TDP-43 regulates retinoblastoma protein
phosphorylation through the repression
of cyclin-dependent kinase 6 expression
Youhna M. Ayala*, Tom Misteli†, and Francisco E. Baralle*‡
*International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy; and†Laboratory of Receptor Biology and Gene
Expression, National Cancer Institute, National Institutes of Health, 41 Library Drive, Building 41, Bethesda, MD 20892
Communicated by Jorge E. Allende, University of Chile, Santiago, Chile, January 21, 2008 (received for review September 3, 2007)
TDP-43 (for TAR DNA binding protein) is a highly conserved
heterogeneous nuclear ribonucleoprotein (hnRNP) involved in spe-
cific pre-mRNA splicing and transcription events. TDP-43 recently
has been identified as the main component of cytoplasmic inclu-
sions in frontotemporal lobar degeneration (FTLD) and amyotro-
phic lateral sclerosis (ALS), two neurodegenerative disorders. The
cellular role of this protein remains to be identified. Here, we show
that loss of TDP-43 results in dysmorphic nuclear shape, misregu-
lation of the cell cycle, and apoptosis. Removal of TDP-43 in human
cells significantly increases cyclin-dependent kinase 6 (Cdk6) pro-
tein and transcript levels. The control of Cdk6 expression mediated
by TDP-43 involves GT repeats in the target gene sequence. Cdk6
up-regulation in TDP-43-depleted cells is accompanied by an in-
crease in phosphorylation of two of its major targets, the retino-
blastoma protein pRb and pRb-related protein pRb2/p130. TDP-43
silencing also is followed by changes in the expression levels of
several factors that control cell proliferation. Morphological nu-
clear defects and increased apoptosis upon TDP-43 loss are medi-
ated via the pRb pathway because pRb-negative cells (Saos-2) do
not undergo programmed cell death or nuclear shape deformation
upon TDP-43 removal. Our results identify a regulatory target of
TDP-43 and show that TDP-43 depletion has important conse-
quences in essential metabolic processes in human cells.
via its N-terminal RNA recognition motif (1). Members of the
hnRNP family serve multiple roles in the generation and pro-
cessing of RNA, including transcription, splicing, transport, and
stability. TDP-43 inhibits exon recognition during splicing upon
recruitment to the 3? splice site of the cystic fibrosis transmem-
brane conductance regulator (CFTR) and apolipoprotein AII
transcripts via a sequence of GU repeats (2–5). The binding
affinity of the recombinant human, worm, and fly homologues
for this target sequence is remarkably high, measured in the low
nanomolar range (6). TDP-43 also has been implicated in the
transcription regulation of HIV and the spermatid-specific gene
SP-10 through promoter association (7, 8). More recently,
TDP-43 was identified as the main ubiquitinated component of
cytoplasmic inclusions in neurodegenerative diseases, specifi-
cally frontotemporal lobar degeneration (FTLD) and amyotro-
phic lateral sclerosis (ALS) (9, 10). Abnormal aggregation of
TDP-43 in the cytoplasm now is thought to define a class of
frontotemporal dementias termed TDP-43 proteinopathies.
Mislocalization and the consequent loss of TDP-43 function in
neuronal cells may represent a common event in FTLD patho-
genesis. Despite the increasing awareness of processes involving
TDP-43, the cellular role of the protein still is poorly defined.
We depleted TDP-43 by RNA interference (RNAi) to identify
TDP-43-regulated transcripts. Our results point to cyclin-
dependent kinase 6 (Cdk6) as a unique target of TDP-43
regulation and suggest that TDP-43 inhibits Cdk6 expression
through recruitment to the GU-rich transcript. Simultaneously,
DP-43 belongs to the family of heterogeneous nuclear ribo-
nucleoproteins (hnRNPs) and binds single-stranded RNA
we found that TDP-43 silencing alters cell cycle distribution and
TDP-43 Down-Regulation Alters the Expression of pRb-Related Fac-
tors. TDP-43 was depleted from HeLa cells by RNAi routinely
achieving ?90% silencing as measured by Western blot, 48 h
after small interfering RNA (siRNA) transfection (3, 5). RNA
microarray analysis was performed on TDP-43 depleted and
control treated cells. The data obtained indicated altered levels
of several cell proliferation factors in TDP-43-silenced cells.
Table 1 lists 16 of these proteins whose functions have been
associated with retinoblastoma protein (pRb) activity. The tu-
mor suppressor pRb is essential for the control of cell cycle
progression, cellular differentiation, and maintenance of ge-
nome integrity. Inactivation of pRb occurs through its gradual
phosphorylation by Cdks during the G1phase of the cell division
cycle resulting in the activation of transcription factors that
promote cell proliferation and enable transition on to the S
phase (see ref. 11 for review). Our RNA microarray analyses
showed altered levels of transcripts coding for proteins whose
functions are related to the control of cell cycle progression
(Cdk6, POLD4, cyclin B1, Cdk2, UBE2C, and SKP2). In addi-
tion, some of these factors are known to either directly interact
with pRb (e.g., HDAC1, RBBP4, and CRI1), or act in response
to pRb modulation (e.g., E2F8 and NAP1L1) (12–22).
TDP-43 Inhibits Cdk6 Expression. Among the transcripts whose
levels were altered upon TDP-43 loss, the levels of Cdk6 showed
the highest increase in the absence of TDP-43, specifically
10-fold. Cdk6 belongs to the family of Cdks and along with Cdk4
regulates the early G1phase transition during cell growth (23,
24). In addition to pRb, Cdk6 substrates include pRb2/p130,
histone H1, and Bcl-2 (25–27). The appropriate regulation of
Cdk6 activity determines differentiation of many cell types and
a variety of tumors have been associated with elevated levels of
Cdk6 expression (28–33). Careful inspection of human Cdk6
revealed a peculiar gene structure with particularly long introns
and a predicted 3? untranslated region (UTR) spanning nearly
11 Kb (Fig. 1A). Remarkably, its pre-mRNA contains multiple
(GU)nrepeats, the TDP-43 target sequence. The repeat tracts
are present once or twice in all of the larger introns and the 3?
UTR contains a (GU)25sequence 180 nt upstream of the first
predicted polyadenylation site. In contrast, introns in other
human Cdk genes, e.g., Cdk4 and Cdk2, are significantly shorter
Author contributions: Y.M.A., T.M., and F.E.B. designed research; Y.M.A. performed re-
search; and Y.M.A., T.M., and F.E.B. wrote the paper.
The authors declare no conflict of interest.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
March 11, 2008 ?
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and lack (GT)n (Fig. 1A). We observed that the repeats are
conserved in primates and rat and mouse genes. The abundance
of the TDP-43 target sequence within the Cdk6 gene urged us to
confirm the up-regulation of Cdk6 expression upon loss of
TDP-43 in human cells. We quantified Cdk6 protein and mRNA
levels by immunoblotting and quantitative PCR amplification,
respectively (Fig. 2). In agreement with the microarray results,
we obtained a significant increase in Cdk6 protein and transcript
in response to TDP-43 loss. The use of a different dsRNA
protein levels [supporting information (SI) Fig. 5A]. Moreover,
Cdk6 up-regulation upon RNAi treatment was studied in cells
transiently expressing a FLAG-tagged TDP-43 mutant resistant
to siRNA depletion (TDP-43siR). Cdk6 protein levels after
depletion of TDP-43 in three independent experiments were 35
to 45% lower in the presence of TDP-43siRcompared with
control treated cells as seen by quantification of immunoblotting
experiments, using tubulin as loading control (SI Fig. 6). Taken
together these results strongly suggest that Cdk6 levels are
specifically regulated by TDP-43.
To address whether TDP-43 regulates Cdk6 expression via the
GT tracts, we took advantage of the fact that chicken Cdk6 lacks
GT repeats completely, despite its otherwise highly similar exon
and intron structure. Although depletion of TDP-43 leads to
Cdk6 up-regulation in human cells, loss of TDP-43 has no effect
on Cdk6 protein levels in chicken cells (DF-1) (Fig. 2B). This
human specific increase in Cdk6 was confirmed with real time
PCR quantification of Cdk6 mRNA derived from RNAi treated
HeLa and DF-1 cells. The analysis showed a 20-fold increase in
Cdk6 mRNA upon TDP-43 depletion from human cells com-
pared with control transfected cells (Fig. 2C). However, TDP-
43-depleted and control treated DF-1 chicken cells showed no
variation. These data suggest that human Cdk6 expression is
modulated by TDP-43 recruitment to (GU)n repeats in the
TDP-43 Depletion Increases pRb and pRb2/p130 Phosphorylation. We
next tested whether Cdk6 misregulation in the absence of
TDP-43 extends to aberrant pRb phosphorylation. The phos-
phorylation levels of pRb and of the pRb-related protein pRb2/
p130 were determined after TDP-43 silencing. To avoid artifacts
deriving from disruptions of the pRb pathway caused by papil-
loma virus functional genes present in HeLa cells (34), we have
carried out our experiments in human osteosarcoma U2OS cells.
Immunoblot analysis showed that TDP-43 silencing resulted in
a 7 and 6-fold increase in the phosphorylation of pRb and
pRb2/p130, respectively (Fig. 3A). A modest increase in pRb and
Table 1. pRb pathway group microarray transcripts
Cyclin-dependent kinase 6 (Cdk6)
Nucleosome assembly protein 1-related protein (NAP1-L1)
Cyclin-dependent kinase 2 delta T (d-HSCdk2)
Cdk adapter protein (CKS1B)
Polo-like kinase 1 (PLK1)
CREBBP/EP300 inhibitory protein 1 (CRI1)
Polymerase delta 4 (PolD4)
Topoisomerase I (Top1)
Ribonucleotide reductase M2 (RRM2)
Retinoblastoma binding protein 4 (RBBP-4)
Ubiquitin-conjugating enzyme E2-C (UBE2C)
S-phase kinase-associated protein (SKP2)
Histone deacetylase 1 (HDAC1)
Elongation factor E2F8
Shown are transcripts identified by RNA microarray analysis whose levels
either increased or decreased in the absence of TDP-43 compared to control-
mammals. Schematic representation of different Cdk genes, including coding
exons (■) UTRs (?) and introns. (A) Human Cdk6 and the closely related Cdk4
Two stretches ranging from 10 to 40 repeats are found in introns 1, 3, and 4.
The repeats located at the 3? UTR of the gene are 400 nt downstream of the
stop codon and 180 nt upstream of the first polyadenylation site. In contrast
GT repeat sequences. (B) TG repeats are present in primates, mouse, and rat
Cdk6 genes. The length of the introns is more or less conserved, i.e., introns 1,
2, 3, and 4 are ?35-Kb long, whereas introns 5 and 6 range from 1 to 5 Kb.
Although chicken Cdk6 gene structure is similar to that of other vertebrates
analyzed, the sequence lacks GT repeats entirely.
GT repeats are unique to Cdk6 and are conserved in different
chicken cells. (A) Immunoblot analysis of HeLa and chicken embryo fibroblast
(DF-1) cell extracts. Human and chicken cells were transfected with control or
human/chicken TDP-43-targeted siRNA to check for changes in Cdk6 expres-
sion levels. Equal loading was confirmed by tubulin detection. Other human
cell lines tested showed a similar increase in Cdk6 upon TDP-43 depletion. (B)
Real-time RT-PCR analysis of Cdk6 cDNA in TDP-43 depleted (■) and control-
treated (?) HeLa and DF-1 cells. Values shown are the average of three
independent experiments. Error bars indicate SD.
TDP-43 depletion causes up-regulation of Cdk6 in human but not in
www.pnas.org?cgi?doi?10.1073?pnas.0800546105Ayala et al.
pRb2/p130 protein levels (2- and 1.5-fold) was observed in
phosphatase treated samples from TDP-43-depleted cells (lanes
3 and 4 of Fig. 3A). These results indicate that the higher
of TDP-43 were primarily attributable to increased levels of
phosphorylation, corresponding to the increase in Cdk6 levels in
these cells. Moreover, pRb or pRb2/p130 phosphorylation levels
did not change after depletion of TDP-43 in chicken embryo
fibroblasts, where Cdk6 levels are unresponsive to TDP-43
silencing (SI Fig. 7). These data strongly suggest that changes in
pRb and pRb2/p130 posttranslational modification, observed in
U2OS after TDP-43 removal, are mediated by the up-regulation
of Cdk6 in these cells.
We next investigated the impact of silencing TDP-43 and the
consequent alteration of the pRb pathway on cell cycle distri-
bution by Fluorescent-activated cell sorting (FACS) and BrdU
cycle pattern after removal of TDP-43 (Fig. 3B) resulting in a
60% decrease of cells in G0/G1accompanied by a corresponding
increase in S and G2/M cells. The disruption of cell cycle
distribution caused by the loss of TDP-43 is consistent with
alterations in pRb phosphorylation and misregulation of factors
that control cell growth.
Loss of TDP-43 Affects Nuclear Membrane Stability and Increases
Apoptosis. In addition to these changes we observed that TDP-43
depletion caused increased cell death and aberrant nuclear
morphology throughout our experiments. Visualization of the
nuclear lamina using antibodies against integral nuclear enve-
lope proteins lamin A/C and emerin showed membrane bleb-
bling and a significant disruption of the usual smooth and round
nuclear shape (Fig. 4A). Similar results were observed upon
depletion of TDP-43 using a different siRNA oligonucleotide
sequence, T1 (SI Fig. 5B). Such nuclear membrane disruption
was observed in nearly 50% of TDP-43 depleted cells and its
of TDP-43 led to an uneven distribution of the nuclear envelope
protein emerin along the membrane, with discrete regions of
either protein accumulation or lack of protein (Fig. 4A Lower).
TDP-43 silencing did not lead to changes, as judged by immu-
noblotting, in the quantity or processing of the nuclear envelope
proteins lamin A/C, lamin B, emerin, and Lap2? (data not
We investigated whether the effect on cell survival was
attributable to the activation of apoptosis or DNA damage. Loss
of TDP-43 in HeLa cells resulted in increased double strand
DNA breaks as indicated by the presence of ?H2AX foci (35):
?15% of TDP-43-depleted cells contained ?H2AX foci com-
pared with 1% of control treated cells (SI Fig. 8A). At the same
time, U2OS showed an increase in TUNEL staining and nuclear
fragmentation upon TDP-43 depletion (Fig. 4B). Typically, 30%
of TDP-43-depleted cells were TUNEL positive in contrast to
?1% of the control treated U2OS. U2OS cell extracts from
TDP-43-depleted samples were also positive for poly(ADP-
ribose) polymerase-1 (PARP-1) cleavage, consistent with the
activation of programmed cell death (SI Fig. 8B). Remarkably,
TDP-43 silencing in cells that lack functional pRb (Saos-2) did
not cause defects in nuclear envelope shape or apoptotic cell
death (Fig. 4C). Because Saos-2 cells are also p53 negative, it was
necessary to rule out a role of p53 deficiency in the Saos-2
cells, an osteosarcoma cell line deficient in p53 but not defective
in pRb, and observed increased apoptosis upon loss of TDP-43
in these cells, as seen with U2OS (SI Fig. 9). These results
strongly suggest that the morphological defects and increased
apoptosis upon TDP-43 loss are tied to disruption of the pRb
We have shown here that the major component of cytoplasmic
inclusions in frontotemporal dementias TDP-43 is involved in a
key cellular pathway that has ramifications in essential metabolic
processes. Loss of TDP-43 in human cells causes genomic
instability and increased apoptosis as seen by ?H2AX staining
and TUNEL. These results underscore a critical role of TDP-43
for cell survival. Moreover, we identify the cyclin-D dependent
kinase 6 as a target for TDP-43 control and provide a possible
mechanism of regulation. TDP-43 strongly represses Cdk6 ex-
pression as seen from the large increase in protein and transcript
levels upon TDP-43 depletion. We propose that TDP-43 recruit-
ment to the GU-rich Cdk6 transcript reduces RNA levels. This
mechanism is supported by the lack of change in Cdk6 levels
upon TDP-43 depletion in chicken cells, which express a Cdk6
transcript devoid of GU repeats. The involvement of TDP-43 in
the control of RNA processing has been seen in other systems
where GU repeats are involved, i.e., CFTR and ApoAII alter-
native splicing (2–5). The elucidation of the precise step of RNA
processing controlled by TDP-43 in the case of Cdk6 needs
further investigation. The implications of such studies could be
of great relevance because similar mechanisms of RNA process-
in neurodegenerative pathologies.
The hyperphosphorylation of pRb and pRb2/p130 caused by
Cdk6 misregulation in TDP-43-silenced cells is likely to block
pRb function and change the expression of proliferation
associated factors as seen by our microarray analysis (Table 1).
In chicken cells, TDP-43 loss leaves pRb and pRb2/p130
changes in cell cycle distribution. (A) Western blot analysis to detect pRb and
pRb2/p130 levels in U2OS cell extracts from TDP-43 depleted (lanes 2 and 4) and
(PPTase) (lanes 3 and 4) to compare unphosphorylated pRb and pRb2/p130
of cells in G0/G1, S, and G2/M phases of the cell cycle in proliferating siRNATDP-43
and siRNA control-treated U2OS cells. Propidium- and BrdU-labeled cells were
analyzed by FACS after RNAi treatment. Results show average values of three
independent experiments, and error bars show SD.
TDP-43 depletion promotes pRb and p130 phosphorylation and causes
Ayala et al.
March 11, 2008 ?
vol. 105 ?
no. 10 ?
phosphorylation levels unchanged supporting a direct associ-
ation between the gain in Cdk6 levels upon TDP-43 depletion
in human cells and pRb phosphorylation. The observed de-
crease of cells in G0/G1and the corresponding accumulation
of cells in S and G2/M phases in U2OS TDP-43-depleted cells
coincide with pRb hyperphosphorylation. More detailed stud-
ies will be required to determine whether these observations
involve checkpoint activation and cycle arrest. The aberrant
control of pRb function and cell proliferation may cause the
observed activation of apoptosis in the absence of TDP-43.
This interpretation is in line with findings that deregulation of
the pRb pathway does not only affect proliferation, but also
increases programmed cell death and alters DNA damage
responses (36–38). Moreover, increased activity of Cdks also
has been associated with apoptosis (39). Further investigation
will be required to understand the onset of nuclear membrane
instability upon loss of TDP-43. Interestingly, recent work
showed that nuclear membrane dysfunction, particularly loss
of emerin, is tied to misregulation of pRb pathway components
(40, 41) suggesting that nuclear membrane abnormalities
caused by TDP-43 loss are mediated by pRb. These conclu-
sions are supported by the lack of apoptosis and nuclear
membrane disruption after TDP-43 silencing in Saos-2 cells
that lack a functional pRb pathway. At the same time, TDP-43
may be more directly involved in the activation of programmed
cell death. New findings reveal that reduction of progranulin,
characteristic in familial FTLD with ubiquitin-positive inclu-
sions, causes a caspase-mediated cleavage of TDP-43 and
results in mislocalization of TDP-43 to insoluble cytoplasmic
fractions (42). Collectively, our results may help to shed light
on the pathophysiological consequences of TDP-43 loss of
function in neurodegenerative disorders.
Materials and Methods
RNA Microarray. Biotinylated cRNAs from TDP-43 and control siRNA treated
HeLa were hybridized to Affymetrix GeneChip Human Genome U133A 2.0
arrays (Affymetrix). Values reported are the average of three independent
Vectors and Cloning. FLAG-tagged TDP-43siRwas constructed with the pFLAG-
CMV-2 Expression Vector (Sigma E7398). Mutations in the protein were de-
signed to change the nucleotide TDP-43 sequence to render the transcript
resistant to siRNA oligonucleotide T2. These silent mutations do not alter the
amino acid TDP-43 sequence. The mutant vector was generated by PCR
mutagenesis using the following oligonucleotides: RNAi-forward, cttcctaat-
tctaagcagtcccaggatgagcctttgagaagcagaaaag; RNAi-reverse, cttttctgcttct-
Cell Culture and RNAi. HeLa (ATCC, CCL-2, LGC Promochem, MI, Italy), DF-1
DMEM?GlutaMAX I (Invitrogen, 31966), 10% FBS (Euroclone). TDP-43 was
silenced from human cells as described by using siRNA oligonucleotide T2 (3,
5), the same transfection protocol was used with chicken cells using the
following sequence of double-stranded RNA: GCAAAGUCCCGAUGAGCCU
(Dharmacon). A second dsRNA sequence (T1:GAUGAGAACGAUGAGCCCA)
was also used to silence TDP-43. The luciferase siRNA, sicontrol#2, was used as
Immunoblotting. Cell pellets were resuspended in lysis buffer: 50 mM Tris?HCl,
pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1? protease inhibitors (Roche
(15 mM Hepes, pH 7.5, 0.25 M NaCl, 0.5% Nonidet P-40, 10% glycerol, 1?
protease inhibitor, 25 mM NaF, 10 mM ?-glycerolphosphate, 0.2 mM Na3VO4,
and transferred to nitrocellulose (0.22 ?m, Schleicher & Schuell). Membranes
used to check protein phosphorylation were incubated in Western Blocking
Reagent (Roche). Antibodies used were TDP-43 (2),tubulin (Sigma T5168),
?H2AX (Upstate, 05–636), lamin A/C (Santa Cruz Biotechnology, sc-6215),
emerin (Novocastra), pRb (BD Pharmingen, 554136), p130 (Santa Cruz Bio-
technologies, sc-317), Cdk6 (Santa Cruz Biotechnologies, sc-177), and FLAG-
tagged TDP-43 (TDP-43siR) (Sigma F1804).
RNA Isolation and Real-Time RT-PCR. Cell RNA was isolated with TRI Reagent
(Ambion) and treated with DNase I. After reverse transcription, real-time PCR
was conducted on an ABI 7000 real-time PCR system (Applied Biosystems).
Human Cdk6 was amplified with a probe spanning exons 5–6 (assay ID
cells is shown. (Scale bar, 10 ?m.) (A) HeLa cells were stained with antibodies against lamin A/C and TDP-43 (red and green, Upper). Arrowhead points to a cell
(B) U2OS cells were assayed for DAPI and TUNEL staining to detect apoptosis. (Scale bar, 10 ?m.) (C) The nuclei of U2OS and Saos-2 cells were visualized by lamin
A/C detection after TDP-43 depletion. (D) Down-regulation of TDP-43 in U2OS and Saos-2 cells and the consequent Cdk6 increase were verified by immuno-
blotting. Tubulin was used as loading control.
www.pnas.org?cgi?doi?10.1073?pnas.0800546105Ayala et al.
Hs01026372?m1, Applied Biosystems), and chicken Cdk6 was amplified with
CTCTTCTTCT, and the TaqMan probe CTTTGATGTAATTGGACTCC (Applied
Biosystems). GADPH and 18S rRNA were used as endogenous controls.
Immunofluorescence Microscopy. After fixation in 4% buffered paraformal-
dehyde in PBS for 15 min at room temperature, cells were permeabilized by
using 0.5% Triton in PBS for 5 min on ice and blocked with 2% BSA/PBS for 15
min at room temperature. Immunolabeling using specific antibodies was
carried out at room temperature for 1 h in 2% BSA/PBS. Cells were incubated
with conjugated secondary antibodies at room temperature for 1 h in PBS.
Nuclei were stained with 4?,6-diamidino-2-phenylindole (DAPI).
FACS Analyses and BrdU Labeling. RNAi treated cells were pulsed by using 10
anti-BrdU-FITC (Becton Dickinson), followed by fluorescein goat anti-mouse
(Jackson Laboratories), and propidium iodide. FACS analysis using CellQuest
software (Becton Dickinson) was used to quantify BrdU-positive cells and
determine the DNA content of the different samples.
ACKNOWLEDGMENTS. We thank Andrea D’Ambrogio and Emanuele Buratti
useful discussions and for providing the TDP-43siRvector, Marı ´a Elena Lo ´pez
for help with tissue culture, Ramiro Mendoza for his help with FACS analyses,
and Lawrence Banks (ICGEB) for useful discussions and U2OS and Saos-2 cells.
This work was supported by National Science Foundation Postdoctoral Mi-
F.E.B.); Eurasnet Grant LSHG-CT-2005-518238 (to F.E.B.); and the Intramural
Research Program of the National Institutes of Health, National Cancer Insti-
tute, Center for Cancer Research.
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