosome. Cell 127, 1093–1095.
Shinkura, R., Tian, M., Smith, M., Chua, K., Fujiwara, Y., and Alt, F.W. (2003).
The influence of transcriptional orientation on endogenous switch region func-
tion. Nat. Immunol. 4, 435–441.
Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A., and Bhagwat, A.S. (2003).
Human activation-induced cytidine deaminase causes transcription-depen-
dent, strand-biased C to U deaminations. Nucleic Acids Res. 31, 2990–2994.
Staals, R.H., Bronkhorst, A.W., Schilders, G., Slomovic, S., Schuster, G.,
Heck, A.J., Raijmakers, R., and Pruijn, G.J. (2010). Dis3-like 1: a novel exoribo-
nuclease associated with the human exosome. EMBO J. 29, 2358–2367.
Stillman, B.W., and Gluzman, Y. (1985). Replication and supercoiling of simian
virus 40 DNA in cell extracts from human cells. Mol. Cell. Biol. 5, 2051–2060.
Tian, M., and Alt, F.W. (2000). Transcription-induced cleavage of immunoglob-
ulin switch regions by nucleotide excision repair nucleases in vitro. J. Biol.
Chem. 275, 24163–24172.
Tomecki, R., Kristiansen, M.S., Lykke-Andersen, S., Chlebowski, A., Larsen,
K.M., Szczesny, R.J., Drazkowska, K., Pastula, A., Andersen, J.S., Stepien,
P.P., et al. (2010). The human core exosome interacts with differentially local-
ized processive RNases: hDIS3 and hDIS3L. EMBO J. 29, 2342–2357.
Vuong, B.Q., Lee, M., Kabir, S., Irimia, C., Macchiarulo, S., McKnight, G.S.,
and Chaudhuri, J. (2009). Specific recruitment of protein kinase A to the immu-
noglobulin locus regulates class-switch recombination. Nat. Immunol. 10,
420–426.
Wang, L., Wuerffel, R., Feldman, S., Khamlichi, A.A., and Kenter, A.L. (2009). S
region sequence, RNA polymerase II, and histone modifications create chro-
matin accessibility during class switch recombination. J. Exp. Med. 206,
1817–1830.
Wuerffel, R.A., Du, J., Thompson, R.J., and Kenter, A.L. (1997). Ig Sgamma3
DNA-specifc double strand breaks are induced in mitogen-activated B cells
and are implicated in switch recombination. J. Immunol. 159, 4139–4144.
Xue, K., Rada, C., and Neuberger, M.S. (2006). The in vivo pattern of AID
targeting to immunoglobulin switch regions deduced from mutation spectra
in msh2-/- ung-/- mice. J. Exp. Med. 203, 2085–2094.
Yang, S.Y., and Schatz, D.G. (2007). Targeting of AID-mediated sequence
diversification by cis-acting determinants. Adv. Immunol. 94, 109–125.
Yu, K., Chedin, F., Hsieh, C.L.,Wilson, T.E., and Lieber, M.R. (2003). R-loops at
immunoglobulin class switch regions in the chromosomes of stimulated B
cells. Nat. Immunol. 4, 442–451.
Yu, K., and Lieber, M.R. (2003). Nucleic acid structures and enzymes in the
immunoglobulin class switch recombination mechanism. DNA Repair (Amst.)
2, 1163–1174.
Zarrin, A.A., Alt, F.W., Chaudhuri, J., Stokes, N., Kaushal, D., Du Pasquier, L.,
and Tian, M. (2004). An evolutionarily conserved target motif for immunoglob-
ulin class-switch recombination. Nat. Immunol. 5, 1275–1281.
Cell 144, 353–363, February 4, 2011 ª2011 Elsevier Inc. 363
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