|
Branched chain α-ketoacid dehydrogenase (BCKD) is expressed in mitochondria of all human cells. The amount of BCKD within different tissue cells varies but always functions to commit leucine, isoleucine, and valine to catabolism. What makes the concentration of BCKD different in different tissue cells is not understood. Recent experiments in Drosophila demonstrate that expression of a microRNA (miR), which is controlled by the fly’s protein uptake, regulates the amount of BCKD produced. This study seeks to determine if humans have retained this mechanism of miR expression to control the amount of BCKD produced. The interest is based on the increasing understanding of how human tissues use miR expression to control protein formation by blocking messenger RNA (mRNA) translation without destroying the mRNA. Previous experiments in our laboratory reveal that the miR-29b-2 expression decreases the translation of the mRNA core protein for BCKD, known as dihydrolipoamide branched chain transactylase (DBT.) Current experiments investigate whether miR-192, which is implied to bind to the mRNA for DBT, also functions similarly to miR-29b-2. The two miRs potentially bind to different but neighboring regions of DBT mRNA. Results have demonstrated that miR-192 when present will decrease DBT protein in the cell along with a reduction in total BCKD activity. Additionally, experiments address whether both miR-29b-2 and miR-192 can work together to further reduce DBT expression. Genetic changes in BCKD expression are known to cause maple syrup urine disease in humans (MSUD,) making individuals sensitive to protein intake. If the expression of miRs influence BCKD amounts, mutation in the genes for the miRs may be contributors to the disease. Later studies will investigate the relationship of miR expression to this disease expression.
A form of small non-coding single-stranded RNA molecules called microRNAs (miR) (approximately 20-25 nucleotides long) are present in many different cell types and are expressed in several species ranging from the nematode C. Elegans to plants and humans. These short RNA molecules can regulate the expression of protein-coding genes through complementary binding to that protein’s messenger RNA (mRNA,) thereby suppressing mRNA translation. The first 7-9 nucleotides at the 5’-end of the miR most likely pair with bases in the 3’ UTR of the target mRNA. Perfect pairing of a miR with its target leads to target degradation, while imperfect pairing inhibits mRNA translation. Thus, microRNAs directly and specifically affect the levels of proteins in the cell, serving as an important post-transcriptional regulation mechanism.
MicroRNAs originate from within introns of protein-coding transcripts or exist as a product of their own gene. Cut by the RNase III enzyme Drosha in the nucleus, the primary transcript is processed to a 70-100 nucleotide product, which forms a hairpin structure that is transported to the cytosol. Upon entry, these hairpins are cut by an endonuclease known as DICER into a double stranded product of 20-25 base pairs known as precursor miRNA (pre-miRNA.) This dsRNA is taken up by the RNA induced silencing complex (RISC) and the dominant miR is retained to direct RISC to the mRNA target.
Branched chain amino acids (leucine, isoleucine, and valine) are typically catabolized within the cell to maintain a concentration that minimizes toxicity and produces energy. The mitochondrial multienzyme BCKD complex, composed of four protein products, normally catalyzes the second step of branched chain amino acid degradation. A mutation in any three of the genes encoding the complex (E1α, E1β, and DBT) causes the autosomal recessive disorder known as maple syrup urine disease (MSUD.)
We hypothesize that miRs serve as a control mechanism for BCKD activity through post-transcriptional gene silencing. Thus, miR activity may provide the basis for an intermittent form of MSUD, which may be characterized by increased expression of those genes that bind mRNA for subunits of BCKD. This study focuses on specific miRs (miR-192, miR-29b-1, and miR-29b-2) thought to regulate the amount of the BCKD complex within mitochondria. Previous studies have confirmed that miR-29b-1 and miR-29b-2 sequences bind the mRNA for the dihydrolipoamide branched chain transacylase (DBT) component of the BCKD complex and function in its silencing. However, miR-192 has been suggested as an additional regulator of DBT. Our experiments address miR-192’s function and its effects on BCKD levels when combined with miR-29b.
Figure 1: The reactions of BCKD. The branched-chain α-ketoacid and its metabolites are in red. E1-α, E1-β, 2 tetramers, DBT subunits, and E3 homodimers are present in the functional BCKD complex. The α-keto acids derived from Leu, Ile, Val are oxidatively decarboxylated to form CoA derivatives.
Figure 2: Nucleotide sequence for human miR-192 and predicted base pairing with the mRNA for DBT. The paired region in DBT covers nucleotides 1576-1668 that lies within the noncoding region of terminal exon 11.
A. Antisense RNA probes for miR-29b-1 and 29b-2 were end labeled with χ-[32P]-ATP. Following hybridization and RNase digestion, the double-stranded products were resolved on a 15% denaturing polyacrylamide gel and visualized using autoradiography.
B. An antisense RNA probe for miR-192 was in-vitro transcribed and radiolabeled by incorporation of α-[32P]-UTP. HEK 293 cells were grown in standard DMEM media supplemented with 10% fetal bovine serum [FBS] and antibiotics. Glioblastoma (DBTRG) cells were maintained in RPMI-1640 medium supplemented with 10% FBS, antibiotics, sodium pyruvate, and non-essential amino acids. Fibroblast (C504) cells were grown in DMEM media supplemented with 20% FBS. All cells were grown in a 5% CO2 humidified atmosphere at 37°C.
Figure 4: Western blot performed on 4 µg and 8 µg total mitochondrial protein from HEK 293 cells transfected with miR-192. Probed with antisera for DBT (top) and for DLAT, a component of pyruvate dehydrogenase complex (bottom). Proteins were resolved through a 10% SDS-PAGE and transferred to a nitrocellulose membrane by tank transfer in transfer buffer over 2 hours. Mitochondria harvested from 5 x 106 cells/well of a 6-well plate.
Figure 5: BCKD Activity in HEK 293 cells transfected with siRNA of miR-192. Whole cell BCKD activity state was determined by measuring pmol 14CO2 released from [1-14C]-leucine per mg of total cellular protein in 3 hours after a 10 minute pre-incubation with α-chloroisocaproate (an inhibitor of BCKD kinase). Cells were transfected with dsRNA using Lipofectamine 2000 and plated on 6 and 24-well plates containing 5 x 105 and 1 x 105 cells, respectively, in standard growth media without antibiotics. 50 pmol and 100 pmol concentrations of miR-192 were transfected in 6-well plates; 10 pmol and 20 pmol in 24-well plates. A BCKD Assay was conducted from cells grown on 24 well plates: α-chloroisocaproate was added to all wells, excluding triplicate filter blanks, followed by [1-14C]-leucine hot mix. Each well sealed was with caps containing glass fiber filters. After 3 hr incubation, filters sat in scintillation fluid overnight prior to counting.
Figure 6: BCKD Activity in HEK 293 cells transfected with siRNA of miR-192, 29b, combined 192 and 29b, and O-Methyl 192. Antisense RNA labeled with 2’-O-methyl nucleotides for miR-192 were used to block endogenous expression of the microRNAs. Cells were assayed for BCKD activity 48 hr post transfection.
Figure 7: Reverse-Transcriptase PCR. Products from RT-PCR analysis of transfected cells were amplified for DBT mRNA with β-Actin as a loading control. Following 30 cycles with a 51°C annealing temperature, products were analyzed on a 2% Agarose gel. PCR amplification of template cDNA performed using access RT-PCR system from Promega with 1 μg RNA for each condition. Total RNA harvest occurred 48 hours post transfection. Cells lysed with Trizol reagent, extracted with chloroform, and precipitated with isopropyl alcohol.
• RNase protection analysis showed the endogenous presence of miR-29b-2 and miR-192 within HEK 293, DBTRG, and C504 cells. MiR-29b-1 was visible in DBTRG and C504 cells.
• Transfection of in vitro synthesized miR-192 into HEK 293 cells resulted in a decrease in total BCKD activity as demonstrated by a decrease in DBT protein and 14CO2 release.
• Addition of antisense O-methyl RNA against miR-192 led to a small increase in total BCKD activity.
• The amount of mRNA for DBT in 293 cells did not change with the addition of miR, implying that miR blocks DBT expression at the translational level rather than via degradation.
• MiR-192 is expressed in HEK 293, DBTRG, and C504 cell lines.
• Human cell lines have miRs with predicted sequence homology to mRNA encoding subunits of BCKD. MiR-192 targets DBT mRNA of the BCKD complex, demonstrating that control of BCKD expression occurs by microRNAs in HEK 293 cells.
• MiR-192 regulates DBT levels by blocking mRNA translation rather than by target degradation.
• Future studies will investigate whether over expression of miRNA due to mutations in the 5’-UTR leads to an intermittent form of MSUD.
Special thanks to the SURE Program for providing this research opportunity.
Cell culture, snRNA transfections, Protein assays, Mitochondria harvesting, RNA isolation, Western Blots, RNase Protection Assay, RT-PCR
human genetics, branched-chain amino acid catabolism, microRNA, maple-syrup urine disease, BCKD complex in mitochondria
|
