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SCIENCE CHINA Life Sciences, Volume 63 , Issue 10 : 1429-1449(2020) https://doi.org/10.1007/s11427-020-1631-9

Identification of mecciRNAs and their roles in the mitochondrial entry of proteins

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  • ReceivedJan 13, 2020
  • AcceptedJan 19, 2020
  • PublishedJan 21, 2020

Abstract


Funded by

grants to G. S.: the National Key R&D Program of China(2019YFA0802600,2018YFC1004500)

the National Natural Science Foundation of China(31725016,31930019,91940303)

and the Strategic Priority Research Program(Pilot,Study)


Acknowledgment

We thank Lei Liu for technical support of C. elegans experiments. This work was supported by grants to G. S.: the National Key R&D Program of China (2019YFA0802600 and 2018YFC1004500), the National Natural Science Foundation of China (31725016, 31930019, and 91940303), and the Strategic Priority Research Program (Pilot Study) “Biological basis of aging and therapeutic strategies” of the Chinese Academy of Sciences (XDPB10).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Features of mecciRNAs, related to Figure 1.

Figure S2 Experimental verification of mecciRNAs, related to Figure 1.

Figure S3 Analyses of related NGS data, related to Figure 1.

Figure S4 Mitochondrial and cytosolic distributions of mecciRNAs, related to Figure 1.

Figure S5 mecciND1 interacts with RPA70 and RPA32, related to Figure 2.

Figure S6 mecciND5 interacts with hnRNPA proteins, related to Figure 2.

Figure S7 Correlations between mecciND1 and mitochondrial RPA levels, related to Figure 3.

Figure S8 Correlations between mecciND5 and mitochondrial hnRNPA levels, related to Figure 4.

Figure S9 mecciND1 and mecciND5 copy number per cell and in vitro assays, related to Figure 5.

Figure S10 Western blots of proteins in the RNA-IP (for Figure 7A) and knockdown and overexpression of PNPASE, related to Figure 5.

Figure S11 Secondary structure of mecciND1 and mecciND5, related to Figure 5.

Figure S12 mecciND1 levels under stress, mecciND5 levels in HCC, mecciRNAs in S. pombe and C. elegans. Related to Figure 6.

Table S1 List of mecciRNAs from RNA-seq data

Table S2 Mass spectrometry results of mecciND1 and mecciND1 pull-down proteins

Table S3 Oligos used in this study

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Identification and characterization of mecciRNAs. A, Experimental procedures for mitochondrial RNA sequencing to identify mecciRNAs; Western blots showing the quality of the purified mitochondria are presented; ERp70, ER marker; Histone H3, nuclear marker; ACTIN, cytosolic marker; NDUFB8, a mitochondrial inner membrane protein. B, g-circRNAs and mecciRNAs (reads ≥2) identified from the sequencing data of mitochondrial (mito) RNAs in human and murine cells and tissues; HCC, hepatocellular carcinoma. C, Junction motif of human, mouse and zebrafish (adult male) mecciRNAs. D, Northern blots of mecciRNAs. RNase R-treated (+) and untreated (–) total RNAs from HeLa cells were examined; the position of the probe is indicated; actin mRNA served as a linear control. E, Confocal images in single z-sections of TOM20 immunofluorescence (IF) together with FISH of mecciND1 or mecciND5. Boxed areas are enlarged. The colocalization between mecciND1 or mecciND5 (G, green) and TOM20 (R, red) is shown (n=20 randomly selected areas). R/G, proportion of red signal to green signal colocalization; G/R, proportion of green signal to red signal colocalization. Scale bar, 5 µm and 500 nm (enlarged areas) in E; error bars represent standard error of the mean in B; **P<0.01; ***P<0.001 by two-tailed Mann-Whitney U test.

  • Figure 2

    mecciND1 interacts with RPA70 & RPA32, and mecciND5 interacts with hnRNPA proteins. A, Pulldown of MecciND1 with biotin-labelled antisense oligos (AS oligos) in HeLa cells. Proteins that were co-pulled down with mecciND1 were subjected to silver staining; red triangles indicate the bands identified as RPA70 and RPA32 by mass spectrometry. Scra, oligos with scrambled sequences. B, RPA70 and RPA32 co-pulled down with mecciND1 were verified by Western blotting; ACTIN, negative control. C, RNA immunoprecipitation (RIP) against RPA70 (α-RPA70) with whole-cell HeLa cell lysates. Successful IP of the protein was detected by Western blotting; GAPDH, negative control. RNAs from RIP were quantified by real-time qPCR; 18S rRNA, negative control. D, RNA immunoprecipitation (RIP) against RPA32 (α-RPA32) with whole-cell HeLa cell lysates. Successful IP of the protein was detected by Western blotting; GAPDH, negative control. RNAs from RIP were quantified by real-time qPCR; 18S rRNA, negative control. E, Representative structured illumination microscopy images (N-SIM) from single z-sections of RPA32 immunofluorescence (IF) together with TOM40 as well as FISH of mecciND1 in fixed HeLa cells. Boxed areas are enlarged; scale bar, 2 µm and 200 nm (enlarged areas). F, Pulldown of mecciND5 with biotin-labelled antisense oligo in HeLa cells (experimental details were slightly different from pulldown of mecciND1 shown in A, see Methods). Proteins that were co-pulled down with mecciND5 were subjected to silver staining; red triangles indicate the bands identified as hnRNPA1, hnRNPA2B1 and hnRNPA3 by mass spectrometry. Scra, oligos with scrambled sequences. G, hnRNPA1, hnRNPA2B1, and hnRNPA3 co-pulled down with mecciND5 were verified by Western blotting; ACTIN, negative control. H, RNA immunoprecipitation (RIP) against hnRNPA1 (α-hnRNPA1) with whole-cell lysate of HeLa cells. Successful IP of the protein was detected by Western blotting; ACTIN, negative control. RNAs from RIP were quantified by real-time qPCR; Actin mRNA, negative control. I, Representative structured illumination microscopy (N-SIM) images in single z-sections of hnRNPA1 and TOM40 immunofluorescence (IF) as well as FISH of mecciND5 in fixed HeLa cells. Scale bar, 2 µm and 200 nm (boxed areas are enlarged). In C, D, and H, the error bars represent standard error of the mean; n=3 independent experiments; **P<0.01, ***P<0.001, Student’s t-test.

  • Figure 3

    mecciND1 regulates mitochondrial RPA levels. A, Mitochondrial RPA70 and RPA32 protein levels were decreased after knockdown of mecciND1 via siRNA in 293T cells. Western blots of proteins from whole cells (total level) and mitochondria (mito level) were examined; si-NC, siRNA with scrambled sequences; NDUFB8 served as a loading control for mitochondrial protein levels; ERp70, an endoplasmic reticulum marker, served as a loading control for whole-cell protein levels; Histone H3, a nuclear marker. The results of the quantification of RPA proteins are shown. B, Mitochondrial RPA70 and RPA32 protein levels were increased under mecciND1 overexpression in 293T cells. C, Representative structured illumination microscopy images in z-stacks (3D N-SIM) of RPA32 and TOM40 immunofluorescence (IF) as well as FISH of mecciND1 in fixed RPE-1 cells transfected with siRNA (si-NC or si-mecciND1). Plot profiles of representative images of TOM40 and RPA32 IF signals as well as FISH of mecciND1 are shown for si-NC group. Images of single z-sections of the boxed areas are enlarged. Scale bar, 2 µm and 200 nm (enlarged areas). D, The results of quantification of the fluorescence signals of mecciND1 and RPA32 via N-SIM are shown; areas representing single mitochondria were selected, and fluorescence signals overlapping the TOM40 signal or inside the TOM40-enclosed space were quantified; n=30 randomly chosen areas. In A, B, and D, error bars represent standard error of the mean; in A and B, n=3 independent experiments; ns, not significant; *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test.

  • Figure 4

    mecciND5 regulates mitochondrial hnRNPA levels. A, Mitochondrial hnRNPA1, hnRNPA2B1, and hnRNPA3 protein levels were decreased after knockdown of mecciND5 with siRNAs in 293T cells. Western blots of proteins from whole cells (total level) and mitochondria (mito level) are shown; si-NC, siRNA with scrambled sequences; NDUFB8 served as a loading control for mitochondrial protein levels; ERp70, an endoplasmic reticulum marker, served as a loading control for whole-cell protein levels; Histone H3, a nuclear marker. The results of the quantification of hnRNPA proteins are shown. B, Mitochondrial hnRNPA1, hnRNPA2B1, and hnRNPA3 protein levels were increased under mecciND5 overexpression in 293T cells. C, Representative structured illumination microscopy images in z-stacks (3D N-SIM) of hnRNPA1 and TOM40 immunofluorescence (IF) as well as FISH of mecciND5 in fixed RPE-1 cells transfected with siRNA (si-NC or si-mecciND5).Plot profiles of representative images of TOM40 and hnRNPA1 IF signals as well as FISH of mecciND5 are shown for si-NC group. Single z-section images of boxed areas are enlarged. Scale bar, 2 µm and 200 nm (enlarged areas). D, The results of the quantification of the fluorescence signals of mecciND5 and hnRNPA1 via N-SIM are shown; areas representing single mitochondria were selected, and fluorescence signals that overlapped with the TOM40 signal or were inside the TOM40-enclosed space were quantified; n=39 randomly chosen areas.In A, B, and D, error bars represent standard error of the mean; in A and B, n=3 independent experiments; ns, not significant; *P<0.05; **P<0.01; ***P<0.001, Student’s t-test.

  • Figure 5

    mecciRNAs promote mitochondrial importation in in vitro assays and interact with known components of mitochondrial importation. A, Semi-quantitative RT-PCR of in vitro RNA import assays. The linear RNA and the counterpart mecciRNA share the same sequences. circSRSF, a g-circRNA control; RMRP, a nuclear-encoded linear RNA that can be imported into mitochondria, positive control; 16S rRNA, mitochondrion-encoded rRNA, loading control and a marker for mitochondrial integrity. B, mecciND1 added with RPA32 mRNA in the rabbit reticulocyte translational system (co-translation) and the mitochondrial importation of the in vitro-translated RPA32-FLAG protein. Upper, translation products of RPA32-FLAG protein (Input); lower, RPA32-FLAG protein imported into mitochondria. C, mecciND5 added together with hnRNPA1 mRNA in the rabbit reticulocyte translational system (co-translation) and the mitochondrial importation of in vitro-translated hnRNPA1-FLAG protein. Upper, translation products of hnRNPA1-FLAG protein (Input); lower, hnRNPA1-FLAG protein imported into mitochondria. D, Mitochondrial importation of the in vitro-translated RPA32-FLAG protein with mecciND1 added post-translationally. E, Mitochondrial importation of the in vitro-translated hnRNPA1-FLAG protein, mecciND5 added post-translationally. For B–E, circSRSF, a g-circRNA control; TOM40, mitochondrial outer membrane protein, a loading control; the relative importation efficacy of the protein was calculated. F, RNA immunoprecipitation (RIP) against FLAG-tagged TOM40, TOM20 and PNPASE with lysates of transfected 293T cells. The levels of mecciRNA enrichment were examined by real-time qPCR, and the fold changes (log2FC) compared to the IgG control were then converted into a heatmap. G, Mitochondrial mecciRNA levels from mitochondrial RNA-seq data after knockdown of PNPASE. Error bars, standard error of the mean (SEM); ***P<0.001 by the two-tailed Mann-Whitney U test. H, Mitochondrial RPA protein levels and hnRNPA1 protein levels under overexpression of the PNPASE protein in 293T cells; Vec, vector control; PNP, PNPASE overexpression. TIM23, a mitochondrial inner membrane protein, loading control.I, RNA immunoprecipitation (RIP) against FLAG-tagged PNPASE with a cell lysate (293T cells) co-transfected with wild-type (wt) or stem-loop-mutated (mut) mecciND1/mecciND5. For B–E and H, the results of the quantification of protein levels were normalized to mitochondrial loading controls (TOM40 or TIM23), and the relative levels were the ratios to the corresponding control. n=3 independent experiments; data are the mean±SEM; ns, not significant; *P<0.05; **P<0.01; Student’s t-test. In I, error bars, SEM; ns, not significant; **P<0.01; ***P<0.001 by Student’s t-test.

  • Figure 6

    Dynamic expression of mecciND1 and its association with cellular physiology. A, MecciND1 levels and mitochondrial RPA70 and RPA32 protein levels were increased upon UV irradiation in RPE-1 cells. The results of the quantification of RPA proteins are shown (NDUFB8 served as a loading control for mitochondrial proteins; ERp70, an endoplasmic reticulum marker, served as a loading control for whole-cell proteins, and with no UV group as 1.0). B, mecciND1 levels and mitochondrial RPA70 and RPA32 protein levels were increased after H2O2 treatment in RPE-1 cells. The results of the quantification of RPA proteins are shown (NDUFB8 served as a loading control for mitochondrial proteins; ERp70, an endoplasmic reticulum marker, served as a loading control for whole-cell proteins, and with H2O group as 1.0). C, Representative electron microscopy images of immunogold-labelled RPA32 in human RPE-1 cells treated with H2O2 (H2O served as a control). In the H2O control, RPA32-immunogold particles in the mitochondrion are labelled with red arrowheads. M, mitochondrion. D, Knockdown of RPA70 or RPA32 decreased mitochondrial DNA (mtDNA) copy numbers. The knockdown efficiency of RPA70 and RPA32 at the mRNA level is also shown; sh-control, shRNA with scrambled sequence. E, Copy numbers of mtDNA under mecciND1 knockdown or overexpression. F,mecciND1 levels in pairs of tumour samples and adjacent tissues from 21 hepatocellular carcinoma (HCC) patients. In A, B, D, and F, relative RNA levels determined via qRT-PCR were normalized to 18S rRNA; in A, B, D, and E, n=3 independent experiments; in A, B, and D–F, error bars represent standard error of the mean; ns, not significant; *P<0.05; **P<0.01; ***P<0.001 by Student’s t-test.

  • Figure 7

    A working model of mecciRNAs in facilitating mitochondrial protein importation. MecciRNAs (e.g., mecciND1 and mecciND5) interact with nascent polypeptides in the cytosol, and then serve as molecular chaperones to promote mitochondrial importation of proteins through the TOM40 complex (e.g., RPA and hnRNPA proteins). PNPASE interacts with the small stem-loop of mecciND1 and mecciND5, and regulates the distribution of mecciRNAs in the mitochondria and cytosol.

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