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Human Molecular Genetics, 2005, Vol. 14, No. 22
doi:10.1093/hmg/ddi368
Advance Access published on October 3, 2005
3371–3377
Human microRNA (miR29b) expression controls
the amount of branched chain a-ketoacid
dehydrogenase complex in a cell
Benjamin D. Mersey, Peng Jin and Dean J. Danner*
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
Received August 12, 2005; Revised and Accepted September 26, 2005
Branched chain amino acids (BCAAs) play critical roles in cell and tissue functions in addition to being
important components of protein structure. The multifunctional roles speak to the need for maintaining
tight control of their concentration within cells. As the BCAA cannot be made de novo in mammals, their
cellular concentration is a function of dietary intake, endogenous protein turnover and catabolism of the
three amino acids. The branched chain a-ketoacid dehydrogenase (BCKD) complex commits the BCAA to
degradation and thus is vital in controlling their concentration within a cell. In mammals, BCKD activity
state depends on the presence of a covalently bound phosphate on one protein component of the complex.
Phosphate is added to the protein through the action of the complex-specific kinase and results in BCKD
inactivation. Here, we demonstrate that another reaction plays a role in determining the total amount of
BCKD present in a cell. The microRNA (miR29b) molecule tested is targeted to the mRNA for the dihydrolipoamide branched chain acyltransferase component of BCKD and prevents translation when bound. This
is the first demonstration of the use of a microRNA to exert control on a metabolic pathway of amino acid
catabolism in mammals and offers an explanation for the observed differences in the amount of the BCKD
complex present in different tissues and under varying nutritional states.
INTRODUCTION
Expression of small non-coding RNA (ncRNA) molecules is
now recognized to have important roles in development and
maintenance of cells and tissues in organisms from plants
to humans (1). One member group of these ncRNAs is the
microRNAs (miR) whose roles were first recognized in
Caenorhabditis elegans and Drosophila. In mammals, miRs
control gene expression either by interfering with production,
stability or translation of the mRNA target (2 –5). MicroRNAs
can be a product of their own gene or originate from within
introns of protein encoding transcripts (6,7). The primary
miR transcript is cut by the RNaseIII enzyme known as
DROSHA in the nucleus to yield a 70– 100 nucleotide
product that forms a hairpin structure that is exported to the
cytosol by EXPORTIN 5 (8). In the cytosol, these hairpin
structures are cut by the endonuclease DICER to a doublestranded product of 20– 25 bp. This double-stranded RNA is
incorporated into the RNA-induced silencing complex
(RISC) where the dominant miR is retained and used to
direct RISC to the target mRNA (3,9 –11). Identical pairing
of the entire miR with its target results in target degradation,
whereas imperfect pairing beyond the first 7– 9 bases at the
50 end of the miR blocks translation of the targeted mRNA
but does not destroy the mRNA (12).
Surveys to determine tissue distribution and expression
level for miRs in mammals report varying results even with
the same tissues and miRs (13 – 16). This variation remains
to be explained. It has been suggested that miRs can have
multiple targets and the preference is related to the binding
strength to the different targets (17 – 19). Binding of the miR
with its target is strongly influenced by the first 7 – 9 nucleotides at the 50 end of the miR. In mammals, the binding site
in the mRNA target is frequently found in the 30 untranslated
region (UTR) (17). Further, it has been suggested that multiple
miRs may bind to the same mRNA and act in concert with one
another (20,21). The number of miRs that are present and can
recognize a specific target within a cell type remains a
question. Whether these short interfering RNAs are used for
*To whom correspondence should be addressed at: Department of Human Genetics, Emory University School of Medicine, 615 Michael Street,
Room 305C, Atlanta, GA 30322, USA. Tel: þ1 4047275784; Fax: þ1 4047273949; Email: [email protected]
# The Author 2005. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
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Human Molecular Genetics, 2005, Vol. 14, No. 22
control of constitutively expressed genes or to fine tune
expression of inducible genes is not known (1).
The branched chain amino acids (BCAAs), leucine, isoleucine and valine, account for up to 20% of the component
amino acids in most proteins. In addition, BCAAs play important roles in synapse function, insulin secretion and protein
turnover (22 –24). BCAAs are essential components of
the diet of metazoans, because they cannot be synthesized
de novo in these organisms. Cellular concentration of the
BCAA is determined by dietary protein intake, endogenous
protein turnover and the catabolism of the individual BCAA,
which occurs in all cells with mitochondria as the entire catabolic pathway is located in these organelles. Branched chain
a-ketoacid dehydrogenase (BCKD) catalyzes the irreversible
step in BCAA catabolism and thus commits to a decrease in
the concentration of these amino acids within that cell. It
was found that activity of the existing BCKD complex
within a cell is regulated by a complex-specific kinase
(BCkinase) that adds a phosphate group to the E1a subunit.
That portion of BCKD with a phosphorylated subunit is catalytically inactive (25). The activity state of BCKD shows a
direct relationship to the amount of BCkinase present in
mitochondria within the cell. A recent report implicated that
Drosophila use miR to control the expression of the amount
of BCKD complex present in a cell and the authors speculate
that miR expression responds to dietary intake of protein for
the flies (26). A gene for a BCkinase cannot be found in the
Drosophila genomic database. The absence of a BCkinase
supports the idea that the fly BCKD is regulated by miR277
expression. Here we asked if any of the human miRs have
as targets one or more mRNAs that encode human BCKD
subunits. Several miRs were identified with this potential
(http://www.sanger.ac.uk/Software/Rfam/mirna/) (27). Our
results identify that endogenously expressed human miR29b
recognizes the mRNA for the dihydrolipoyl branched chain
acyltransferase (DBT) component of the BCKD complex in
HEK293 cells. These findings suggest that as in Drosophila
the amount of BCKD present within human cells is
determined by the expression of miR29b and potentially
other miRs.
RESULTS
BCKD subunit expression and activity
HEK293 cells express BCKD activity and the activity state
within these cells reflects the expression level of the BCkinase
in human kidney tissue (28). Under normal culture conditions,
about 50% of the BCKD complex in HEK293 cells is catalytically inactive due to BCkinase expression [22.6 + 11.7 pmol
CO2/(mg protein/min) out of 43.1 + 15.7 pmol CO2/(mg
protein/min) total activity] (28). This cell line therefore is
appropriate for assessing whether the miR control for the
amount of BCKD expression reported in flies has been
retained in humans along with the addition of the BCkinase
control of activity state of existing BCKD. Members of the
human miR29 family have, among their predicted mRNA
targets, the one that produces the DBT protein that forms
the core of the BCKD complex. The predicted pairing of
miR29a and miR29b occurs in the 30 end of the mRNA but
Figure 1. Nucleotide sequence for human miRNA29a and miRNA29b and the
predicted base pairing with mRNA for DBT. Different bases are indicated by
the red color of the base. The paired region in DBT mRNA covers nucleotides
1335–1375 that exist within the coding region of terminal exon 11. The
dashes in the pairing indicate the distance between the regions of pairing of
the miR with the mRNA to suggest where the loops are formed in the mRNA.
lies within the coding sequence of the terminal exon 11 and
binds in the region that is 70 nucleotides upstream of the
stop codon (Fig. 1). The pairing for miR29b conforms to the
predicted canonical pattern for a functional miR, as the first
nine bases directly match the nucleotide sequence within the
mRNA for DBT (17). miR29b and miR29a were synthesized
by us and used to transfect HEK293 cells. A fluorescent
labeled, randomly generated double-stranded RNA of 22 bp
was co-transfected along with the specific miR and the percentage of cells with fluorescent expression was estimated by
microscopic visual inspection. Control cells for each protocol
received the tagged randomly generated RNA and served as
mock transfection for the test miR. Data reported were
obtained from cell populations where transfection efficiency
exceeded 85% (Fig. 2).
If transfection of cells with miR29a and/or miR29b alters
the amount of BCKD within a cell, total BCKD activity will
reflect the change. Cells are treated with the BCkinase
inhibitor a-chloroisocaproate to determine the total BCKD
activity within the cells (28,29). Total BCKD activity
decreased over a 6-day period following a single transfection
with either miR29a or miR29b. Control cells contain only the
random 22mer RNA and BCKD activity did not change during
this time course. The decrease in total activity was evident by
24-h post-transfection and continued to decrease over the
144 h tested (Fig. 3). The decrease in total activity amounted
to about 40% by 144 h, suggesting that miR29b has retained
function in humans as in the Drosophila. Growth restrictions
of the cells in the 24-well plates prohibited further extension
of the time course. Transfection with both miRs did not
produce an additive effect and therefore this part of the protocol was not continued. The loss of BCKD total activity implies
a decrease in the amount of total BCKD that is present in
the mitochondria and presumably due to a decrease in DBT
protein. The amount of DBT depends on production and
degradation of this protein. A previous report has predicted
that the half-life of the BCKD proteins is 22– 26 h (30).
Another factor that might play a role in DBT production is
the amount of miR present in a cell and the half-life of
these molecules. No information is available regarding the
stability of exogenously added double-stranded microRNA,
but endogenously produced mature miRs are reported to
exist for more than 3 days within a cell (31).
When the miR exhibits imperfect pairing with its target,
mRNA beyond the first nine bases translation of the
message is blocked, but not destroyed. The antigenic presence
of DBT was determined by western blots using increasing
Human Molecular Genetics, 2005, Vol. 14, No. 22
Figure 2. Photomicrograph of cells transfected with a random 22mer RNA
labeled with FITC (1 and 3) or cells with lipofectamine alone (2 and 4).
Panels 1 and 2 are dark field and 3 and 4 are viewed with a GFP filter. The
figures show the greater than 80% transfection of the cells used in these
studies. Magnification was 50.
Figure 3. Total BCKD activity in HEK293 cells transfected with 20 pmol per
105 cells Block-IT Fluorescent RNA oligo (y), miR29a (n) or miR29b (s), or
10 pmol per 105 cells both miR29a and miR29b (l). Activity was measured at
the indicated time points for cell growth after transfection and is reported as
pmol CO2 released per mg of protein per minute. The numbers of independent
experiments used to create the average values are reported in parentheses.
amounts of mitochondrial protein to estimate the relative
concentration (Fig. 4). A reduced amount of DBT in the mitochondria occurred with the addition of either miR29a or
miR29b by 24-h post-transfection. The amount of DBT
returned to its original level by 144 h. Loss of DBT reduces
the other BCKD subunits, as their presence depends on
binding to DBT. Therefore, even though DBT is found to be
back to normal amount 144 h after transfection with
miR29b, the total activity of BCKD is still lower than
normal because the complex has not been reformed. Control
cells were harvested at different time points in individual
experiments following this protocol. In the example shown
here, control cells were harvested at the 96-h time point;
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Figure 4. Western blot and PCR of DBT gene products in HEK293 cells transfected with miR29a or miR29b. Antisera for BCKD complex was used to
probe mitochondrial proteins (4, 8 and 12 mg of mitochondrial protein at
each time point) after transfection with miR29a (A) and miR29b (B). (C) Products from RT–PCR analysis (28 cycles) of transfected cells for DBT mRNA
with b-actin as a loading control analyzed on a 2% agarose gel. The transfected miRNA and number of hours post-transfection when RNA harvest
occurred are indicated. Ratio of DBT to b-actin density traces is indicated
between the two products. The control value was done at different time
points and does not change. The control value reported here was done at
96 h for (A) and (B) and 48 h for (C) and contain only the non-specific
22mer RNA.
however, the amount of DBT protein in the control cells did
not change over the entire time course. Recall that control
cells always contain the random sequence for a 22mer RNA.
During this same time course, the amount of mRNA for
DBT did not change (Fig. 4C), indicating that mRNA was
not destroyed and suggests that translation of the DBT
mRNA was blocked when miR29b is bound to the mRNA.
To demonstrate that the observed loss of activity and
protein was not through a direct interaction of the miR with
the protein, a western blot for the dihydrolipoyl acyltransferase (DLAT) protein was done with the same membrane used
for western blots to assess DBT in these mitochondrial preparations. DLAT is the core protein of the pyruvate dehydrogenase complex that also functions as a multienzyme
complex in mitochondria. As with the BCKD complex, the
proteins forming the pyruvate dehydrogenase complex are
encoded by nuclear genes. DBT and DLAT are structurally
similar in amino acid sequence, but exhibit a high degree of
substrate specificity in catalytic function (32). Comparison
of the two nucleic acid sequences for the mRNA for DBT
and DLAT revealed no sequence identity. There is no
sequence pairing between the miR29a and miR29b for the
mRNA for DLAT as shown for the mRNA encoding DBT
(Fig. 1). The observed decrease in DBT but not in DLAT
(Fig. 5) supports the function of the miRs used here specifically affect translation of mRNA for DBT. As variation
between membranes prepared from individual experiments
will not allow for averaging of the data, results shown in
Figure 5 are from a single experiment representative of three
repeats of this protocol.
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Human Molecular Genetics, 2005, Vol. 14, No. 22
Figure 6. Endogenous expression of miR29a and miR29b in HEK293 cells.
Radiolabeled antisense RNA against miR29a and miR29b were used to hybridize the isolated cellular RNA from two different cell preparations. RNase
digestion was used to identify the double-stranded RNA products. Controls
contain antisense RNA probe and tRNA, but no cellular RNA during the
RNase treatment.
Figure 5. DBT protein in HEK293 cells relative to DLAT protein after cell
transfection with an miR29b. (A) Western blot for DBT and DLAT in mitochondria after transfection with miR29b at the indicated times after transfection. Control cells were taken after 96 h. (B) Calculations of the concentration
of DBT to DLAT for the time course.
Endogenous expression of miR29a and miR29b
Although demonstration that exogenous addition of these
miRs decreases DBT synthesis and results in a reduced level
of BCKD function, the results do not address whether these
miRs exist and function endogenously within the HEK293
cells. To address this question, RNA was isolated under
conditions that preserve the small RNA from two independent
cultured cell preparations collected on separate days. The
isolated RNA was hybridized in separate reactions with a
radiolabeled antisense RNA against miR29a or miR29b. As
illustrated by the autoradiograph of the RNase-protected products, only miR29b was detected in each of the two separate
preparations of the HEK293 cells (Fig. 6). To begin assessing
a functional role for this miR on BCKD, antisense RNA with
20 -O-methyl oligoribonucleotides was used for transfection
followed by BCKD activity measurements and western blots
as earlier. The O-methyl modification has the ability to inactivate endogenously produced miR function in cultured human
cells (33). With either antisense RNA, a small change in DBT
protein concentration occurred (Fig. 7) that resulted in a small
increase in total BCKD activity (Fig. 8). This increase in total
BCKD activity was 20% above control cells that received only
the random sequence 22mer RNA. Although endogenous
miR29a is not detected, the similarity in nucleotide sequence
for the two antisense RNAs may result in hybridization to
block endogenous miR29b.
The miRNA Registry Web site assigns the human chromosome location for the mi29b-1 gene to 7q32.2 –32.3 and that
for miR29b-2 to 1q32.2. The hairpin loop pre-miR structure
is similar in nucleotide sequence for each gene product with
diversity only in the sequence forming the hairpin. Antisense
RNAs to the loop region were synthesized and used for
RNase protection assays to determine which gene site was producing the miR29b found in these cells. As seen in Figure 9,
only the miR29b-2 showed the predicted hybridization
product in the HEK293 and two other human cell types
implicating expression from the region on chromosome 1.
DISCUSSION
The speculation that Drosophila use miR expression in
response to dietary protein to control BCAA metabolism by
regulating expression of the BCKD complex raised the question of whether this mechanism for controlling the amount
of BCKD in a cell was retained in human cells (26). Numerous
human miRs exist that have as predicted targets mRNAs for
components of the BCKD complex along with a large
number of mRNAs for other proteins (17). The strongest predicted pairing occurs between members of the miR29 family
and the mRNA for DBT. DBT forms the core of the BCKD
complex and provides the binding site for all other proteins
in the complex including the BCkinase (34). Reducing DBT
will therefore reduce the amount of total BCKD, as all
the other subunits must bind to this core component. There
are no reports that describe procedures for formation of the
BCKD complex through subunit interactions.
Our results support predicted function of reduced translation
for an miR with pairing of only the first 7 –9 nucleotides in the
50 end with the targeted mRNA (17). The remaining nucleotides potentially pair further into the mRNA with formation
of loops in the mRNA. As HEK293 cells express BCkinase
to a level that maintains the activity state of BCKD around
50%, these cells provide a model where we can ask if
expression of DBT is reduced and the total activity of the
BCKD complex is decreased. Addition of in vitro synthesized
miR29a or miR29b to HEK293 cells separately or in combination resulted in a linear decrease in total BCKD activity.
If miR expression blocks mRNA translation, the amount of
protein found in the cells reflects a protein’s half-life. Little
is known about the half-life of components of the BCKD
complex, but one report gave a 24-h half-life for all components (30). As the miR was added exogenously, the halflife of the added miR could contribute to the length of time
for the observed change in proteins studied. No data is
available for these in vitro synthesized double-stranded short
RNAs (31).
Although there is no report for a direct interaction of an miR
with a protein rather than an mRNA, we addressed that by
investigating the effect of the miR29a and miR29b transfection on the expression of DLAT (35). Our findings support
the proposal that mRNA for DBT is the target of miR29b
and the resulting duplex blocks translation of this message.
Human Molecular Genetics, 2005, Vol. 14, No. 22
Figure 7. The use of O-methyl antisense RNA to block expression of
endogenous miR29a and miR29b. (A) Western blot for DBT and DLAT for
mitochondrial protein isolated at the indicated times after treatment with
O-methyl antiRNA for 29a and 29b. (B) Relative concentration of DBT
versus DLAT during this time course. The diamond is the control cells that
only have the non-specific fluorescence labeled 22mer RNA. Squares are for
antisense RNA 29a and triangles are for antisense RNA 29b.
Results with the exogenously added miR29a and miR29b do
not address whether HEK293 cells show in vivo expression of
these ncRNAs and when present if the miRs target the DBT
mRNA. RNase protection analysis demonstrated the endogenous presence of only miR29b in HEK293 cells. The
observed decrease in DBT does result in a decrease in total
BCKD activity because the amount of complex will decrease
without the core protein. It appears that other proteins of the
BCKD complex also are lost when DBT is decreased. This
is likely due to the fact that they bind to DBT and without
DBT for binding, other proteins are degraded. It now
becomes important to begin investigations to address factors
that control expression of the miR29b gene. When the cells
were transfected with antisense RNA against miR29a or
miR29b, a modest increase in total BCKD activity was
observed. The amount of increase is consistent with what we
reported when cells were transfected with a plasmid containing the cDNA for DBT that resulted in overexpression of
DBT (36). The amount of DBT protein produced in our previous paper was about 25% more than that in control cells
and total BCKD activity was 15% higher than that in
control cells (36). Increased expression of DBT did not
result in an increased expression of the other subunits and
therefore increase in total BCKD activity was minimal (36).
There are two human genes reported to encode miR29b
(http://www.sanger.ac.uk/Software/Rfam/mirna/). The gene
we expressed is that for miR29b-2 located on chromosome
1q32.2 and the human gene for DBT is also located on this
chromosome but on the opposite arm at 1p31 (27,37).
RNase protection analysis supports the finding that the gene
for miR29b-2 on chromosome 1 is being expressed in the
human kidney, glial and fibroblast cells (Fig. 9). The means
by which expression of the miR29b gene in these cells is
controlled needs to be investigated to better understand
3375
Figure 8. Effect of O-methyl antisense RNA for miR29a and miR29b on
BCKD total activity: (A) graphic representation of the activity; (B) absolute
values for each time point.
Figure 9. Detection of specific endogenous gene products for miR29b in three
different human cell lines. HEK293 cells (1 and 2) represent repeated analysis
of this cell type and DBTRG is a human glial cell line and C504 a human
dermal fibroblast cell line. Conditions are as described in Figure 6, with the
antisense RNA used here is for the unique sequence of the hairpin loops for
the gene encoding miR29b1 on chromosome 7 and the gene encoding
miR29b2 on chromosome 1. Controls are as described in Figure 6.
its observed function for blocking translation of the DBT
mRNA.
Our finding represents the first report demonstrating regulation of the amount of BCKD that is present within a cell
through the action of microRNAs. It is interesting to speculate
that as the BCkinase must associate with the DBT protein to
function efficiently, it may be necessary to regulate the
amount of DBT produced (34). The demonstration that
expression of microRNA can influence the amount of BCKD
presents another site for investigation of inherited defects
affecting BCKD function. Maple syrup urine disease
(MSUD) is a condition in which BCKD activity is impaired
by inherited mutations in the genes for the catalytic subunits
of BCKD. Some individuals with MSUD present with a less
severe clinical course and were missed in newborn screening
programs but present with elevations in plasma BCAAs later
in life (38). A molecular genetic basis for this intermittent
condition remains to be defined. Changes in expression of
the microRNAs that alter the amount of the BCKD present
in a cell offer a possible explanation for this condition. We
need to examine the tissue expression level of these and
other microRNAs in cell lines from the individuals presenting
with the intermittent MSUD.
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Human Molecular Genetics, 2005, Vol. 14, No. 22
MATERIALS AND METHODS
Human HEK293 cells were maintained in standard DMEM
media supplemented with 10% FBS and antibiotics at 378C
and 5% CO2-humidified atmosphere. Prior to transfection
with the miRs in Lipofectamine 2000, the cells were placed
in standard media without antibiotics for 24 h. The individual
miR was used at a concentration of 20 pmol/well for a 24-well
plate or 100 pmol/well for a 6-well plate. When miR29a and
miR29b were co-transfected, 10 pmol of each was used to
produce the 20 pmol total per well. Transfection with the
20 -O-methyl oligoribonucleotides was done at a concentration
of 10 pmol/well for the 24-well plates and 50 pmol/well for
the 6-well plates. To estimate transfection efficiency, the
Block-iT Fluorescent double-strand random 22mer RNA
from Invitrogen (Carlsbad, CA) was co-transfected at onehalf the concentration of the experimental miR. Control
populations of cells contained only this tagged 22mer RNA.
The FITC fluorescence was visualized by lex ¼ 494 nm and
lem ¼ 519 nm to assess the number of cells that were transfected. Cells receiving only the tagged random sequence
double-strand 22mer were used for non-specific reference at
all data points. Cells were harvested and analyzed at indicted
times post-transfection.
buffered saline (PBS) and resuspended in 1 ml mitochondrial
isolation buffer (MIB; 220 mM mannitol, 70 mM sucrose,
2 mM HEPES, pH 7.4 with KOH). Cells were homogenized
by douncing 25 times in a Wheaton Potter-Elvehjem tissue
grinder and nuclei were pelleted by centrifugation at
2000 g for 10 min at 48C. The mitochondria were pelleted
from the supernatant by centrifugation for 10 min at
15 000 g at 48C. Protein concentration in each sample was
determined using the BCA Protein Assay (Pierce Biotechnology, Rockford, IL).
For western blot experiments, identical amounts of mitochondrial proteins for each condition were resolved through
a 10% SDS –PAGE and then transferred to a nitrocellulose
membrane by tank transfer in transfer buffer (24.7 mM Tris,
192 mM glycine and 20% methanol) at room temperature
over 2 h. Equality of protein loading and transfer was determined by Ponceau-S staining of the membrane. Development
of the immunoblot with antisera against the BCKD complex
was done as previously described (36). Antisera against the
dihydrolipoyl acetyltransferase (DLAT) was purchased from
Santa Cruz Biotechnology, Inc. Visualization of the antibody
binding was with West Pico Chemiluminescent Substrate
detection protocol (Pierce). The chemiluminescent signal
was exposed to Blue BioFilm (Denville Scientific, Inc.,
Metuchen, NJ) for up to 5 min.
Synthesis of microRNA
Quantitative analysis of DBT mRNA
MicroRNAs tested in these transfection experiments were
synthesized using the Silencer siRNA Construction Kit from
Ambion (Austin, TX) according to the manufacturer’s
instructions. The primers used for miR29a were 50 -CTAGCA
CCATCTGAAATCGGTTCCTGTCTC-30 (forward) and 50 AAAACCGATTTCAGATGGTGCTCCTGTCTC-30 (reverse)
and for the 29b duplex 50 -TAGCACCATTTGAAATCAGTCC
TGTCTC-30 (forward) and 50 -AAACTGATTTCAAATGGTG
CCCTGTCTC-30 (reverse). Concentration of the final product
was estimated by absorption measurement at 260 nm after suspension in RNase-free water. Antisense oligoribonucleotides
with all but three core nucleotides bearing the 20 -O-methyl modification were purchased from Integrated DNA Technologies
(Coralville, IA).
Total RNA from transfected cells was isolated using TRI
Reagent (Sigma) according to the manufacturer’s protocol.
First-strand cDNA was synthesized using the SuperScript
First Strand Synthesis System (Invitrogen) with Oligo-dT
primers. PCR amplification of template cDNA was performed
using Platinum Taq DNA Polymerase (Invitrogen). Samples
were denatured at 948C for 2 min followed by 28 cycles of
denaturation at 948C for 30 s, annealing at 518C for 30 s and
extension at 728C for 30 s, with a 10-min final extension.
Primers used for amplification of DBT and b-actin were
purchased from Invitrogen and Integrated DNA Technologies,
respectively. Sequences were as follows. DBT: 50 -CTCT
CCGTGGACAGGTTGTT-30 (forward), 50 -GTCTCCCCACA
TAGGCAATA-30 (reverse); b-actin: 50 -GTGGCCGAGGA
(forward), 50 -AGTGGGGTGGCTTTT
CTTTGATTG-30
AGGATG-30 (reverse).
Cell culture
Whole cell BCKD assay
Whole cell BCKD activity state, expressed as percent activity,
was determined by measuring the 14CO2 released from
[1 2 14C]-leucine per mg of total cellular protein under basal
conditions relative to that produced after a 10-min preincubation with 1 mM a-chloroisocaproate, an inhibitor of
BCKD-kinase, as previously described (39). Total activity
values reported are an average + standard error (SE) of
three to four experiments per cell line, each performed in
triplicate.
Mitochondria isolation and western blot
Mitochondria were isolated from approximately 5 106 cells.
Cells were released from the culture plates with trypsin/
EDTA, centrifuged at 300 g, washed with phosphate-
RNase protection assay
Enriched small RNA from 107 non-transfected HEK293 cells
was purified using the miRVana miRNA Isolation Kit
(Ambion). Final concentration and purity of RNA was estimated
using absorbencies at 260 and 280 nm. For microRNA
detection, the miRvana miRNA Detection Kit (Ambion) was
used according to the manufacturer’s instructions. Antisense
RNA probes for miR-29a 50 -AAAACCGAUUUCAGAU
GGGUCUAGAAAACCAGAG-30 and for miR29b 50 -AAAC
UGAUUUCAAAUGGUGCUACCAGAG-30 and for hairpin
loop probes 29b-1 50 -CCCAAGAACACUGAUUUCAAAU
GGUGCUAGACCCAGAG-30 and for 29b-2 50 -AAAAA
were
GGUGCUAGAUACAAAGAUGGAAACCAGAG-30
obtained from Integrated DNA Technologies and end-labeled
Human Molecular Genetics, 2005, Vol. 14, No. 22
using g-[32P]-ATP. After hybridization and RNase treatment,
the double-strand products were resolved in a 15% polyacrylamide 8 M urea denaturing gel and visualized using
phosphoimaging and autoradiography.
Conflict of Interest statement. None declared.
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