2.2. LA IMAGEN DE CHINA EN EL DOCUMENTAL
2.2.1. China en el documental europeo
The CLIP assay was performed in order to assess whether or not SLIRP (SRA stem- loop interacting RNA binding protein) was able to bind mtRNA directly (Figure 3.2). The CLIP data revealed that the predicted RRM domain in SLIRP is functional and acts to bind RNA in human mitochondria. The CLIP reads were located in 12 of the 13 mt- ORFs with the exception of MTND6. This result remains in agreement with now published data showing dependence of mt-mRNAs stability on SLIRP presence in human and mouse cells (Sasarman et al., 2010, Ruzzenente et al., 2011, Chujo et al., 2012). SLIRP was reported to be in high-molecular weight complexes with LRPPRC. The latter also contains RNA binding domains, 22 of which are PPR motifs. The CLIP result for SLIRP shows unequivocally that this protein interacts directly, not just via interaction with LRPPRC, with messenger RNA in vivo in human mitochondria.
6.2 MRPL12
MRPL12 belongs to the group of conserved mitochondrial proteins having the bacterial orthologue called L7/L12. Moreover it is assigned to a unique pool of acidic ribosomal proteins, the analogues of which are present throughout phylogeny. The unique features of these proteins are due to their dynamic character and exchange of location between ribosomal LSU and the free pool, postulated to be a regulatory mechanism of translation process in response to fluctuations in cell metabolism.
Obtaining immortalised fibroblasts from the patient with mutated form of MRPL12 allowed me to study function of this protein in human mitochondria. The point mutation causing substitution of Alanine 181 by Valine in C terminal domain of MRPL12 (Figure 4.1) caused a reduction in the steady state level to only 30% of control value (Figure 4.3) with the free pool determined as absent via the sucrose gradient fractionation of mitochondrial lysate (Figure 4.6). Furthermore, a reduced number of MRPL12
150 molecules per mt-LSU was observed (Figure 4.12). However, the reduction in the level of MRPL12 did not affect overall mitoribosome assembly, but a visible decline in mitochondrial translation was detected in the in vivo 35S-metabolic labelling assay (Figure 5.7). Interestingly, the decrease in the rate of translation differentiated between polypeptides with highest decrease observed in products for COX and ATP6 subunits whilst cyt b was unaffected. As the steady state levels of MTCOX1, MTND1 and cyt b transcripts were not decreased (Figure 4.9) the reduced level of MRPL12/LSU can be postulated as the main reason for mitoribosome dysfunction, although the reduction in translational efficiency for different mitochondrially encoded subunits varied. Overall, my investigation revealed that although the stoichiometry of MRPL12 mutant/LSU is affected in the patient cell line, the mitoribosomes are assembled but their efficacy is lowered. The main technique, which could be applied to further investigate MRPL12 function, is ribosome profiling. It would allow to establish whether mitoribosomes with reduced numbers of MRPL12 molecules stall on specific sequences of mt-mRNAs, which slows down the translation.
6.3. RBFA.
This protein has not yet been characterised in the literature. The studies by Ricarda Richter, during her doctoral studies in my host laboratory, demonstrated an inhibitory effect of RBFA depletion on the HEK293 rate growth and clear localisation to the mt- SSU. None of the other analyses performed clarified is the function of this protein in mitoribosome assembly. The profile of mt-SSU and mt-LSU location was not changed in sucrose gradient fractions after RBFA silencing (for 3 and 6 days in HEK293 cells; Appendix 1.3). Furthermore, no decline was observed in mitochondrial RNA species or mtDNA, with only marginal decrease of mitochondrial translation rate in cells grown in the presence of RBFA si-RNA for 6 days. My project continued with characterisation and the results presented in my thesis indicate that RBFA:
Interacts directly with 3’ terminal stem loop of 12S mt-rRNA (Figure 5.1). The same sequence as already described for ERAL1 protein, by me in my host laboratory (Dennerlein et al., 2010).
Participates in the methylation process of two neighbouring adenines located in the 3’ terminal stem loop of 12S mt-rRNA (Figure 5.13).
The primer extension assay performed for 12S mt-rRNA immunoprecipitated with MRPS27-FLAG or ICT1-FLAG (Figure 5.14) from HEK293 cell lysates where RBFA was silenced for 5.5 days indicate that:
151 The level of methylation at the 3’ terminus of 12S incorporated into small
subunit of mitoribosome (28S) was decreased by 26%.
The mitoribosomes (55S) contained almost exclusively 28S with methylated 12S at the 3’ end, even after 5.5 days of RBFA depletion. This observation clearly indicates that fully assembled mitoribosomes favour modified 12S and are able to recycle those mt-SSU, which were assembled before RBFA depletion. The primer extension assay applied for 12S mt-rRNA immunoprecipitated with
ERAL1-FLAG or RBFA-FLAG (Figure 5.17) to investigate the order in which ERAL1 and RBFA binds to the 3’ terminus of the 12S presents clearly that:
ERAL1 binds mostly unmethylated 12S (74%)
whereas RBFA interacts almost exclusively with 12S methylated at the 3’ terminus (96%).
This experiment strongly suggests that RBFA succeeds ERAL1 binding at the 3’ terminus of 12S mt-rRNA (helix 45).
Overall, the results described above assign RBFA as a new member of maturation factors of the mammalian mt-SSU.
Finally, the CLIP assay revealed that RBFA binds second rRNA species 5S rRNA (Table 5.2). Identification of chimeric CLIP reads containing both 5S and 12S rRNA fragments suggest that RBFA interacts with both RNA species simultaneously in the mitochondrial matrix. Resemblance of the 5S rRNA secondary structure to the snoRNA, which guides modifications on cytosolic rRNA led to the hypothesis of a novel function for 5S rRNA to guide methylation at the 3’ terminus of mt-12S rRNA.
Further investigation aims to test this hypothesis:
Gel shift experiments will be performed to potentially show simultaneous interaction of RBFA with 5S and 12S rRNA in vitro.
In order to assess influence of 5S rRNA on the level of methylation at the terminal stem loop of 12S rRNA, siRNA silencing of 5S rRNA in HEK293 cells will be attempted followed by primer extension assays.
In conclusion, obtained data strongly assign RBFA to the group of factors responsible for maturation of the mt-SSU and mitoribosome assembly.
152
153 Adams, K.L., Palmer, J.D., 2003. Evolution of mitochondrial gene content: gene loss
and transfer to the nucleus. Mol. Phylogenet. Evol. 29, 380–395.
Agirrezabala, X., Frank, J., 2009. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu. Q. Rev. Biophys. 42, 159–200.
Agrawal, R.K., Sharma, M.R., 2012. Structural aspects of mitochondrial translational apparatus. Curr. Opin. Struct. Biol. 22, 797–803.
Alam, T.I., Kanki, T., Muta, T., Ukaji, K., Abe, Y., Nakayama, H., Takio, K., Hamasaki, N., Kang, D., 2003. Human mitochondrial DNA is packaged with TFAM. Nucleic Acids Res. 31, 1640–1645.
Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 90, 7915–7922. Amunts, A., Brown, A., Bai, X. -c., Llacer, J.L., Hussain, T., Emsley, P., Long, F.,
Murshudov, G., Scheres, S.H.W., Ramakrishnan, V., 2014. Structure of the Yeast Mitochondrial Large Ribosomal Subunit. Science 343, 1485–1489. Anderson, S., Bankier, A.T., Barrell, B.G., De Bruijn, M.H., Coulson, A.R., Drouin, J.,
Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J., Staden, R., Young, I.G., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465.
Antonicka, H., Ostergaard, E., Sasarman, F., Weraarpachai, W., Wibrand, F., Pedersen, A.M.B., Rodenburg, R.J., Van der Knaap, M.S., Smeitink, J.A.M.,
Chrzanowska-Lightowlers, Z.M., Shoubridge, E.A., 2010. Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. Am. J. Hum. Genet. 87, 115–122.
Antonicka, H., Sasarman, F., Nishimura, T., Paupe, V., Shoubridge, E.A., 2013. The mitochondrial RNA-binding protein GRSF1 localizes to RNA granules and is required for posttranscriptional mitochondrial gene expression. Cell Metab. 17, 386–398.
Asin-Cayuela, J., Gustafsson, C.M., 2007. Mitochondrial transcription and its regulation in mammalian cells. Trends in Biochemical Sciences 32, 111–117.
Barciszewska, M.Z., Szymański, M., Erdmann, V.A., Barciszewski, J., 2001. Structure and functions of 5S rRNA. Acta Biochim. Pol. 48, 191–198.
Barrientos, A., Korr, D., Barwell, K.J., Sjulsen, C., Gajewski, C.D., Manfredi, G., Ackerman, S., Tzagoloff, A., 2003. MTG1 codes for a conserved protein required for mitochondrial translation. Mol. Biol. Cell 14, 2292–2302.
Bar-Yaacov, D., Blumberg, A., Mishmar, D., 2012. Mitochondrial-nuclear co-evolution and its effects on OXPHOS activity and regulation. Biochim. Biophys. Acta 1819, 1107–1111.
Baughman, J.M., Nilsson, R., Gohil, V.M., Arlow, D.H., Gauhar, Z., Mootha, V.K., 2009. A Computational Screen for Regulators of Oxidative Phosphorylation Implicates SLIRP in Mitochondrial RNA Homeostasis. PLoS Genetics 5, e1000590.
Benard, G., Karbowski, M., 2009. Mitochondrial fusion and division: Regulation and role in cell viability. Seminars in Cell & Developmental Biology 20, 365–374. Bensaad, K., Tsuruta, A., Selak, M.A., Vidal, M.N.C., Nakano, K., Bartrons, R.,
Gottlieb, E., Vousden, K.H., 2006. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 126, 107–120.
Bentley, R.C., Keene, J.D., 1991. Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A’ protein. Mol. Cell. Biol. 11, 1829–1839.
154 Berk, V., Cate, J.H., 2007. Insights into protein biosynthesis from structures of bacterial
ribosomes. Current Opinion in Structural Biology 17, 302–309.
Boehringer, D., O’Farrell, H.C., Rife, J.P., Ban, N., 2012a. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis. Journal of Biological Chemistry 287, 10453–10459.
Boehringer, D., O’Farrell, H.C., Rife, J.P., Ban, N., 2012b. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis. Journal of Biological Chemistry 287, 10453–10459.
Bonawitz, N.D., Clayton, D.A., Shadel, G.S., 2006. Initiation and Beyond: Multiple Functions of the Human Mitochondrial Transcription Machinery. Molecular Cell 24, 813–825.
Borowski, L.S., Dziembowski, A., Hejnowicz, M.S., Stepien, P.P., Szczesny, R.J., 2013. Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci. Nucleic Acids Res. 41, 1223–1240.
Borowski, L.S., Szczesny, R.J., Brzezniak, L.K., Stepien, P.P., 2010. RNA turnover in human mitochondria: More questions than answers? Biochimica et Biophysica Acta (BBA) - Bioenergetics 1797, 1066–1070.
Bowmaker, M., Yang, M.Y., Yasukawa, T., Reyes, A., Jacobs, H.T., Huberman, J.A., Holt, I.J., 2003. Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J. Biol. Chem. 278, 50961–50969.
Brot, N., Weissbach, H., 1981. Chemistry and biology of E. coli ribosomal protein L12. Mol. Cell. Biochem. 36, 47–63.
Bruni, F., Gramegna, P., Oliveira, J.M.A., Lightowlers, R.N., Chrzanowska-
Lightowlers, Z.M.A., 2013. REXO2 Is an Oligoribonuclease Active in Human Mitochondria. PLoS ONE 8, e64670.
Brzezniak, L.K., Bijata, M., Szczesny, R.J., Stepien, P.P., 2011. Involvement of human ELAC2 gene product in 3’ end processing of mitochondrial tRNAs. RNA Biology 8, 616–626.
Bullerwell, C.E., Gray, M.W., 2004. Evolution of the mitochondrial genome: protist connections to animals, fungi and plants. Current Opinion in Microbiology 7, 528–534.
Burd, C.G., Dreyfuss, G., 1994. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621.
Bylund, G.O., Wipemo, L.C., Lundberg, L.A., Wikström, P.M., 1998. RimM and RbfA are essential for efficient processing of 16S rRNA in Escherichia coli. J.
Bacteriol. 180, 73–82.
Cairns, C.A., White, R.J., 1998. p53 is a general repressor of RNA polymerase III transcription. EMBO J. 17, 3112–3123.
Cámara, Y., Asin-Cayuela, J., Park, C.B., Metodiev, M.D., Shi, Y., Ruzzenente, B., Kukat, C., Habermann, B., Wibom, R., Hultenby, K., Franz, T., Erdjument- Bromage, H., Tempst, P., Hallberg, B.M., Gustafsson, C.M., Larsson, N.-G., 2011. MTERF4 regulates translation by targeting the methyltransferase NSUN4 to the mammalian mitochondrial ribosome. Cell Metab. 13, 527–539.
Chan, D.C., 2006. Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241–1252.
Chatre, L., Ricchetti, M., 2013. Large heterogeneity of mitochondrial DNA
transcription and initiation of replication exposed by single-cell imaging. J. Cell. Sci. 126, 914–926.
Chen, H.-W., Rainey, R.N., Balatoni, C.E., Dawson, D.W., Troke, J.J., Wasiak, S., Hong, J.S., McBride, H.M., Koehler, C.M., Teitell, M.A., French, S.W., 2006. Mammalian polynucleotide phosphorylase is an intermembrane space RNase that maintains mitochondrial homeostasis. Mol. Cell. Biol. 26, 8475–8487.
155 Chen, T., Damaj, B.B., Herrera, C., Lasko, P., Richard, S., 1997. Self-association of the
single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain. Mol. Cell. Biol. 17, 5707–5718.
Cheng, N., Mao, Y., Shi, Y., Tao, S., 2012. Coevolution in RNA Molecules Driven by Selective Constraints: Evidence from 5S rRNA. PLoS ONE 7, e44376.
Cheung, E.C.C., Joza, N., Steenaart, N.A.E., McClellan, K.A., Neuspiel, M., McNamara, S., MacLaurin, J.G., Rippstein, P., Park, D.S., Shore, G.C.,
McBride, H.M., Penninger, J.M., Slack, R.S., 2006. Dissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and
apoptosis. EMBO J. 25, 4061–4073.
Chow, C.S., Lamichhane, T.N., Mahto, S.K., 2007. Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications. ACS Chem. Biol. 2, 610–619.
Christian, B., Haque, E., Spremulli, L., 2009. Ribosome shifting or splitting: it is all up to the EF-G. Mol. Cell 35, 400–402.
Christian, B.E., Spremulli, L.L., 2010. Preferential Selection of the 5’-Terminal Start Codon on Leaderless mRNAs by Mammalian Mitochondrial Ribosomes. Journal of Biological Chemistry 285, 28379–28386.
Christian, B.E., Spremulli, L.L., 2012. Mechanism of protein biosynthesis in mammalian mitochondria. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1819, 1035–1054.
Chujo, T., Ohira, T., Sakaguchi, Y., Goshima, N., Nomura, N., Nagao, A., Suzuki, T., 2012. LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and
promotes polyadenylation in human mitochondria. Nucleic Acids Research 40, 8033–8047.
Ciganda, M., Williams, N., 2011. Eukaryotic 5S rRNA biogenesis. Wiley Interdiscip Rev RNA 2, 523–533.
Claros, M.G., Perea, J., Shu, Y., Samatey, F.A., Popot, J.L., Jacq, C., 1995. Limitations to in vivo import of hydrophobic proteins into yeast mitochondria. The case of a cytoplasmically synthesized apocytochrome b. Eur. J. Biochem. 228, 762–771. Clatterbuck Soper, S.F., Dator, R.P., Limbach, P.A., Woodson, S.A., 2013. In Vivo X-
Ray Footprinting of Pre-30S Ribosomes Reveals Chaperone-Dependent Remodeling of Late Assembly Intermediates. Molecular Cell 52, 506–516. Cohen, B.H., Naviaux, R.K., 2010. The clinical diagnosis of POLG disease and other
mitochondrial DNA depletion disorders. Methods 51, 364–373.
Connolly, K., Culver, G., 2013. Overexpression of RbfA in the absence of the KsgA checkpoint results in impaired translation initiation: Quality control of small subunit biogenesis. Molecular Microbiology 87, 968–981.
Connolly, K., Rife, J.P., Culver, G., 2008. Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA. Molecular Microbiology 70, 1062–1075.
Cotney, J., McKay, S.E., Shadel, G.S., 2009. Elucidation of separate, but collaborative functions of the rRNA methyltransferase-related human mitochondrial
transcription factors B1 and B2 in mitochondrial biogenesis reveals new insight into maternally inherited deafness. Human Molecular Genetics 18, 2670–2682. Cotney, J., Shadel, G.S., 2006. Evidence for an Early Gene Duplication Event in the
Evolution of the Mitochondrial Transcription Factor B Family and Maintenance of rRNA Methyltransferase Activity in Human mtTFB1 and mtTFB2. Journal of Molecular Evolution 63, 707–717.
Cotney, J., Wang, Z., Shadel, G.S., 2007. Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-
156 mtTFB2 in mitochondrial biogenesis and gene expression. Nucleic Acids Res. 35, 4042–4054.
Cox, C.J., Foster, P.G., Hirt, R.P., Harris, S.R., Embley, T.M., 2008. The
archaebacterial origin of eukaryotes. Proceedings of the National Academy of Sciences 105, 20356–20361.
Dairaghi, D.J., Shadel, G.S., Clayton, D.A., 1995a. Addition of a 29 residue carboxyl- terminal tail converts a simple HMG box-containing protein into a
transcriptional activator. J. Mol. Biol. 249, 11–28.
Dairaghi, D.J., Shadel, G.S., Clayton, D.A., 1995b. Human mitochondrial transcription factor A and promoter spacing integrity are required for transcription initiation. Biochim. Biophys. Acta 1271, 127–134.
Dammel, C.S., Noller, H.F., 1993. A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. Genes Dev. 7, 660–670. Dammel, C.S., Noller, H.F., 1995. Suppression of a cold-sensitive mutation in 16S
rRNA by overexpression of a novel ribosome-binding factor, RbfA. Genes Dev. 9, 626–637.
Datta, K., Fuentes, J.L., Maddock, J.R., 2005. The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2. Mol. Biol. Cell 16, 954–963.
Datta, P.P., Wilson, D.N., Kawazoe, M., Swami, N.K., Kaminishi, T., Sharma, M.R., Booth, T.M., Takemoto, C., Fucini, P., Yokoyama, S., Agrawal, R.K., 2007. Structural aspects of RbfA action during small ribosomal subunit assembly. Mol. Cell 28, 434–445.
Dawson, V.L., Dawson, T.M., 2004. Deadly conversations: nuclear-mitochondrial cross-talk. J. Bioenerg. Biomembr. 36, 287–294.
Demirci, H., Murphy, F., 4th, Belardinelli, R., Kelley, A.C., Ramakrishnan, V., Gregory, S.T., Dahlberg, A.E., Jogl, G., 2010. Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function. RNA 16, 2319–2324.
Dennerlein, S., Rozanska, A., Wydro, M., Chrzanowska‑Lightowlers, Z.M.A., Lightowlers, R.N., 2010. Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit. Biochemical Journal 430, 551–558.
Deroo, S., Hyung, S.-J., Marcoux, J., Gordiyenko, Y., Koripella, R.K., Sanyal, S., Robinson, C.V., 2012. Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G. ACS Chem. Biol. 7, 1120–1127. Desai, P.M., Rife, J.P., 2006. The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit. Arch. Biochem. Biophys. 449, 57–63.
Dey, D., Bochkariov, D.E., Jokhadze, G.G., Traut, R.R., 1998. Cross-linking of selected residues in the N- and C-terminal domains of Escherichia coli protein L7/L12 to other ribosomal proteins and the effect of elongation factor Tu. J. Biol. Chem. 273, 1670–1676.
Dey, D., Oleinikov, A.V., Traut, R.R., 1995. The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis. Biochimie 77, 925–930.
Diaconu, M., Kothe, U., Schlünzen, F., Fischer, N., Harms, J.M., Tonevitsky, A.G., Stark, H., Rodnina, M.V., Wahl, M.C., 2005. Structural Basis for the Function of the Ribosomal L7/12 Stalk in Factor Binding and GTPase Activation. Cell 121, 991–1004.
Diedrich, G., Spahn, C.M., Stelzl, U., Schäfer, M.A., Wooten, T., Bochkariov, D.E., Cooperman, B.S., Traut, R.R., Nierhaus, K.H., 2000. Ribosomal protein L2 is
157 involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer. EMBO J. 19, 5241–5250.
Donati, G., Peddigari, S., Mercer, C.A., Thomas, G., 2013. 5S Ribosomal RNA Is an Essential Component of a Nascent Ribosomal Precursor Complex that Regulates the Hdm2-p53 Checkpoint. Cell Reports 4, 87–98.
Ekstrand, M.I., Falkenberg, M., Rantanen, A., Park, C.B., Gaspari, M., Hultenby, K., Rustin, P., Gustafsson, C.M., Larsson, N.-G., 2004. Mitochondrial transcription