Interest-ingly, both miRNAs are capable of binding directly to TDP-43 in different positions: within the miRNA sequence itself let-7b or in the hairpin pre-cursor miR-663.. 2D, upper pan
Trang 1Emanuele Buratti1, Laura De Conti1, Cristiana Stuani1, Maurizio Romano2, Marco Baralle1and Francisco Baralle1
1 International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
2 Department of Life Sciences, University of Trieste, Italy
Introduction
TDP-43 is a protein belonging to the hnRNP class
of nuclear factors that has been described to play a
role in a variety of cellular processes, including gene
transcription, pre-mRNA splicing and mRNA
stabil-ity [1] Recently, it has been identified as the major
protein component of neuronal inclusions in
neurode-generative diseases such as frontotemporal dementias
and amyotrophic lateral sclerosis [2] The impact of
TDP-43 in the neurodegeneration field has been so
pervasive that disease nomenclature consensus is
cur-rently being modified to reflect the new clinical and
pathological findings originating from recent research
better [3,4] This finding has promoted studies to
characterize better the functional role(s) played by this protein inside the cell As a result, apart from its historical involvement in splicing and transcription [5–7], several recent observations have successfully highlighted new biological characteristics of this protein, such as acting as a neuronal response activity factor and an in vitro mRNA translational repressor [8], an mRNA stability factor for neurofila-ments [9,10] and as a regulator of Rho family GTPase expression [11] and HDAC6 [12] All of these observations may be conducive to under-standing the potentially pathogenic role of TDP-43 in neurodegeneration
Keywords
amyotrophic lateral sclerosis; let-7b;
microRNAs; miR-663; TDP-43
Correspondence
F E Baralle, Padriciano 99, 34012 Trieste,
Italy
Fax: +39 040 3757361
Tel: +39 040 3757337
E-mail: baralle@icgeb.org
(Received 2 September 2009, revised 26
February 2010, accepted 8 March 2010)
doi:10.1111/j.1742-4658.2010.07643.x
TDP-43 has recently been described as the major component of the inclu-sions found in the brain of patients with a variety of neurodegenerative dis-eases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis TDP-43 is a ubiquitous protein whose specific functions are prob-ably crucial to establishing its pathogenic role Apart from its involvement
in transcription, splicing and mRNA stability, TDP-43 has also been described as a Drosha-associated protein However, our knowledge of the role of TDP-43 in the microRNA (miRNA) synthesis pathway is limited to the association mentioned above Here we report for the first time which changes occur in the total miRNA population following TDP-43 knock-down in culture cells In particular, we have observed that let-7b and miR-663 expression levels are down- and upregulated, respectively Interest-ingly, both miRNAs are capable of binding directly to TDP-43 in different positions: within the miRNA sequence itself (let-7b) or in the hairpin pre-cursor (miR-663) Using microarray data and real-time PCR we have also identified several candidate transcripts whose expression levels are selec-tively affected by these TDP-43–miRNA interactions
Abbreviations
DYRK-1A, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A; EPHX1, epoxide hydrolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; LAMC1, laminin, gamma 1 (formerly LAMB2); miRNA, microRNA; siRNA, short inhibitory RNA; STX3, syntaxin 3; VAMP3, vesicle-associated membrane protein 3.
Trang 2With regards to the wider biological properties of
TDP-43, a new indication has been provided by its
presence in both the human and the mouse
micropro-cessor complexes, suggesting a potential involvement
in microRNA (miRNA) biogenesis [13,14] Further
support for a role in miRNA biogenesis for TDP-43
is its localization in perichromatin fibres [15], a
nuclear region specifically associated with this process
[16] The Drosha nuclear complex is one of the key
enzymes involved in the biogenesis of miRNAs and
has the function of converting pri-miRNA molecules
to 70 nucleotide-long pre-miRNA molecules, which
are then exported to the cytoplasm and further
pro-cessed in mature miRNAs by Dicer [17] These small
RNA molecules can then bind to their target
mRNAs through sequence complementarity and
affect gene expression by regulating either mRNA
levels or translation [18–21] Recently, hnRNP
pro-teins were shown to be involved in miRNA
process-ing [22,23] It was therefore of interest to investigate
the consequences on the cellular miRNA population
of removing TDP-43
Results
An analysis of Drosha levels by western blot in
TDP-43-depleted Hep-3B cells did not reveal any significant
changes in Drosha migration pattern or signal intensity
with respect to mock-treated cells (Fig 1A), pointing
to specific miRNA targets for TDP-43 To investigate
this possibility, miRNA profiling in TDP-43-depleted
Hep-3B cells from three independent samples was
per-formed by Exiqon (Vedbaek, Denmark) The
micro-array experiment tested for 607 known and proprietary
miRNA sequences (438 and 169, respectively) In this
triplicate experiment, 146 miRNA sequences could be
detected in our samples and 90 of these miRNA
signa-tures could be quantitatively tested in all three short
interfering RNA (siRNA) and control experiments (a
list of the 67 registered ones can be found in Fig S1)
The eight miRNAs that were either down- or
upregu-lated in a statistical significant manner following
deple-tion of TDP-43 in Hep-3B cells are shown in Fig 1B
For the three most statistically significant miRNAs
(let-7b, miR-663 and miR-744), the results were
vali-dated using the commercial miRvana kit, which is
based on a hybridization procedure with small
radioac-tive probes based on the miRNA of interest (Fig 1C,
D) In this experiment, the changes in these miRNA
expression levels as detected by the microarray
experi-ment were confirmed in three cell lines: HeLa
(adeno-carcinoma), Hep-3B (hepatocarcinoma) and SH-S-5Y
(neuroblastoma)
As microarray experiments represent an indirect way
of measuring TDP-43 effects on the general miRNA population, it was not possible, on the basis of these data alone, to rule out the possibility that a lack of TDP-43 may have affected the levels or activity of another factor involved in miRNA processing (for example, hnRNP A1 or other miRNA processing fac-tors) Therefore, in order to establish a direct link between TDP-43 and any of these miRNAs, we focused on TDP-43 RNA binding properties that have been previously characterized in our laboratory [24,25] Looking at the miRNA sequences it was interesting
to note that let-7b contained in its sequence a discrete number of (GU)nrepeats, the preferred target sequence
of TDP-43 [24] (Fig 1C) A band shift analysis per-formed using recombinant GST–TDP-43 confirmed that both the let-7b and the let-7b hairpin sequence (Fig 2A) could bind these sequences (Fig 2B) Most interestingly, variations in the levels of both let-7a and let-7c did not appear to be statistically significant in the microarray assay (Fig 2C) By comparing the let-7a, -7b and -7c sequences (Fig 2D, upper panel) we observed that a critical guanosine residue in the let-7b sequence at position +17 had the effect of creating a new GU repeat, suggesting that this miRNA could be particularly sensitive to TDP-43 cellular levels as opposed to the other let-7 family members A band shift experiment using labelled let-7a, -7b and -7c sequences confirmed that recombinant GST–TDP-43 could only bind the let-7b sequence (Fig 2D, lower panel) The critical importance of the +17 residue is highlighted by the observation that introducing
a + 17a > g substitution in the let-7a sequence can promote TDP-43 binding (Fig 2D, lower panel) It should be noted that the importance of this critical res-idue has also been confirmed using pulldown analysis
by immobilizing these miRNA sequences on adipic acid dehydrazide beads and incubating with total HeLa nuclear extracts The results of this experiment confirmed that introducing a + 17a > g nucleotide in the let-7a sequence gave it the ability to bind TDP-43, even in the presence of all other nuclear competing proteins (Fig S2)
We also examined the sequences of all the other miRNAs, and noted that within the sequence of the miR-663 precursor (the second most statistically affected miRNA after let-7b) there was an almost per-fect GU repeated sequence localized in the apical por-tion of the hairpin (Fig 3A) A band shift analysis with recombinant TDP-43 confirmed binding to the precursor hairpin, but not to the miR-663 sequence itself (Fig 3B, left and central panels, respectively) Deletion of the GU-rich sequence in the hairpin also
Trang 3abolished TDP-43 binding (Fig 3B, right panel).
Finally, neither the miR-744 sequence and its hairpin
(Fig S3) nor all the other identified miRNA sequences
could bind TDP-43 in band shift analyses (data not
shown) These data are consistent with the observation
that the sequence of this miRNA does not contain a
sufficient number of (ug)nrepeats
From a TDP-43–miRNA interaction point of view,
these results also suggest that there may be several
other potential miRNA targets of TDP-43 that could
not be detected in our analysis because they were not
expressed at sufficient levels (or at all) in Hep-3B cells
In order to obtain some indication in this regard, we examined the primary sequence of all known miRNAs present in miRBase for GU-repeated regions This analysis identified two other miRNAs that could potentially bind TDP-43: miR-574-5p in the miRNA sequence itself (Fig 3C) and miR-558 in the hairpin element (Fig 4A) Nothing is known regarding the expression profile or importance of these miRNAs, with the exception of miR-558, which has been described to be transiently upregulated in fibroblasts
Drosha
kDa
175
TDP-43
siRNA + –
siRNA + –
siRNA + –
TDP-43 47.5
175
TDP 43
Tubulin
Let-7b
siRNA
siRNA
Coomassie 47.5
miR-663
miR-744
Statistical significance (T-test) let-7b 0.0039
0.0069
miR-629 0.019
0.017
0.0053
0.032 0.039
#3 #1 #2 #2 #1 #3 siRNA Mock
Down-regulated
miR-23a miR-744 miR-373*
miR-663 miR-572
depletion
Up-regulated
following TDP-43
depletion
–2.0 –1.0 0 1.0 2.0
B
D
Fig 1 Effect of TDP-43 depletion on Drosha and selected miRNA expression levels in Hep-3B cells (A) Western blot assay of Hep-3B cells treated with a control siRNA (mock) and a specific TDP-43 siRNA (siRNA) The protein extracts were normalized by Coomassie intensity (lower panel) and hybridized with a polyclonal antibody against TDP-43 and a rabbit polyclonal antibody against Drosha (B) Heat map showing all the miRNAs (P < 0.05) differentially expressed in TDP-43-depleted Hep-3B cells with respect to mock-siRNA-treated cells The blue labels indicate downregulated miRNAs, the red labels indicate upregulated ones The clustering is reported as log2(Hy3 ⁄ Hy5) ratios (C) TDP-43 knockdown levels achieved in three cell lines: HeLa, Hep-3B and SH-SY-5Y cells (D) Quantification of let-7b, miR-663 and miR-744 expression levels in HeLa, Hep-3B and SH-SY-5Y cell lines using the commercial miRvana kit Undigested probe (p).
Trang 4following high doses of radiation [26] Band shift
assays confirmed that TDP-43 could bind efficiently to
the miR-574-5p sequence (Fig 3D, left) but, unlike
let-7b, could not bind anymore to the miR-574-5p
sequence when it was embedded in the RNA
second-ary structure (compare Figs 2B and 3D, right) The
reason for this probably resides in the inability of
TDP-43 to compete for RNA secondary structure
formation in the miR-574-5p sequence This structure,
in fact, is more extended and GC-rich than the
corresponding let-7b structure element As expected,
TDP-43 could bind to the miR-558 hairpin sequence,
but not to the miR-558 miRNA (Fig 4B)
In order to confirm the functional significance of the
TDP-43 let-7b⁄ miR-663 interactions we then used a
heterologous assay based on a luciferase reporter Four
complementary target sequences for let-7b and miR-663 were subcloned in the pGL3 vector, to obtain pGL3-mir-let-7b and pGL3-mir-663 (Fig 4C) Both constructs, together with a pRL-TK Renilla luciferase vector, were transiently transfected into Hep-3B cells and assayed for luciferase activity in both the presence (mock) or the absence (siRNA) of TDP-43 according
to the manufacturer’s instructions The results were normalized according to the firefly⁄ Renilla luciferase ratios obtained in each sample As expected, no signifi-cant difference could be detected in the firefly⁄ Renilla ratios of the pGL3 empty vector following knockdown
of TDP-43 in Hep-3B cells (Fig 4D, left) However, a significant increase in reporter gene activity was observed following transfection of the pGL3-mir-let-7b sequence following TDP-43 knockdown (Fig 4D,
let-7a let-7b let-7c let-7a+17a>g
let-7a let-7b let-7c
Statistical significance (T-test)
#1 #2 #3 #1 #2 #3 siRNA Mock
*let-7d-7e-7f-7g-7i - No detectable levels
let-7 family*
let-7b stem loop:
let-7b:
let-7b let-7b
stem-loop
–1.0 –0.5 0 0.5 1.0
let-7b let-7a
let-7a +17a>g let-7c
Fig 2 Specific interaction of TDP-43 with let-7b (A) Schematic diagram of the let-7b miRNA sequence and of its precursor hairpin (B) Band shift assay with recombinant GST–TDP-43 using the labelled let-7b sequence itself (left) and the let-7b hairpin element (right) (C) Heat map profile for all detected members of the let-7 family found in our assay, together with their statistical significance (D) The upper panel shows the sequence comparison (the GU dinucleotides are highlighted in bold), the lower panel shows a band shift analysis of labelled let-7a, let-7a+17a>g, let-7b and -7c miRNA sequences incubated with recombinant GST–TDP-43.
Trang 5centre) This is the result that should have been
expected if depletion of TDP-43 was associated with
lower expression levels of let-7b (as this would have
meant lower translational inhibition on the
pGL3-mir-let-7b construct) Exactly the opposite effect was
observed when we transfected the pGL3-mir-663
construct in depleted or control cells (Fig 4D, right)
Also, this result was completely consistent with
increased miR-663 expression following TDP-43
depletion, as such an outcome would have caused a
higher translational inhibition on the pGL3-mir-663
construct One important issue that should be
mentioned is the fact that these two GU-rich regions
in the let-7b miRNA and miR-663 do not exactly
match the optimal TDP-43 binding consensus
represented by perfect GU-repeated sequences and this
may well explain why in both cases TDP-43 has only
modulating effects on their expression rather than an
all or nothing phenomena
Most importantly, it was interesting to determine
the potential consequences of these changes in terms of
cellular transcript alterations It was originally
thought, in fact, that miRNA-mediated regulation was
mainly at the level of translation and not at the level
of mRNA degradation It is now clear, however, that this view is only partially correct and that, depending
on a variety of factors still only partially understood, many miRNA targets are regulated by degradation (as recently reviewed by Nilsen [20]) This has enabled the identification of miRNA targets by mRNA microarray analysis but, of course, it still remains very difficult to determine the proportion of mRNA targets affected in this way as opposed to strictly translation regulatory pathways (at least until large-scale proteomic approaches reach the level of sensitivity now available for mRNA microarray approaches)
Keeping in mind these limitations, we took advan-tage of our previously determined microarray evalua-tion of the cellular transcripts that were either
down-or upregulated following TDP-43 knockdown in HeLa cells [27] These transcripts (a total of 786) were com-pared with a set of transcripts (numbering 838) that have been observed to be downregulated following let-7b overexpression in a culture of primary human fibroblasts and which contained a let-7b seed target region in their 3¢ UTRs [28] The 23 common hits
miR-663:
miR-663 stem loop:
miR-663 miR-663
Stem loop
miR-663 Stem loop delta-UG
miR-574-5p:
miR-574-5p stem-loop:
miR-574-5p miR-574-5p
Stem-loop
Fig 3 Interaction of TDP-43 with miR-663 and functional analysis (A) Potential TDP-43 binding site to the miR-663 precursor hairpin element (highlighted in bold) (B) Band shift assay with recombinant GST–TDP-43 using the labelled miR-663 sequence itself (left), the miR-663 hairpin element (middle) and a miR-663 gucugugu-deleted sequence (right) (C) Potential TDP-43 binding site to the miR-574-5p sequence and the sequence of its precursor hairpin element (D) Band shift assay with recombinant GST–TDP-43 using the labelled miR-574-5p sequence itself (left) and the miR-574-5p hairpin element (right).
Trang 6between the two lists are reported in Table 1 First of
all, it should be noted that in the microarray
experi-ment, 16 of the 23 hits were upregulated following
TDP-43 removal This situation was therefore largely
consistent with the downregulatory effect on let-7b
expression levels following TDP-43 removal (Fig 1B)
More interestingly, among the most upregulated
tran-scripts were several with a potentially important
function in neuronal and synapse development: the
dual-specificity tyrosine-(Y)-phosphorylation regulated
kinase 1A (DYRK-1A), syntaxin 3 (STX3), the
vesicle-associated membrane protein 3 (cellubrevin; VAMP3)
and laminin, gamma 1 (LAMC1, formerly LAMB2)
Interestingly, this list also contained the enzyme
cyclin-dependent kinase 6, which we previously found to be
upregulated following TDP-43 removal [27]
Upregula-tion of these transcripts was confirmed by real-time
PCR (Figs 5A, 6A) using six independent siRNA
knockdown and siRNA control batches The results
showed that all these transcripts were significantly
up-regulated from a minimum of 1.7- to 3-fold following
TDP-43 removal (Fig 5A) In parallel to this analysis
we wanted to rule out the possibility that upregulation
of these transcripts could be due to changes in their mRNA splicing profiles owing to the presence of sev-eral putative TDP-43 binding sites in their intronic ele-ments (Fig 5B) Normal RT-PCR analysis of the coding regions, however, also ruled out this possibility
by showing that the splicing profile of these transcripts did not specifically change following TDP-43 removal (Fig 5C)
In the case of miR-663, no data are currently avail-able regarding the variation in cellular transcripts fol-lowing its overexpression⁄ removal In order to find an alternative solution, our list of microarray targets fol-lowing TDP-43 removal was compared with a list of more than 1000 putative miR-663 targets obtained using the miranda software and downloaded from miRBase (http://microRNA.sanger.ac.uk/) Only three putative common transcripts were identified through this comparison (Table 2) It can be seen that in this reduced sample obtained by indirect methods we had two cases that showed the expected decrease in tran-script levels that could follow miR-633 increase due to
SV40 promoter
SV40 polyA
Fir.Luc AAAAA pGL3-mir-let-7b
XbaI
Fir.Luc AAAAA pGL3-mir-663
pGL3 pGL3-mir-let-7b
0.2 0.4 0.6 0.8 1.0
pGL3-mir-663
1.2
0.2 0.4 0.6 0.8 1.0 1.2
0.5 1.0 1.5 2.0 2.5 3.0
miR-558:
miR-558 stem-loop:
miR-558 miR-558
Stem-loop
C A
D B
Fig 4 Interaction of TDP-43 with miR-558 and miR-574-5p (A) Potential TDP-43 binding site to the miR-558 sequence and the sequence precursor hairpin element (B) Band shift assay with recombinant GST–TDP-43 using the labelled 558 sequence itself (left) and the
miR-558 hairpin element (right) (C) Schematic diagrams of the constructs pGL3, pGL3-mir-let-7b and pGL3-mir-663 Each construct contained four copies of the complementary target sequence of let-7b and miR-663, respectively (D) Results of a luciferase assay performed on TDP-43-depleted and mock-depleted Hep-3B cells following transfection of these constructs In this type of experiment, the level of the interac-tion between the endogenous let-7b and miR-663 and the expression vector determined the levels of luciferase expression Transfecinterac-tion efficiencies were normalized using the Renilla luciferase internal control Standard deviation values from three independent experiments are indicated.
Trang 7TDP-43 depletion We have analysed in more detail
the enzyme epoxide hydrolase (EPHX1) because of its
putative role as an antagonist of oxidative stress [29]
The decrease in EPHX1 levels was confirmed by
real-time PCR (Fig 6A) and RT-PCR ruled out any effect
of TDP-43 removal on the splicing process of this
enzyme (Fig 6B–C)
Finally, we also began to investigate the regulatory
pathways that may be controlled by TDP-43 At least
for TDP-43, we decided to measure the pre-miRNA
levels in TDP-43-depleted and mock-depleted cells
For this reason, we measured the levels of pri-let-7b
miRNAs according to established protocols [30] As
shown in Fig 6D, upper panel, following TDP-43
removal, the levels of pri-let-7b were significantly
increased to a level that was comparable with the loss
of mature let-7b miRNA within the cell Moreover,
these changes were statistically significant These
results demonstrate that TDP-43 actively participates
in the Drosha processing mechanisms and its absence
in the case of let-7b leads to a block in the maturation
of pri-let-7b miRNA Finally, we also measured the
levels of pri-miR-663 using a similar procedure In this
case, however, the difference in miR-663 precursor
levels did not reach statistical significance (Fig 6D,
lower panel)
Discussion
The biological function of TDP-43 in the eukaryotic cell
is far from being fully understood Even more obscure
is its role in the pathogenesis of amyotrophic lateral sclerosis⁄ frontotemporal lobar degeneration and other neurodegenerative diseases In particular, several
gain-or loss-of-function mechanisms have been put fgain-orward
in recent times The gain-of-function mechanisms focus
on the generation of potentially toxic C-terminal frag-ments [31–33], its toxicity in a yeast cellular model [34] and increased aggregation properties in the presence of missense mutations in the C-terminal region [35] On the other hand, loss-of-function mechanisms are sup-ported by indications that TDP-43 may be playing a fundamental role in a variety of nuclear processes, such
as splicing regulation [5], transcription [36], chromatin organization [37] and a variety of other processes, such
as cell death and nuclear shape [27] Loss-of-function mechanisms are also supported by a recent Drosophila animal model that has shown that removal of the fly homologue of TDP-43 can recapitulate several features
of motoneuron disease [38] These two different patho-physiological mechanisms are not mutually exclusive and may indeed take place at the same time, although determining their relative importance may be especially
Table 1 List of altered cellular transcripts in TDP-43 knockdown experiments that have also been found to be downregulated following let-7b overexpression.
DYRK1A NM_001396 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A +4.6
SLC5A6 NM_021095 Solute carrier family 5 (sodium-dependent vitamin transp.), mem 6 )3.2
a Fold expression difference according to Ayala et al [27].
Trang 8important with regards to planning and developing
suc-cessful therapeutic strategies
To understand these pathological processes better, it
is of course important to define TDP-43 functional
properties as much as possible In this regard, the
effects of TDP-43 on the miRNA population are
par-ticularly interesting, considering previous observation
that TDP-43 itself is a minor component of the
Drosha enzyme complex [13] and the increasing role
played by aberrant miRNA expression in a variety of
neurodegenerative diseases, as recently reviewed in sev-eral publications [39–43]
However, to date no studies are yet available regard-ing the potential role played by TDP-43 in miRNA processing In general, Drosha-associated factors are required to help or inhibit the processing of particular subsets of miRNA molecules Indeed, this has been shown to be the case for the p68 and p72 helicases [14] and, more recently, for the KH-type splicing regula-tory protein (KSRP) protein [44] Of course, this regu-latory role is not solely confined to Drosha-associated proteins Indeed, one of the best characterized example
of miRNA regulatory proteins is represented by
Lin-28, which can regulate let-7 processing [45–48] by inducing uridylation of its precursor and cause its deg-radation [49] In a situation probably more similar to TDP-43, miRNA regulating properties have also been described for the well-known splicing factor hnRNP A1 This protein has been shown to regulate the expression of miR-18a by binding to the loop of pri-miR-18a and inducing a relaxation at the stem,
creat-0 0.5 1 1.5 2 2.5 3
0 0.5 1 1.5 2 2.5
0
0.5
1
1.5
2
0 0.5 1 1.5 2
SD = 0.07
SD = 0.06
SD = 0.03
SD = 0.1
SD = 0.09
SD = 0.1
DYRK1A (exons 1-13)
DYRK1A (150 kb)
STX3 (50 kb)
VAMP3 (10 kb)
*
non-coding exons coding exons
(ug) 6 repeats
****
LAMC1 (exons 1-14) LAMC1 (exons 14-28)
LAMC1 (120 kb)
**
STX3 (exons 1-9) VAMP3 (exons 2-5)
LAMC1 (P < 0.001)
SD = 0.05
SD = 0.1
A
B
C
Fig 5 Real-time PCR levels of let-7b regulated transcripts (A) Real-time PCR quantification analysis of the DYRK1A, LAMC1, STX3 and VAMP3 transcript levels following TDP-43 knockdown in HeLa cells based on the results of Table 1 Six independent experiments were anal-ysed and both standard deviations and P-values are shown for each transcript (B) Schematic diagram of the intron ⁄ exon architecture of these genes with the presence of potential TDP-43 binding motifs, (ug)6, indicated (C) Standard RT-PCR of each transcript to rule out the effects of TDP-43 on their RNA splicing process.
Table 2 List of altered cellular transcripts in TDP-43 knockdown
experiments that also represent putative miR-663 targets.
Gene
Accession
Microarray variationa
AAMP NM_001087 Angio-associated,
migratory cell protein
)2.3
a Fold expression difference according to Ayala et al [27].
Trang 9ing a more favourable cleavage site for Drosha
[22,23,50] Our results have shown that TDP-43 has
the potential to affect the levels of four miRNAs,
let-7b, miR-663, miR-574-5p and miR-558, by potentially
binding to their sequence and⁄ or precursor elements
(schematically summarized in Fig 7) With regards to
the potential importance of the interaction between
TDP-43 and miRs 574-5p⁄ 558 a cautionary note is
represented by the fact that, owing to the lack of cell
lines expressing these miRNAs, we were unable to
functionally validate them Therefore, this is an issue
that will have to be addressed in future studies
We then asked what kind of processing steps in the
biogenesis of these miRNAs may be affected In the
case of the let-7b family, the data that let-7a, which
originates from the same precursor as let-7b, is not
affected by TDP-43 support that the regulation is
post-transcriptional In particular, the observation that
TDP-43 depletion leads to an increase in pri-let-7b
lev-els suggests that for this miRNA, TDP-43 helps to
keep⁄ recruit the pri-miRNA sequences in place during
Drosha processing In the case of miR-663, we should
consider the fact that for several miRNAs, such as
miR-30 and miR-21, efficient processing is dependent
on the presence of a terminal loop more than 10 nucleotides long [51] However, the measurement of miR-663 precursor levels in TDP-43 minus and mock-depleted cells has failed to find a statistically significant difference This suggests that miR-663 regulation by TDP-43 may take place in steps subsequent to Drosha cleavage, an observation that may be consistent with the opposite effect of TDP-43 on miR-663 levels (upregulated) as opposed to let-7b (downregulated) The function of these different up- or downregulatory mechanisms is, of course, still an open question The most probable explanation is that there might be two sets of transcripts whose expression has to be upregulated (in the case of let-7b) and downregulated (in the case of miR-663) at the same time to achieve a functionally specific effect At the moment, identifying these hypothetical effects is hampered by our incom-plete knowledge of TDP-43 general functions and its expression regulation within the cell (especially in nor-mal, nonpathological conditions)
With regards to the miRNA we have identified, nothing is known about the functions of miR-663, miR-558 and miR-574-5p On the other hand, the let-7b family is an abundant, highly conserved family
0
0.2
0.4
0.6
0.8
1
1.2
EPHX1 (P < 0.01)
+Mock +siRNA
ls SD = 0.08
SD = 0.09
EPHX (20 kb) non-coding exons
coding exons
(ug) 6 repeats
EPHX (exons 2-9)
0.0 0.5 1.0 1.5 2.0
0.0 0.5 1.0 1.5 2.0
+Mock +siRNA
hsa-let7b precursor levels (P < 0.05)
SD = 0.05
SD = 0.03
+Mock +siRNA
miR-663 precursor levels (P > 0.05)
SD = 0.42 SD = 0.22
A
B
C
D
Fig 6 Real-time PCR levels of let-7b and miR-663 regulated transcripts (A) Real-time PCR quantification analysis of the EPHX1 transcript levels following TDP-43 knockdown in HeLa cells based on the results of Table 2 Six independent experiments were analysed and both standard deviations and P-values are shown for each transcript (B) Schematic diagram
of the intron ⁄ exon architecture of these genes with the presence of potential TDP-43 binding motifs indicated (C) Standard RT-PCR of each transcript to rule out the effects of TDP-43 on their RNA splicing process (D) Measurement by real-time PCR of the let-7b and miR-663 precursor levels following TDP-43 depletion and mock depletion in HeLa cells Standard deviations are shown above each bar and P-values are indicated.
Trang 10of miRNAs that are important in cellular
differentia-tion processes and their misreguladifferentia-tion may lead to
can-cer formation, as recently reviewed by Roush and
Slack [52] However, Drosophila let-7 has been
described as being essential for correct neuromuscular
development in the transition from larva to adult [53],
suggesting that members of this family may also
par-ticipate in neuronal and developmental processes
In keeping with this hypothesis, we provide evidence
that the removal of TDP-43 from the cell nucleus
causes specific downregulation of let-7b, and this can
in turn influence the expression levels of several
poten-tially important transcripts involved in
neurodegenera-tion and synapse formaneurodegenera-tion (Fig 7) These transcripts
include DYRK1A, a kinase that has been found to be
upregulated in patients affected by Down syndrome
and whose increased expression correlates with the
neuronal defects [54,55] They also include components
of synapse formation, such as STX3, which is
impor-tant for the growth of neurite processes [56], and
VAMP3, which can functionally substitute for
syna-ptobrevin in synaptic exocytosis [57] The upregulation
of LAMC1, on the other hand, is particularly
interest-ing in light of previous observations that dysmorphic
nuclear shape phenotypes are produced upon removal
of TDP-43 [27] Finally, another interesting transcript
that is downregulated following TDP-43 knockdown (but this time due to miR-663 upregulation) is repre-sented by the EPHX1 enzyme, a detoxifying enzyme that functions to regulate oxidative stress and has been previously shown to be significantly elevated in the hippocampal region of patients suffering from Alzhei-mer’s disease [29]
Taken together, these results provide an experimen-tal basis suggesting that TDP-43 can play a role in miRNA expression pathways Of course, how these changes relate to TDP-43¢s other normal biological properties (splicing, transcription, mRNA export⁄ translation) and, most importantly, to an eventual dis-ease context, will require future analyses Finally, as TDP-43 is also a splicing factor, it will also be interest-ing to explore the potential role of TDP-43 in Drosha-free miRNA synthesis (miRtrons) [58] At the moment, going through the list of miRtron genes recently com-piled by Berezikov et al [59], the consensus sequences
of the small introns responsible for miRtron formation
in vertebrates display a G-rich sequence at the 5¢ end and a U⁄ C-rich sequence at the 3¢ end None of these two sequences contains a number of GU repeats that may resemble (at least visually) potentially strong TDP-43 binding sites However, it is a possibility that warrants experimental testing in the future
let-7b
RNA
Pol II
miR-663
RNA
Pol II
miR-574-5p
RNA
Pol II
miR-558
RNA
Pol II
m7G
AAAAA
m7G
AAAAA
m7G
AAAAA
m7G
AAAAA
pri-let-7b
pri-miR-663
pri-miR-574-5p
pri-miR-558
pre-miRNA miRNA
TDP -43
Gene(s) potentially affected in neuro degeneration: DYRK1A STX3 VAMP3 LAMC1
Effect of TDP-43 removal on cellular concentration of the miRNA
TDP-43 binding to:
TDP -43
TDP -43
TDP -43
TDP -43
EPHX1
Fig 7 Schematic diagram of TDP-43–miRNA interactions This figure shows a summary of TDP-43 interactions with the various miRNA sequences and precursors identified in the present study Moreover, it summarizes the effects of its removal on miRNA expression levels and on potentially important transcripts for neuronal development or degeneration.