After RNA extraction, 50 ng RNA was mixed in DEPC ddH2O to reach a total volume of 6 µL and added with 1 µL 10 mM dNTP and 6 µL 2 µM reverse primer mixture containing MF17, MF19, MF85, MF87, MF89 and MF91 dissolved in DEPC ddH2O. To anneal the primers, the RNA mixture underwent 65°C heating using thermocycler for 5 minutes before being cooled down on ice for 2 minutes. Next, reverse transcription (RT) reaction mixture which consists 4 µL 5X SSIV buffer, 1 µL 0.1 M DTT, 1 µL RNase OUT and 1 µL SuperScript IV Reverse Transcriptase (#18090050, Thermo Fisher Scientific) was premixed and added into the RNA mixture, making a total volume of 20 µL. RT reaction was conducted on Mastercycler 6325 Pro S (Eppendorf) at 60°C for 50 minutes and 80°C for 10 minutes to obtain cDNA.
After RT, cDNA was diluted from 2500 to 10 ng/µL by molecular ddH2O. Primers used for RT-qPCR are listed in Table 1. To quantify RNA levels of the indicated transcripts in whole cell extract or purified mitochondria, qPCR was conducted in triplicate for all samples by using Power SYBR Green Master Mix (#4367659, Thermo Fisher Scientific) on the StepOnePlusTM Real-Time PCR System (Applied Biosystems) as following.
Firstly, SYBR Green/primer mixture was premixed on ice according to the number of samples. Take the relative abundance of sgRNA over endogenous 5S rRNA in 10 samples for instance, a total volume of 399 µL SYBR Green/primer mixture was prepared in one 1.5 mL Eppendorf tube before being distributed as 37.5 µL aliquot for ten 1.5 mL Eppendorf tubes (samples) and as 13.5 µL aliquot for one 1.5 mL Eppendorf tube (no template control, NTC) (see Appendix 2). Next, 12.5 µL of 10 ng/µL indicated cDNA samples or 4.5 µL molecular water (for NTC) were loaded into individual tubes, respectively. The mixture was thoroughly vortexed at the lowest amplitude for three times (~1 second each) and spinned down. This vortex process was repeated for another two times before the mixture was aliquoted as 15 µL into optical tubes or 96-well for qPCR using low retention tips. After being loaded, sample was centrifuged at 300 x g under room temperature for 1 minute and put onto the StepOnePlusTM Real-Time machine for qPCR.
Figures and Tables
Figure 1. Comparison between current methods for mtDNA manipulation
Restriction enzymes were firstly introduced into the field of mtDNA manipulation. Despite its high efficiency on targeted mtDNA cleavage, restriction enzyme is hard to use due to its limitation on target selection. One the other hand, artificial DNA endonucleases, like ZFN and TALEN, involve a nonspecific restriction enzyme FokI and different combinations of DNA binding domains to enable specific cleavage on targeted mtDNA region. Despite the high specificity on DNA targeting, ZFN and TALEN take time to rearrange and recombine various domains when switching onto a different target. Compared to the other two methods, CRISPR-Cas9 gene editing technology is user-friendly because it depends on one single protein CRISPR-Cas9 and is able to cleave different mtDNA by simply changing its guide RNA.
Figure 2. Schematic design for mito-EGFP transfection
(A) To test our hypothesis whether we could send an MTS-tagged Cas9 RNP into mitochondria through electroporation, we used a relatively smaller MTS-tagged EGFP to simplify the condition. By electroporating either a plasmid form or a purified protein form of COX8A MTS-EGFP, we wanted to see which form of EGFP can localize to mitochondria after the transfection.
(B) Schematic flow: mito-EGFP is transiently expressed in HeLa cells 2 days after transfection with 1 µg plasmid or 500 µM purified protein through electroporation. MitoTracker Deep Red is used to stain mitochondria one day ahead of confocal microscopy imaging.
Figure 3. The intracellular localization of mito-EGFP
Mito-EGFP (COX8A MTS-EGFP) is transiently expressed in HeLa cells 2 days after nucleofection with 1 µg plasmid (upper row) or 500 µM purified protein (lower row) through electroporation. Mitochondria were marked by MitoTracker Deep Red (red, see panels A and D).
(B) The well-folded purified EGFP in the protein group spreads all over the cells at the indicated timing, showing no concentration effect even with the help of COX8A MTS. (C) Merged image shows protein form EGFP even localize into nucleus, where mitochondrial pseudogenes exist and may cause gene instability if a folded MTS-tagged Cas9 RNP directly enter nucleus. (E) The plasmid form of EGFP, after transcription and translation, can generate concentrated EGFP signal only within mitochondria. (F) Co-localization appears yellow on digitally merged image.
Figure 4. Co-translational mechanism for mitochondrial protein import
According to our data along with studies from other scientists, mitochondrial protein import mechanism is largely coupled with translation. Firstly, mRNA approaches the outer membrane of mitochondria through the help of its 3’ UTR, which would be recognized by proteins in charge of this process. Furthermore, once the ribosome is combined with the mRNA and starts translation, the polypeptide generated would be identified by TOM through the MTS signal on the N terminal of the polypeptide. The polypeptide is transported through TOM-TIM channel and finally undergoes refolding to form functional protein within the mitochondrial matrix.
Referring this theory, a well-folded Cas9 RNP cannot be used in this study. Cas9 must be transfected in either plasmid, viral vector, or mRNA form, and later transported to mitochondria coupling with translation. To be noticed, since polypeptide form of Cas9 can no longer bind guide RNA, we postulate that guide RNA needs to enter mitochondria separately through a different pathway.
Figure 5. Plasmid map for pSL127 series
A series of Cas9 harboring either without or with an MTS was generated through this study.
Triple repeats of FLAG motifs were cloned following the Cas9 sequence to enhance FLAG signal.
Figure 6. Schematic flow to determine localization of mito-Cas9s
After electroporation of the different versions of pSL127 series, we hope to see that Cas9 cannot enter mitochondria but Cas9 with MTS can show a concentration effect within mitochondria.
Figure 7. Common MTSs are not enough for mito-Cas9 to enter mitochondria
(A) Untreated HeLa cells serve as negative control. (B) Cas9 without or with different MTSs in HeLa cells 2 days after transfection was detected by anti-FLAG antibody (see panels B-H).
(B) Pure Cas9 spreads all over the cytosol and cannot enter mitochondria. (C) Cas9 with double-repeats of reported MTS from COX8A, 1-21 aa, shows no concentration effect within mitochondria, suggesting that COX8A MTS is not sufficient for Cas9. (D) Cas9 fused with a reported MTS from S. cerevisiae COX4P, 1-24 aa, cannot enter mitochondria, indicating this MTS is not suitable for Cas9. Blue signal, nuclei stained by DAPI. Red signal, mitochondria stained by MitoTracker Deep Red. Green signal, Cas9 determined by anti-FLAG antibody.
Figure 8. Candidate MTSs from large nuclear-encoded mitochondrial proteins on UniProt To search a suitable MTS for mitochondrial import of such a large Cas9, which is about 160 kDa, we found all the nuclear-encoded mitochondrial proteins on online protein database UniProt and prioritize them by their size. We chose four candidate MTS from the first four largest proteins with reported or predicted MTS. Each MTS was named by the gene its corresponding protein is encoded. To be noticed, each MTS is composed of various length of amino acids.
Figure 9. Most candidate MTSs from UniProt are not suitable for mito-Cas9
(A) Cas9 with an MTS from Acetyl-CoA carboxylase 2 (UniProt ID: O00763), 1-40 aa, shows no concentration effect within mitochondria, suggesting that this MTS is not sufficient for Cas9. (B) Cas9 fused with a predicted MTS from mitochondrial glycine dehydrogenase (UniProt ID: P23378), 1-35 aa, cannot enter mitochondria, indicating this MTS is not suitable for Cas9 as well. (C) Cas9 fused with an MTS from DNA polymerase subunit gamma-1 (UniProt ID: O00763), 1-38 aa, can neither enter mitochondria, showing this MTS is inappropriate for Cas9. Blue signal, nuclei stained by DAPI. Red signal, mitochondria stained by MitoTracker Deep Red. Green signal, Cas9 determined by anti-FLAG antibody.
Figure 10. MTHFD1L MTS is suitable for mito-Cas9
Cas9 fused with an MTS from mitochondrial monofunctional C1-tetrahydrofolate synthase (UniProt ID: Q6UB35), 1-38 aa, can localize into mitochondria, suggesting this MTS is able to bring a protein as large as Cas9 into mitochondria. (A) After transfection for 2 days, the nuclei were stained by DAPI, showing blue signal, and the mitochondria were stained by MitoTracker Deep Red, showing red signal. (B) mito-Cas9 was firstly located by primary anti-FLAG antibody and an Alexa Flour 488-conjugated secondary antibody, thereby showing green signal. (C-F) Merged images from different samples all indicate co-localization of mito-Cas9 and mitochondria, showing yellow signals.
Figure 11. PNPase as a putative RNA transporter
Polynucleotide phosphorylase (PNPase) was previously published to function as a RNA exonuclease and polymerase in both bacteria and plants. This multifunctional protein which had been found over 60 years ago was recently uncovered a new function to import several nuclear-encoded non-coding RNAs, including 5S rRNA, RNase P RNA, and RNase MRP RNA, into mitochondria. These non-coding RNAs are postulated to involve numeral crucial mitochondrial metabolisms like transcription and translation. Nevetheless, questions like how PNPase, which is mainly located at the mitochondrial intermembrane space, can facilitate RNA import from cytosol and whether there are other mitochondrial RNA import mechanisms existing are still under debate.
Figure 12. PNPase exists in HeLa mitochondria
Untreated HeLa cells were observed under confocal microscope with immunofluorescent signals to determine the localization of PNPase (A-C). (A) DAPI and MitoTracker Deep Red locate nuclei and mitochondria, respectively. (B) PNPase was firstly located by primary anti-PNPase antibody and emits fluorescent signal using Alexa Fluor 488-conjugated secondary antibody. (C) Merged image illustrates co-localization of PNPase with mitochondria, thus showing yellow signal.
Figure 13. Schematic flow for mitochondrial RNA extraction
(A) Simplified work flow demonstrates how total RNA and mitochondrial RNA were extracted from one HeLa sample. After sonication, a portion of whole cell lysate was collected and underwent TRIzol-mediated RNA recovery to generate total RNA. The rest of the cell lysate went through mitochondrial purification and RNase A/T1 treatment before adding TRIzol to ensure RNA purity. Both total RNA and mitochondrial RNA underwent RT-qPCR to analyze mitochondrial import rates of the three PNPase-related nuclear-encoded non-coding RNAs. (B) Cartoon illustration simplifies the process for mitochondrial purification. After sonication, the mitochondria were released and pulled down by anti-TOM22-antibody-conjugated magnetic beads. After discarding supernatant which contains cell debris and intact cells, the mitochondrial pellet was resuspended by storage buffer and treated with RNase A/T1 under room temperature
Figure 14. Calculation for mitochondrial translocation rates of PNPase-related RNAs To calculate the mitochondrial translocation ratio of endogenous nuclear-encoded RNAs, we adopted the concept that the ratio equals to the amount of a specific RNA within the purified mitochondria over its total amount within cells. To calculate the real value from qPCR results, the Ct value of target RNA was firstly normalized to its corresponding M16S rRNA Ct value in both total RNA and mitochondrial RNA fractions. Then, the delta-delta Ct value equals to the delta Ct of mitochondrial RNA group taken away the delta Ct of total RNA group. Finally, the translocation ratio equals to 2 to the power of minus delta-delta Ct value. For example, a real data set was written above to calculate the translocation ration of negative control 5.8S rRNA.
The result shows its ratio is 0.1%, indicating no import as expected.
Figure 15. PNPase-related nuclear-encoded RNAs exist in HeLa mitochondria
Total RNA and mitochondrial RNA were extracted from untreated HeLa cells according to the methods aforementioned. After reverse transcription with a mixture of specific reverse primers for M16S rRNA, 5.8S rRNA, 5S rRNA, RNase P RNA and RNase MRP RNA (see Table 1), qPCR was conducted to evaluate the translocation ratio of each RNA. According to our data, 5.8S rRNA, a nuclear-encode cytosolic ribosomal RNA, expresses the lowest import rate as expected, showing our RNase treatment was adequate to eliminate RNA contamination outside of purified mitochondria. All the three PNPase-related nuclear-encoded RNAs exist in our HeLa mitochondria.
Figure 16. Reported hairpin-shaped motifs for mitochondrial RNA transportation
All the hairpin-shaped RNA motifs used in this study have been experimentally proven that they have the ability to import RNA fragments into human mitochondria (A-E). (A) The hairpin structure from the gamma domain of 5S rRNA, 65-110 nt. (B) The hairpin structure form RNase P RNA, 1-20 nt. (C) The hairpin structure form RNase MRP RNA, 150-169 nt. (D) The double hairpin structure from F-form of yeast tRNALys. (E) The single hairpin structure from F-form of yeast tRNALys.
Figure 17. Hypothesis for PNPase-mediated mitochondrial import of mito-sgRNA
Studies have revealed that the hairpin-shaped motifs on PNPase-related non-coding RNAs, like 5S rRNA, RNase P RNA and RNase MRP RNA may be involved in the recognition of PNPase and their import into mitochondrial matrix. Without the motifs, studies have shown loss of expression of these RNAs within mitochondria. Hence, we collected all these hairpin-shaped RNA motifs and fused them onto our single guide RNAs to form a series of 15 mito-sgRNAs.
Theoretically, we hope that, with the help of these RNA motifs, our mito-sgRNAs can enter mitochondria through PNPase-mediated pathway or other unknown pathways.
Figure 18. Cartoon illustration of predicted mito-sgRNA structures
15 mito-sgRNAs generated through this study were in vitro transcribed and treated with CIP to remove the triphosphate on the 3’ end in order to reduce their toxicity toward cells. Mito-sgRNA36 is the original guide RNA targeting ND4 gene on mtDNA without any modification.
Since any modification on guide RNA may change its conformation and further influences its binding affinity with Cas9, we put a 10-nucleotide linker to extend the motif away from the guide RNA scaffold. Mito-sgRNA51 was added a 10-nucleotide linker. Mito-sgRNA52, 53, 56, 68 and 73 were generated by adding different hairpin-like RNA motifs in front of the 10-nucleotide linker of mito-sgRNA51. Mito-sgRNA69, 70, 71, 72 and 74 were generated through adding hairpin motifs onto the internal region of guide RNA scaffold. Mito-sgRNA54, 55 and 57 possess hairpin motifs on the 5’ end as well as a 3’ UTR sequence from mRNA, which have the ability to recruit cytosolic RNA to proximity of mitochondrial outer membrane.
Figure 19. Modifications on all mito-sgRNAs maintain Cas9 cleavage
(A) Simplified structures of mito-sgRNAs synthesized in this study. Modifications are marked with different colors. Brown indicates 10-nucleotide linker sequence. Red is putative RNA hairpin structures which assist the import of RNA through PNPase. Green is the 3’ untranslated region of mitochondrial ribosomal protein S12, which confer RNA localization to mitochondrial outer membrane. (B) Schematic demonstration of in vitro cleavage assay. The higher the Cas9 cleavage efficiency is, the more cleaved products are shown in the lower bands. (C) ND4 DNA subtract was incubated with purified Cas9 and respective in vitro transcribed mito-sgRNA at 37°C for 30 minutes. The products were separated through agarose electrophoresis. Mito-sgRNAs with modifications on the internal RNA scaffold show the best cleavage efficiency, as good as the nonmodified one and the one with only 10-nucleotide linker motif.
Figure 20. Schematic flow to determine timing for mito-sgRNA transfection
(A) mito-sgRNA51 served as representative for all the mito-sgRNA generated through this study and was transfected into cell through Lipofectamine 2000. After mixing mito-sgRNA51 with Lipofectamine 2000, small lipid micelles surrounding the mito-sgRNA form in Opti-MEM reagent. The mixture was added into the cell medium and engulfed by cells through either fusion or endocytosis. Once a mito-sgRNA reached cytosol, it would be further recruited into mitochondria according to the modification on it. (B) To determine the best timing when the most amount of mito-sgRNA accumulates within cytosol, we transfected mito-sgRNA51 with increasing time span from 3 to 12 hours. At the indicated timing, total RNA of each group was extracted through TRIzol-mediated RNA recovery and was analyzed by RT-qPCR. According to our data, the amount of mit-sgRNA51 increased as the transfection time was expanded from 3 to 9 hours, but started to decrease afterwards, indicating that incubation for 9 hours is the best timing for mito-sgRNA transfection.
Figure 21. Schematic flow for in vivo mito-sgRNA screening and calculation
(A) To determine the in vivo relative abundance of all the mito-sgRNAs generated throughout this study, we conducted a 9-hour transfection of individual mito-sgRNA before the mitochondrial purification and RNase A/T1 treatment. The RNA was extracted from purified mitochondria by TRIzol-mediated recovery and analyzed by RT-qPCR. (B) The concept for the relative abundance of individual mito-sgRNA is the amount of target mito-sgRNA within mitochondria over the amount of RNase P RNA within mitochondrial fraction. To calculate from the real data, the delta Ct value equals to the Ct value of targeted mito-sgRNA taken away the Ct value of RNase P RNA. The result of 2 to the power of minus delta Ct value will be the relative abundance of targeted mito-sgRNA over RNase P RNA in mitochondria.
Figure 22. Mito-sgRNA with only linker on 5’ end has the highest import efficiency
In vivo test for mitochondrial abundance of target mito-sgRNAs relative to 5S r RNA by reverse
transcription-qPCR of mitochondrial RNA extracts. The abundance of mito-sgRNA51, which has no modification but a short 10-nucleotide linker, is over one hundred times as high as that of endogenous 5S rRNA level within mitochondria. All the other mito-sgRNAs, which possess MTSs either at their 5’ ends, internal region, or both ends, is much less than mito-sgRNA51.
Figure 23. Schematic flow for expression of mito-Cas9 in vivo
Since the expression level of mito-Cas9 (MTHFD1L MTS-Cas9) was low after electroporation of corresponding plasmids, we used Lipofectamine 3000 in hope that we could enhance the transfection efficiency. In this part of experiment, the plasmid not only has MTHFD1L MTS in front of the Cas9 sequence, but was also fused with a signal-enhanced turboGFP sequence (Lonza). In order to test the in vivo influence of transfection with mito-Cas9, after 2 days of transfection for mito-Cas9 through Lipofectamine 3000, GFP-positive cells were sorted out and seeded onto culture dish for 1 day. Later, cellular and mitochondrial morphology were determined by confocal microscope imaging.
Figure 24. Impaired cellular physiology after transfection of Cas9-GFP
Cellular and mitochondrial morphology detected by immunofluorescence and confocal microscopy after transfection of plasmid which expresses either normal Cas9 or mito-Cas9 with MTHFD1L MTS. (A) The cells transfected with normal Cas9 showed typical phenotype of HeLa mitochondria structure. (B) Cas9 without MTS could only localize at the cytosol. (C) Merged image indicates no co-localization between Cas9 and mitochondria. (D) The cells transfected with mito-Cas9 (MTHFD1L MTS-Cas9) showed abnormal mitochondrial phenotype, suggesting that mitophagy was induced by overexpression of mito-Cas9 and GFP. (E) Mito-Cas9 showed no concentration effect within mitochondria, but spread all over the cytosol. (F) Merged image indicates no co-localization between mito-Cas9 and mitochondria.
Figure 25. Physiological anomaly occurs after transfection of mito-Cas9-GFP
Cellular and mitochondrial morphology detected by immunofluorescence under a larger confocal microscopy field after transfection of plasmid which expresses either normal Cas9 or mito-Cas9 with MTHFD1L MTS. (A-C) The majority of the cells transfected with normal Cas9 (pSL294) showed atypical phenotype of HeLa cells. Cells became smaller and apoptotic bubbles were even observed surrounding the cells. (D-F) Similarly, most of the cells transfected with mito-Cas9 (MTHFD1L MTS-Cas9) showed abnormal cellular phenotype, like spindle- or sphere-shaped outlooks, suggesting that mitophagy was induced by overexpression of mito-Cas9-GFP.
Figure 26. Plasmid map for pSL296
A plasmid harboring human PNPase sequence (PNPT1 gene), following CMV enhancer and chicken β-acting promoter, was generated through this study.
Figure 27. Plasmid map for pSL307 which has not been cloned yet
A plasmid harboring human PNPase sequence (PNPT1 gene), followed by self-cleavable peptide
A plasmid harboring human PNPase sequence (PNPT1 gene), followed by self-cleavable peptide