RNA processing in human mitochondria

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Jun 24, 2011 - Manam S, Van Tuyle GC. Separation and characteriza- tion of 5'- and 3'-tRNA processing nucleases from rat liver mitochondria. J Biol Chem ...

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Cell Cycle 10:17, 2904-2916; September 1, 2011; © 2011 Landes Bioscience

RNA processing in human mitochondria Maria I.G. Lopez Sanchez,1 Tim R. Mercer,2 Stefan M.K. Davies,1 Anne-Marie J. Shearwood,1 Karoline K.A. Nygård,1 Tara R. Richman,1 John S. Mattick,2 Oliver Rackham1 and Aleksandra Filipovska1,* Western Australian Institute for Medical Research and Centre for Medical Research; The University of Western Australia; Perth, WA Australia; 2 Institute for Molecular Bioscience; The University of Queensland; Brisbane, QLD Australia

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Key words: RNA processing, tRNA, transcriptomics, mitochondria

Mammalian mitochondrial DNA is transcribed as precursor polycistronic transcripts containing 13 mRNAs, 2 rRNAs, punctuated by 22 tRNAs. The mechanisms involved in the excision of mitochondrial tRNAs from these polycistronic transcripts have remained largely unknown. We have investigated the roles of ELAC2, mitochondrial RNase P proteins 1 and 3, and pentatricopeptide repeat domain protein 1 in the processing of mitochondrial polycistronic transcripts. We used a deep sequencing approach to characterize the 5' and 3' ends of processed mitochondrial transcripts and provide a detailed map of mitochondrial tRNA processing sites affected by these proteins. We show that MRPP1 and MRPP3 process the 5' ends of tRNAs and the 5' unconventional, non tRNA containing site of the CO1 transcript. By contrast, we find that ELAC2 and PTCD1 affect the 3' end processing of tRNAs. Finally, we found that MRPP1 is essential for transcript processing, RNA modification, translation and mitochondrial respiration.

Introduction

necessary and sufficient to give this complex an activity apparently identical to other members of the RNase P family. Interestingly, two of the subunits are proteins with additional functions and exist as a stable subcomplex, suggesting that recruitment into a mitochondrial RNase P complex represents an additional role for these proteins. MRPP1 is thought to be a m1G9 methyltransferase responsible for a common methylation modification of tRNAs, and may be involved in the tRNA binding capacity required for the RNase P complex, but these functions have not been shown experimentally to date. MRPP2 is a member of the short-chain dehydrogenase/reductase family,11 and has a NAD + -binding domain that may be required for RNase P activity. MRPP2 does not contain RNA-binding domains and is predominantly cytoplasmic and consequently not exclusively mitochondrial.7 MRPP3 is a pentatricopeptide repeat (PPR) containing protein, of previously unknown function that contains a putative metallonuclease domain. All three MRPP subunits appear to be required for RNase P activity at the 5' end of three mitochondrial tRNAs, with combinations of only 1 or 2 of the subunits resulting in accumulation of some tRNA precursors.7 However, it is not clear if RNase P is responsible for the 5' processing of all mitochondrial tRNAs. An additional RNA-containing RNase P has been reported, which may function in mitochondria with an alternative or complementary processing specificity.12 Recently we have shown that manipulating the expression of another PPR domain protein, pentatricopeptide repeat domain protein 1 (PTCD1), alters the abundance of some tRNAs.13 ELAC2 is thought to be the mitochondrial RNase Z due to its mitochondrial localization14,15 and RNase Z activity in vitro.8 Although the ELAC2 gene is a strong candidate for prostate cancer susceptibility,15 its

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The mitochondrial genome is a compact, circular, doublestranded genome encoding only 13 proteins that are all subunits of the electron transport chain, as well as 2 rRNAs and 22 tRNAs required for their translation.1,2 Mitochondrial genes for proteins and tRNAs are located on both the heavy and light strands of the genome, which are transcribed as large polycistronic transcripts covering almost the entire length of each strand.3-5 A third transcript covering the start of the heavy strand and the two rRNA genes is also produced.6 The punctuation model, described in 1981, proposes that genes encoding protein or rRNA interspersed by one or more tRNAs can act as “punctuation” marks for processing.1 This processing is thought to involve cleavage at the 5' end of tRNAs by RNase P,7 and cleavage of the 3' end by the mitochondrial RNase Z, which has been thought to be ELAC2.8 A CCA triplet is added to the 3' ends of tRNAs and specific bases within both tRNAs and rRNAs are often modified, while mRNAs are generally polyadenylated at their 3' ends.9 Although previously not investigated it has been proposed that regulation of the processing of mitochondrial tRNAs might have profound effects on mitochondrial gene expression, affecting the levels of mature species, the final processing of the different RNAs, and the overall level of translation. RNase P has recently been identified as a mitochondria targeted RNase composed of three protein subunits, mitochondrial RNase P protein 1 (MRPP1), MRPP2 and MRPP3.7,10 The mitochondrial RNase P differs from previously described RNase P enzymes, as it lacks an RNA component typically required for catalytic function. The three protein subunits of the RNase P are

*Correspondence to: Aleksandra Filipovska; Email: [email protected] Submitted: 06/24/11; Accepted: 06/27/11 DOI: 10.4161/cc.10.17.17060 2904

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sequencing combined with functional analysis to generate a detailed map of mitochondrial tRNA processing sites that provides new insights into the regulation, processing and expression of mitochondrial RNAs. Results ELAC2, MRPP1, MRPP3 and PTCD1 are mitochondrial proteins that regulate the processing of mitochondrial RNAs. All four proteins ELAC2, MRPP1, MRPP3 and PTCD1 have a predicted mitochondrial targeting signal (MTS) at their N termini suggesting they are all targeted to mitochondria. To investigate their localization in HeLa cells we fused the C-terminus of the ELAC2, MRPP1, MRPP3 and PTCD1 proteins to EYFP and co-localized them with Mitotracker Orange to show that they are all mitochondrial proteins (Fig. 1). Recently it was shown that ELAC2 can also localize to the nucleus when the endogenous translation initiation context is preserved;14,16 however, when an optimised translation initiation site is used, as we have done in our study, ELAC2 is localized to mitochondria (Fig. 1).14 We next determined the contribution of these proteins to the regulation of mitochondrial RNA processing by measuring the abundance of mitochondrial precursor transcripts. To achieve this, we performed qRT-PCR spanning the mitochondrial RNA junctions between each coding gene (Fig. 2A) in HeLa cells where ELAC2, MRPP1, MRPP3 or PTCD1 were knocked down to 5.6% ± 0.3, 1.8% ± 0.8, 1.8% ± 0.7 and 9.7% ± 1.7, respectively, by siRNAs compared with cells treated with non-targeting (NT) siRNAs as a control. In addition, we determined the effects of ELAC2, MRPP1, MRPP3 and PTCD1 by qRT-PCR compared with an EYFP control in cells transfected with plasmids expressing each of these proteins. For all four proteins, we observed striking differences in the abundance of mitochondrial precursor transcripts (Fig. 2B–E). Reduction of ELAC2 in cells resulted in the significant accumulation of several different junctions, particularly the ND1-ND2, ND2-tRNA Ala, tRNACys-COI, COI-COII and ND4-ND5 transcripts (Fig. 2B). Knockdown of MRPP1 caused the most significant increase in almost all mitochondrial precursor transcripts (Fig. 2C). Reduction in MRPP3 caused accumulation of several similar precursor junctions as those observed for MRPP1. However, MRPP1 knockdown caused the accumulation of junctions that were not affected by MRPP3 knockdown, such as ND2-tRNA Ala, tRNACys-COI, ND3-ND4L and ND6-Cyt b, suggesting that the roles of these two proteins may be different or in some instances MRPP1 and MRPP3 may act independently of each other (Fig. 2D). Although MRPP1 and MRPP3 exist in the same RNase P complex7 and their mRNAs were reduced significantly, the observed effects may also reflect potential differences in the stability of these two proteins. When we reduced the level of PTCD1 in cells, we observed a unique distribution of changes compared with the other three proteins thought to be involved in 5' and 3' mitochondrial tRNA processing (Fig. 2E). Decreasing PTCD1 abundance resulted in a moderate increase of most junctions, the most significantly abundant of which were the ND4–5 and tRNA Ala-COI transcripts. In contrast, increasing PTCD1 abundance reduced most

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Figure 1. ELAC2, MRPP1, MRPP3 and PTCD1 are mitochondrial proteins with RNA-binding or modifying domains as shown in their gene structures. HeLa cells transfected with ELAC2, MRPP1, MRPP3 or PTCD1 fused to EYFP at their C-termini were incubated with 100 nM Mitotracker Orange and fixed. The EYFP fusion proteins (green) were co-localized to mitochondria stained with Mitotracker Orange (red) directly by fluorescence microscopy and in the overlaid images yellow indicates their co-localization with mitochondria. Scale bar is 5 μm.

protein function or role as a mitochondrial RNase Z had not been investigated previously. Contemporaneously with our presented work, an additional study reported that ELAC2 was responsible for processing the 3' ends of mitochondrial tRNAs.16 Given that these four proteins, ELAC2, MRPP1, MRPP3 and PTCD1, all contain RNA-binding or modifying protein domains, we investigated their roles in mitochondrial tRNA processing using deep

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Figure 2. Change in the level of ELAC2, MRPP1, MRPP3 or PTCD1 affects the processing of precursor RNAs. RNA isolated from cells transfected with ELAC2, MRPP1, MRPP3 or PTCD1 siRNA, non-targeting (NT) siRNA, plasmids that overexpress ELAC2, MRPP1, MRPP3 or PTCD1, or a plasmid that overexpresses EYFP (pEYFP) were reverse transcribed and mitochondrial precursor RNA transcripts were analyzed by qRT-PCR. The locations of primers are indicated with arrows in (A). The data are expressed as a ratio of transcripts from experimental samples compared with control samples. The effects of ELAC2 (B), MRPP1 (C), MRPP3 (D) or PTCD1 (E) knockdown and overexpression on precursor mitochondrial transcripts are shown. Data are means ± SEM of four separate biological experiments.

of the precursor transcripts and the same precursor ND4–5 and tRNA Ala-COI transcripts were the most affected (Fig. 2E). We have observed similar effects following PTCD1 knockdown and overexpression previously in 143B cells for the ND4–5 transcript,13 with some additional changes that may reflect differences in the activities of the processing proteins between these cell lines. The overexpression of ELAC2, MRPP1 and MRPP3 resulted either in no change or moderate decrease of most precursor transcripts (Fig. 2B–E). Interestingly, changes in the abundance of all four proteins did not affect the junction levels of

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the two transcripts ATP8/6 and ND4L/4, further confirming that they are bi-cistronic transcripts, which are not processed into individual mRNAs. The qRT-PCR data showed that reduction of all four proteins results in the accumulation of unprocessed polycistronic transcripts, further confirming their role in mitochondrial RNA processing. To determine the sites of action of each protein in tRNA processing, we investigated 5' and 3' cleavage events by RNA sequencing on RNA isolated from purified mitochondria of cells where ELAC2, MRPP1, MRPP3 or PTCD1 were decreased

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by siRNA as described above. In total, 5.6 million reads uniquely aligned to the mitochondrial genome. In addition, we also noted a large number of reads (56%) corresponding to co-purifying nuclear encoded transcripts, mostly cytoplasmic rRNA, that were used to normalize between libraries. We determined the effect of these proteins by exploiting the distinctly enriched accumulation of the 5' termini of sequenced reads at mitochondrial tRNA 5' processing sites (Fig. 3A). We confirmed that the 5' ends of mitochondrial tRNA are processed at their previously annotated sites (Fig. 3A).1,17 To distinguish the processing of 5' termini from unprocessed precursor transcripts, we derived the ratio of 5' end reads to total mitochondrial tRNA expression, observing a significant decrease in this 5' ratio in response to MRPP1 (p = 0.0006, paired t-test) or MRPP3 (p = 0.0196) knockdown, confirming the involvement of these proteins in 5' tRNA cleavage (Fig. 3B). In contrast, we did not observe a significant response for ELAC2 (p = 0.4277) or PTCD1 (p = 0.172) knockdown. Next, we identified sequenced reads containing non-template 3'-CCA trinucleotide additions as evidence of mature 3' tRNA processing events, which occurred at their canonical sites (Fig. 3C). We derived the ratio of processing events to total tRNA expression to discern the contribution of the four proteins to 3' tRNA processing. We observed a significant depletion in 3' processed reads when ELAC2 (p = 0.0581, paired t-test) or PTCD1 (p = 0.0171) were knocked down in cells (Fig. 3D). Conversely, we did not observe a significant response for 3' processed reads when MRPP1 (p = 0.706) or MRPP3 (p = 0.3723) were knocked down. These data confirm that MRPP1 and MRPP3 process the 5' ends of mitochondrial tRNAs, and provide the first experimental evidence to confirm that ELAC2 is responsible for 3' processing of mitochondrial tRNAs as well as evidence for an unanticipated role for PTCD1 in 3' tRNA cleavage. Although we observe that MRPP1 and MRPP3 are largely responsible for 5' processing (Fig. 3E) and ELAC2 and PTCD1 contribute to tRNA 3' processing (Fig. 3F), we do note instances where MRPP1 and MRPP3 affect processed 3' end accumulation and ELAC2 and PTCD1 affect processed 5' end accumulation. This suggests a potential context-dependent contribution for each protein possibly depending on whether the processed tRNA is adjacent to other tRNAs or mRNAs. Furthermore, MRPP1 and MRPP3 affect some 3' tRNA processing, which may be an indirect effect of reduced 5' processing. The observation that 5' processing of mitochondrial tRNAs can precede 3' processing has been shown for mitochondrial tRNATyr in an in vitro study.18 Nevertheless, these data show a strong correlation between the increased abundance of unprocessed mitochondrial transcripts and the decreased number of correctly processed 5' and 3' ends of mitochondrial tRNAs when each of the four proteins are knocked down in cells, confirming their importance to mitochondrial RNA processing. ELAC2 and PTCD1 associate in cells. The association between MRPP1 and MRPP3 in the RNase P complex is necessary for the 5' processing activity of this enzyme.7 As ELAC2 and PTCD1 had similar effects on 3' tRNA processing, we next investigated if these proteins associate with each other within cells. We fused the C-terminus of ELAC2 or PTCD1 to a tandem

affinity purification (TAP) tag.19 We used the TAP tag to isolate the ELAC2-TAP or PTCD1-TAP protein from cells and used immunoblotting to determine if each of these proteins was associated with the endogenous PTCD1 or ELAC2 proteins, respectively. When ELAC2 was isolated from cells using its TAP tag we confirmed by immunoblotting an association with endogenous PTCD1 (Fig. 3G). We also found reciprocally that ELAC2 was enriched when PTCD1 was isolated from cells using its TAP tag (Fig. 3G), but we did not observe enrichment of these proteins when we isolated EYFP-TAP as a control (data not shown). Like MRPP1 and MRPP3, this association may underscore their shared roles in processing of 3' tRNA ends. The effect of ELAC2, MRPP1, MRPP3 and PTCD1 knockdown and overexpression on mitochondrial tRNA abundance. We next considered the effect of protein knock down on mitochondrial tRNA abundance. In our deep sequencing data, we found that most of the tRNAs were reduced when each of the four proteins was knocked down, further illustrating the involvement of these proteins in tRNA metabolism (Fig. 4A and B). Northern blotting showed that decreasing ELAC2 levels lowered the levels of several mitochondrial tRNAs including, tRNATrp, tRNA Ala and tRNAGlu but not all tRNAs, such as tRNA Asp and tRNA His (Fig. 4C). The decrease in these tRNAs correlated well with an increase in the junctions containing these tRNAs when ELAC2 was knocked down in cells that we investigated by qRT-PCR (Fig. 2A). The levels of mature mRNAs, bicistronic mRNAs and rRNAs were not significantly reduced when ELAC2 was knocked down or overexpressed (Fig. 4C). By contrast, we observed a dramatic loss of most mitochondrial tRNAs including tRNATrp, tRNA Ala, tRNA Asp, tRNA His and tRNAGlu when MRPP1 was reduced in HeLa cells (Fig. 4D), which correlated with the increase in the junctions containing these tRNAs observed previously (Fig. 2B). Similarly we observed a significant, but not as striking decrease of the same tRNAs investigated when MRPP3 was knocked down. The different roles and efficiency of these proteins in the cleavage of individual tRNAs, observed in our deep sequencing data (Fig. 4B), may control the overall abundance of functional tRNAs in mitochondria. Unlike ELAC2 knockdown, diminished MRPP1 and MRPP3 levels resulted in the dramatic reduction of mature and bicistronic mRNAs and a minor decrease in the rRNAs (Fig. 4D), suggesting these proteins affect tRNA processing with a subsequent impact on mature transcript production. Finally, we observed that PTCD1 reduction can cause either a subtle increase or decrease in the abundance of different tRNAs, while mRNA and rRNA levels were unaffected (Fig. 4D). In additon, we found that overexpression of PTCD1 in HeLa cells reduced ND5 mRNA (Fig. 4B and D) that we have not previously observed in 143B cells.13 The overexpression of MRPP1, MRPP3 and ELAC2 did not cause significant changes to mature mitochondrial transcripts and tRNA levels suggesting the physiological abundance of these proteins is not rate-limiting for mitochondrial RNA processing in HeLa cells. Impaired processing of mitochondrial transcripts leads to decreased protein synthesis. Changes to mitochondrial tRNA levels have been shown to affect translation of

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Figure 3 (See opposite page). MRPP1, MRPP3, ELAC2 and PTCD1 mediate the processing of 5' and 3' ends of mitochondrial tRNAs. (A) Fraction of reads that align to the canonical 5' ends of mitochondrial tRNAs. (B) Level of 5' reads relative to total tRNA expression. Data are shown as a box and whisker plot, showing the distribution of reads within each knockdown. The horizontal line in the box represents the mean, the top and bottom of the box represent the lower and upper quartiles, respectively, and the top and bottom of the whiskers represent the minimum and maximum values in the data set. (C) Fraction of reads that align to the canonical 3' ends of mitochondrial tRNAs. (D) Level of 3' reads relative to total tRNA expression, shown as a box and whisker plot. Fold change in 5' (E) and 3' (F) end reads relative to control for individual mitochondrial tRNAs as a result of MRPP1, MRPP3, ELAC2 or PTCD1 knockdown. (G) ELAC2 or PTCD1 were isolated from HeLa cells expressing ELAC2-TAP or PTCD1-TAP using IgG agarose followed by TEV protease cleavage. The endogenous proteins associated with the TAP tagged proteins were detected using αELAC2 and αPTCD1 antibodies.

mitochondrial-encoded genes.20 We investigated the effect on de novo mitochondrial protein synthesis by pulse (Fig. 5A–C, left hand parts) or chase (Fig. 5A–C, right hand parts) incorporation of 35S-labeled methionine and cysteine in mitochondrial proteins upon changes in ELAC2, MRPP1, MRPP3 or PTCD1 abundance. We observed an overall decrease of mitochondrial protein synthesis upon MRPP1 knock down, relative to non-targeted siRNAs (Fig. 5A–C), suggesting that a decrease in MRPP1 that causes the accumulation of unprocessed mitochondrial transcripts and a consequent decrease in mature RNAs results in significantly decreased mitochondrial translation. In contrast, we did not observe any significant changes to mitochondrial protein synthesis upon ELAC2, MRPP3 or PTCD1 knock down, nor when all four proteins were overexpressed (Fig. 5A–C). Although mitochondrial RNAs were lowered upon MRPP3 knockdown, this did not significantly affect protein synthesis. This may be because MRPP3 knockdown did not affect RNA abundance as dramatically as MRPP1 knockdown. The dramatic loss of mitochondrial protein synthesis upon MRPP1 knockdown suggests that there may be additional functions of this protein in mitochondrial gene expression. Decrease in MRPP1 causes ribosomal protein instability. The loss of core mitochondrial small ribosomal subunit proteins can cause a loss or reduction of other small ribosomal subunit proteins.21 We investigated if impaired mitochondrial RNA processing leads to mitochondrial ribosome instability by measuring the abundance of marker proteins for the small and large mitochondrial ribosomal subunits (MRPS15 and MRPL11) after ELAC2, MRPP1, MRPP3 or PTCD1 knockdown and compared them to control siRNAs after 3 and 6 days, respectively. We found the levels of these proteins were unaffected by the reduction of ELAC2, MRPP3 and PTCD1. However a decrease in MRPP1 levels results in a decrease in the abundance of ribosomal proteins that is more pronounced following a 6-day knockdown (Fig. 5D) indicating that ribosome stability is affected by MRPP1 levels. This is likely a consequence of the inability to process mitochondrial rRNAs, which cannot subsequently assemble into ribosomes and thereby affect translation and the stability of the ribosomes. Therefore mitochondrial RNA processing is intimately linked to translation via the provision of processed rRNAs that are integral components of mitochondrial ribosomes. Functional effects of knockdown and overexpression. We next considered whether the observed changes to mitochondriaencoded protein levels affected respiratory complex activity. Complex IV activity was measured by respiration of digitoninpermeabilized cells on ascorbate/TMPD. Decreased MRPP1 levels resulted in reduced mitochondrial respiration (Fig. 5E and F). By contrast, mitochondrial oxygen consumption increased when

PTCD1 was knocked down, confirming our previous findings with 143B cells.13 Mitochondrial respiration was unaffected by the changes in ELAC2 and MRPP3 abundance (Fig. 5E and G). MRPP1 mediates tRNA modification. During our analysis of sequenced reads corresponding to mitochondrial tRNAs, we noted an average 19.3-fold increase in the sequencing error rate at known modified nucleotides (mean 21.2%) compared with nonmodified nucleotides (mean 1.1%). By using this sequencing error rate as a proxy for the presence of nucleotide modifications,22,23 we next analyzed the contributions of MRPP1, MRPP3, ELAC2 and PTCD1 to tRNA nucleotide modifications. Given that MRPP1 has a conserved guanine methyl-transferase domain, we determined whether it may modify the guanine at position 9 of mitochondrial tRNAs in addition to its role as a subunit of RNase P (Fig. 6A). We noted a significant sequencing error rate deviation (p < 0.001) for both guanine and adenine methylated residues at this site in numerous tRNAs in cells where MRPP1 was knocked down, but not when the other three proteins were knocked down and show an example selection of these tRNAs (Fig. 6B–F). The modification of guanine at position 9 in the tRNA Leu(UUR) has been observed before, but the enzyme responsible was not known.24 Together, this approach to identify modified nucleotides in mitochondrial transcripts suggests a role for MRPP1 in guanosine and adenine modification of tRNAs, supporting the existence of a functional guanine methyl-transferase domain within MRPP1. The methyl-transferase role of MRPP1 has previously only been speculated and here we have found evidence for its activity in cells. Processing of non conventional mitochondrial precursor transcripts. Contrary to the tRNA punctuation model, the mitochondrial transciptome contains four non-canonical cleavage sites that are not adjacent to tRNAs. It is currently unclear whether these non-canonical sites are processed by a mechanism shared with tRNAs or subject to an alternative processing pathway. To investigate if any of the four proteins were responsible for the processing of these non-canonical cleavage sites, we performed strand specific qRT-PCR across junctions in cells where ELAC2, MRPP1, MRPP3 or PTCD1 were knocked down (Fig. 7A). We first validated this approach by analyzing the processing of a 5' site of tRNAVal, confirming our RNA sequencing results and previous studies in reference 7, which indicated this site to be a target for RNase P (Fig. 7A). We then examined the processing of the 3' end tRNA Leu(UUR) as our next generation sequencing data indicated that this site is affected by the RNase P proteins, ELAC2 and PTCD1. Strand specific qRT-PCR confirmed that knockdown of the ELAC2, MRPP1 and MRPP3 proteins caused an accumulation of this unprocessed RNA (Fig. 7A), suggesting that 5' processing of this tRNA affects its subsequent 3' cleavage

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Figure 3. For figure legend, see page 2908.

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Figure 4. Decrease in ELAC2, MRPP1, MRPP3 or PTCD1 lowers the abundance of mitochondrial transcripts. (A) Fold change of total mitochondrial mature tRNAs relative to control. (B) Fold change of individual mitochondrial tRNAs relative to control. (C and D) RNA isolated from cells transfected with ELAC2, MRPP1, MRPP3 or PTCD1 siRNA, non-targeting (NT) siRNA, plasmids that overexpress ELAC2, MRPP1, MRPP3 or PTCD1, or a plasmid that overexpresses EYFP (pEYFP) was analyzed for tRNA, mRNA and rRNA abundance by Northern blotting. The data are typical of results repeated on three separate RNA preparations. Each Northern blot was probed for all tRNAs by stripping the blot and re-probing.

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Figure 5. MRPP1 affects mitochondrial protein synthesis, ribosome assembly and mitochondrial respiration. HeLa cells were transfected for 3 d with ELAC2, MRPP1, MRPP3 or PTCD1 siRNA, non-targeting (NT) siRNA as control, plasmids that overexpress ELAC2, MRPP1, MRPP3 or PTCD1, or a plasmid that overexpresses EYFP as control. The protein synthesis of cytoplasmic proteins was inhibited with emetine for pulse analyses (A–C, left hand parts) or with anisomycin for chase analyses (A–C, right hand parts), and mitochondrial protein synthesis was analyzed by incorporating 35S-labeled methionine and cysteine in the 13 mitochondrial proteins synthesized by the mitochondrial ribosomes. Equal amounts of cell lysate protein (20 μg) were separated on 12.5% SDS polyacrylamide gels and visualized on film. Data are representative of five independent experiments with the same outcome. (D) HeLa cells were transfected with siRNAs targeting ELAC2, MRPP1, MRPP3 or PTCD1 and non-targeted siRNAs as controls for 3 and 6 d, mitochondria were isolated and the abundance of protein markers for the mitochondrial small and large ribosomal subunit were detected by immunoblotting using antibodies for the MRSP15 and MRLP11 proteins. The abundance of nuclear encoded subunit NDUFA9 of Complex I was used as a control. (E–G) State 4 respiration on ascorbate/TMPD was measured in digitonin-permeabilized HeLa cells transfected with ELAC2, MRPP1, MRPP3 or PTCD1 siRNA, non-targeting (NT) siRNA as control, plasmids that overexpress ELAC2, MRPP1, MRPP3 or PTCD1, or a plasmid that overexpresses EYFP as control using an OROBOROS oxygen electrode. Data are means ± SEM of four separate experiments; *p < 0.05 compared with all treatments by a two-tailed paired Student’s t-test.

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or that it may be a target for all of these proteins. We found that the 5' end of the CO1 mRNA was processed by RNaseP, because MRPP1 and MRPP3 knockdown resulted in several fold enrichment of this junction (Fig. 7A). This confirms the observation that MRPP1 is required for cleavage of the 5' end of CO1,16 likely because the adjacent sequence, which encodes a tRNA on the opposite strand, may fold to form a tRNA like structure that can act as a substrate for RNase P. The remaining three non-conventional junctions were not significantly affected by the knockdown of the four proteins (Fig. 7A). We validated these findings using rapid amplification of cDNA ends (RACE) and confirmed that MRPP1 is responsible for processing the 5' end of the CO1 mRNA (Fig. 7B). Interestingly none of the four proteins were responsible for processing of the junctions between the ATPase8/6 and CO3 mRNAs, the ND5 and Cyt b mRNAs and the 3' end of the ND6 mRNA (Fig. 7B), supporting the existence of an independent pathway by which these junctions are processed. Discussion The processing of tRNAs that punctuate mitochondrial polycistronic precursor transcripts is essential for the release and maturation of mitochondrial mRNAs and rRNAs.25 Here we investigated the role of four mitochondrial proteins in mitochondrial RNA processing with the aid of an RNA sequencing approach that specifically investigated the 5' ends and 3' CCA additions of mitochondrial tRNAs. We found that MRPP1 and MRPP3 are responsible for 5' processing of all mitochondria encoded tRNAs. Furthermore we identified ELAC2 as the human RNase Z responsible for 3' end processing of mitochondrial tRNAs in association with the PPR protein PTCD1. Taken together our data confirm the primary proteins responsible for mitochondrial 5' and 3' tRNA processing, a critical step in mitochondrial gene expression. Our data also supports previous findings that MRPP1 is responsible for 5' processing of mitochondrial tRNAs.7,16 The newly identified RNase P protein complex is not related to the well characterized RNA-containing nuclear and bacterial RNase P enzymes, and consequently little is known about its mechanism of tRNA substrate recognition. For instance it is not known whether the methyltransferase domain of MRPP1 is responsible for tRNA recognition and if its methylase activity, which we have identified using deep sequencing, is required for subsequent processing. This is of particular importance, since methylation of the mitochondrial tRNA Lys is necessary for the correct folding of this tRNA.26 Misfolding of tRNAs in the absence of methylation at position 9 may result not only in aberrant tRNA processing, but also impaired aminoacylation or transfer to the ribosome, which may explain the pleiotropic effects of MRPP1 knockdown that causes accumulation of precursor transcripts and decreased protein synthesis. Unlike RNase P, ELAC2 encodes a single protein that, given its homology to other RNase Z enzymes, in vitro RNase Z activity,8 mitochondrial localization and the discovery of its role in the 3' end processing of tRNAs, here and by Brzezniak et al. indicate its

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Figure 6. MRPP1 affects guanine and adenine methylation of mitochondrial tRNAs. (A–F) The role of the tRNA processing enzymes in tRNA nucleotide modification at position 9 was determined by analyzing increased sequencing error rate deviation in each library and are shown for representative tRNAs. Sequences obtained for tRNALeu(UUR) are shown as an example in (A).

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Figure 7. Non-canonical role of the MRPP1 protein. The effects of ELAC2, MRPP1, MRPP3 or PTCD1 knockdown in HeLa cells were analyzed for the unconventional junctions of mitochondrial transcripts by strand specific qRT-PCR (A) and by rapid amplification of cDNA ends (B).

function as a mitochondrial RNase Z. Future studies may indicate other enzymes that have RNase Z activity that may not share structural similarities with the ELAC family proteins. Whether PTCD1 represents such an enzyme is unclear. Previously we have observed that PTCD1 is a negative regulator of leucine tRNAs13 and here we have observed that PTCD1 affects 3' processing of mitochondrial tRNAs, although it is still not clear how these two effects are related. The PTCD1 protein sequence consists predominantly of PPR domains and does not contain any regions with predicted nuclease activity. Many PPR domain proteins in plants are thought to exert their effects by binding to specific RNA sequences in order to stabilize particular RNA structures.27 Whether PTCD1 can regulate the stability or facilitate the cleavage of tRNAs by ELAC2, by making favorable RNA substrates available, remains an attractive model for future testing. The levels of functional tRNAs available for protein synthesis could provide an important mechanism to regulate mitochondrial

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translation. We have shown that modulating the levels of different tRNA processing proteins can dramatically alter the levels of mature tRNAs, revealing a level of important physiological control. Interestingly, because of the organization of tRNA genes in the mitochondrial genome, the effect on some tRNAs could be quite different to others, depending on whether they are flanked by other tRNAs or mRNAs and rRNAs. For instance it has been proposed that the 5' ends of tRNAs with 5' flanking tRNAs can be processed independently of RNase P,28 perhaps by RNase Z acting at the 3' end of the flanking tRNA.29 Furthermore our data indicate that tRNA processing is not entirely predictable with our current understanding of human mitochondrial tRNA recognition by RNase P, ELAC2 and PTCD1. Although it has been suggested that cleavage by RNase P may precede that of RNase Z in some cases,18 it will be important to investigate the exact hierarchy of mitochondrial RNA processing by these two enzymes. This is particularly important, since knockdown of the

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components of RNase P, but not ELAC2 and PTCD1, led to loss of mature mRNAs and decreased rRNA levels, ribosome instability, decreased mitochondrial protein synthesis and respiration. There are four exceptions to the “tRNA punctuation” processing model where there are no intervening tRNA genes that separate the mRNA coding regions. One of these occurs between the ND2 and the CO1 mRNAs and we and Brzezniak et al. have identified that this site is processed by RNase P. Recently we have shown that the strand complementary to this site, which encodes five tRNAs, has a strong secondary structure,17 and this may be recognized by RNaseP at the 5' end. The remaining unconventional junctions were not processed by the four proteins investigated here, indicating that there are alternative enzymes that process these sites. Recently it has been shown that disrupted expression of another PPR protein, PTCD2, decreased the abundance of the Cyt b and ND5 mRNAs and increased the levels of the pre-processed ND5-Cyt b transcript.30 This suggests that PTCD2 may be a potential candidate protein that has a role in the processing of the non tRNA containing 5' end of the Cyt b mRNA. It remains to be determined which enzymes are responsible for the processing of the 5' end of CO3, to release it from the ATP8/6 bi-cistronic mRNA, and the 3' end of ND6. This work has provided insight into the role of four mitochondrial proteins in processing of human mitochondrial tRNAs and developed new deep sequence analyses that provide a valuable resource and methodologies for the future study of mitochondrial processing. The levels of nuclear tRNAs are regulated according to the cell cycle and in response to apoptotic stimuli.31-34 It will be interesting to determine if altered mitochondrial RNA processing and consequently changes in mitochondrial tRNA levels occur during cell growth, development and in response to different stimuli. This is of particular importance given that many mutations in mitochondrial tRNAs affect their processing and can play a crucial role in the pathophysiology of mitochondrial diseases.

antibody to detect the TAP tag of ELAC2, MRPP1, MRPP3 and PTCD1. Fluorescence cell microscopy. HeLa cells were plated onto 13 mm diameter glass coverslips and allowed to attach overnight. Cells were transfected with pELAC2-EYFP, pMRPP1EYFP, pMRPP3-EYFP and pPTCD1-EYFP for 48 h, and at the end of the incubation treated with 100 nM Mitotracker Orange for 15 min. The cells were fixed with 4% paraformaldehyde in PBS for 30 min and washed with PBS. Cells were mounted in DABCO/PVA medium and images were acquired using a Nikon Ellipse Ti fluorescent inverted microscope using a Nikon 60x objective. Mitochondrial isolation. Mitochondria were prepared from 5 x 106 cells grown overnight in 15 cm2 dishes. Cells in PBS were sedimented (150 g for 5 min at 4°C), resuspended in 4 ml of ice-cold 10 mM NaCl, 1.5 mM MgCl2 and 10 mM TRIS-HCl, pH 7.5. Cells were allowed to swell for 5 min on ice and briefly homogenized with a 7 ml glass homogenizer. The sucrose concentration was then adjusted to 250 mM by adding 2 M sucrose in 10 mM TRIS-HCl and 1 mM EDTA, pH 7.6 (T10E20 buffer). The nuclei from this suspension were sedimented (1,300 g for 3 min at 4°C), and the centrifugation step was repeated for the supernatant once more. Mitochondria were sedimented from the supernatant by centrifugation (15,000 g for 15 min at 4°C). The mitochondrial suspension was layered on a discontinuous sucrose gradient (1.0 and 1.7 M) in T10E20 buffer and centrifuged at 70,000 g for 40 min at 4°C. The mitochondrial fraction was recovered from the interface between the two sucrose cushions, diluted with 400 μl of 250 mM sucrose in T10E20 buffer and washed once in the same solution. The final mitochondrial pellet was resuspended in 100 μl of 250 mM sucrose in T10E20 buffer and protein concentration as determined by the bicinchoninic acid (BCA) assay35 using bovine serum albumin (BSA) as a standard. RNA harvesting from purified mitochondria. Total RNA was harvested from HeLa cells or isolated mitochondria using a miRNeasy RNA extraction kit according to manufacturer’s instructions (Qiagen). Contaminating genomic DNA was eliminated by an on-column RNase-free DNase treatment. RNA purity and integrity were confirmed by BioAnalyser (Agilent, CA). Quantitative RT-PCR. The transcript abundance of mitochondrial genes and pre-processed junctions was measured on RNA isolated from HeLa cells using the miRNeasy RNA extraction kit (Qiagen). cDNA was prepared using ThermoScript reverse transcriptase (Invitrogen) with random hexamers and used as a template in the subsequent PCR that was performed using a Corbett Rotorgene 3000 using Platinum UDG SYBR Green mastermix (Invitrogen), normalized to 18S rRNA. Control reactions were performed by omitting the reverse transcriptase in the cDNA synthesis; qRT-PCR using this material as a template revealed no detectable amplification. Strand-specific qRT-PCR. To quantify the levels of unprocessed junctions from a specific strand of mitochondrial RNA, total cellular RNA was denatured at 65°C for 5 min and reverse transcribed using SuperScript III (Invitrogen) at 55°C for 1 h in the presence of a gene-specific primer that incorporates additional

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Materials and Methods Cell culture. HeLa human cervical cancer cells were cultured at 37°C under humidified 95% air/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing glucose (4.5 gl-1), 1 mM pyruvate, 2 mM glutamine, penicillin (100 Uml-1), streptomycin sulfate (100 μgml-1) and 10% fetal bovine serum (FBS). Transfections. HeLa cells were plated at 60% confluence in 6-well plates or 10 cm dishes and transfected with annealed siRNAs or mammalian expression plasmids in OptiMEM media (Invitrogen). 125 nM (for 6-well plates) or 145 nM (for 10 cm dishes) of ELAC2, MRPP1, MRPP3, PTCD1 or control, off-target siRNAs (Dharmacon) were transfected using Lipofectamine 2000 (Invitrogen). 158 ng/cm2 of ELAC2, MRPP1, MRPP3, PTCD1 or control EYFP plasmid DNA was transfected using Fugene HD (Roche). Cell incubations were performed for up to 72 h following transfection. Protein expression was detected by immunoblotting using polyclonal ELAC2 and PTCD1 antibodies or using a secondary HRP

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adaptor sequence. The reverse transcriptase was inactivated by incubation at 70°C for 15 min. qRT-PCR was performed with a gene specific primer designed to anneal at the adjacent side of the junction of interest and another primer complementary to the additional adaptor sequence incorporated in the primer used for reverse transcription. Rapid amplification of cDNA ends (RACE). We polyadenylated the 3' ends of RNAs isolated from HeLa cells where MRPP1, MRPP3, ELAC2 or PTCD1 were knocked down, with Escherichia coli poly(A) polymerase, ligated RNA adaptors to the RNA 5' ends and reverse transcribed it with an oligo d(T). Rapid amplification of cDNA ends (RACE) was then performed to determine whether these proteins acted at the labeled 5 or 3' ends of unconventional junctions between mitochondrial genes that were not separated by tRNA genes. Northern blotting. RNA (20 μg) was resolved on 1.2% agarose formaldehyde gels, then transferred to 0.45 μm Hybond-N+ nitrocellulose membrane (GE Lifesciences) and hybridized with biotinylated oligonucleotide probes specific to mitochondrial tRNAs. The hybridizations were performed overnight at 50°C in 5x SSC, 20 mM Na 2HPO4, 7% SDS and 100 μgml-1 heparin, followed by washing. The signal was detected using a streptavidin-linked horse radish peroxidase (diluted 1:2,000 in 3x SSC, 5% SDS, 25 mM Na 2HPO4, pH 7.5) by enhanced chemiluminescence (GE Lifesciences). RNA sequencing. RNA was isolated from highly purified, RNase A-treated mitochondria using a miRNeasy RNA isolation kit (Qiagen), to preserve the tRNA content of the sample. Contaminating genomic DNA was eliminated by an on-column RNase-free DNase treatment. Deep sequencing of mitochondrial RNA was performed by GeneWorks (Adelaide, Australia) on the Illumina GAII platform (Illumina) according to the Illumina Directional RNA-Seq protocol. Notably, to preferentially capture tRNA sequences we size selected RNA between 60 and 90 nucleotides in length. Samples were ligated to RNA barcodes, combined and sequenced with a read length of 100 bases. Affinity purification and RNA isolation. HeLa cells (2 x 106) were lysed in 50 mM Tris/HCl (pH 7.5), 125 mM NaCl, 5% glycerol, 1% Igepal CA-630, 1.5 mM MgCl2, 1 mM DTT, 25 mM NaF, 1 mM Na 3VO4, 1 mM EDTA, 1x Complete protease inhibitors (Roche), 200 Uml-1 RNaseOUT (Invitrogen) at 4°C. The lysate was cleared by centrifugation and incubated with rabbit-IgG agarose (Sigma) at 4°C for 2 h. The agarose was washed with lysis buffer and then with cleavage buffer (10 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 1% Igepal CA-630, 200 Uml-1 RNaseOUT) and protein was eluted by addition of 0.25 Uμl-1 of AcTEV protease (Invitrogen, RT for 1 h). The TAP fusion proteins and endogenous ELAC2 and PTCD1 proteins were detected by immunoblotting.

Immunoblotting. Specific proteins were detected using rabbit αMRPL11 (NEB, diluted 1:1,000), αMRPS15 antibodies (Abcam, diluted 1:1,000), αELAC2 (Sigma, diluted 1:500) and αPTCD1 (Sigma, diluted 1:500) and mouse αNDUFA9 antibodies (MitoSciences, diluted 1:2,000) in 1% skim milk powder in phosphate-buffered saline (4.3 mM sodium phosphate, dibasic, 137 mM sodium chloride, 2.7 mM potassium chloride, 1.4 mM potassium phosphate, monobasic, PBS). The primary antibodies were detected using goat anti-mouse or goat anti-rabbit horse-radish peroxidase (Biorad, diluted 1:10,000). Proteins fused with the TAP tag were detected using goat anti-mouse HRP. Mitochondrial protein synthesis. HeLa cells were grown in six-well plates until 60% confluent, three or six days later transfected and de novo protein synthesis was analyzed. The growth medium was replaced with methionine and cysteine free medium containing 10% dialysed FBS for 30 min before addition of 100 μgml-1 emetine for 5 min for pulse analyses or 100 μgml-1 anisomycin for chase analyses. In addition, for the chase analyses, 40 μgml-1 of chloramphenicol was added 24 h prior to the addition of the labeling mix. Next, 200 μCi Expres35S Protein Labeling Mix [35S] (14 mCi, Perkin Elmer) was added and incubated at 37°C for 1 h then washed in PBS and centrifuged. Chase labeled cells were incubated for further 17 h in growth medium before collection. The cells were collected, suspended in PBS and 20 μg of proteins were separated on 12.5% SDS PAGE and the radiolabelled proteins were visualized on film. Respiration. Permeabilized cell respiration on glutamate/ malate was measured according to Kuznetsov et al. Briefly, 1 x 106 cells were washed in PBS, resuspended in 0.25 ml mitomedium B (0.5 mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 1 gl-1 fatty acid-free BSA, 60 mM lactobionate, 110 mM mannitol, 0.3 mM DTT, pH 7.1 with KOH) and added to 0.5 ml mitomedium B in a 1 ml OROBOROS high resolution respirometer thermostatically maintained at 37°C. The system was left to equilibrate for 5 min, before adding digitonin (50 μgml-1) and waiting for 5 min for the oxygen consumption to decline. Respiration on 0.5 mM TMPD and 2 mM ascorbate was measured in the permeabilized cells.

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Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

This work was supported by grants and fellowships from the Australian Research Council, the National Health and Medical Research Council of Australia and the Cancer Council of Western Australia. We thank Rob King from GeneWorks (Adelaide, SA) for advice with RNA sequencing.

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