Romanticismo y determinismo biológico (S.XIX)

The reviews concerning one of the most sophisticated machineries found in all types of cells, the ribosome, usually starts with the basic description of it addressing the

heterogeneity in its composition and asymmetry in the structure. The sentence that always is present in summaries of established knowledge confirmed through analysis of the crystal structures of LSUs and complete ribosomes from different organisms inform that a 5S rRNA is present in virtually all ribosomes except mitoribosomes of some fungi, mammals and most protists (Barciszewska et al., 2001, Szymański et al., 2003). Interestingly, the function of eubacterial and eukaryotic cytosolic 5S rRNA despite years of research remains unclear. Published results on studies in bacteria localise 5S rRNA in the central protuberance of LSU, where it interacts with three proteins L5, L18

(the bacterial homologue of eukaryotic L5) and L25. However,in eukaryota this

interaction is limited only to L5 protein. Structural analyses of ribosomes from many species, positions 5S rRNA in the junction between small and large subunit, which allows it a broad interaction with many centres crucial for translation (Kiparisov et al., 2005, Kouvela et al., 2007). Indeed, its absence causes a decline in protein synthesis in both bacterial and eukaryotic cells (Ciganda and Williams, 2011). Furthermore, the most recent publication, presenting cryo-EM structures of the final-stage assembly precursors of the bacterial LSU shows involvement of 5S rRNA in the maturation process of 50S (Li et al., 2013). In addition, data on silencing of TFIIIA in human U2OS cells reports an inhibition of 5S rRNA transcription and impairment of ribosome biogenesis (Donati et al., 2013).

49 The 5S rRNA is the smallest ribosomal RNA molecule, built of 120 nt (40 kDa), with a secondary (Figure 1.10) and tertiary structures largely preserved across

phylogeny (Ciganda and Williams, 2011, Cheng et al., 2012).

Figure 1.10. Secondary structure of human 5S rRNA. Evolutionary changes in the sequence of the 5S rRNA are indicated with yellow, green and purple. Image taken from Cheng et al., 2012.

Intriguingly, the 5S gene is present in a different number of tandem or multiple repeats spread throughout the genome. Recently, it has been shown that each mammalian species has a greatly conserved 5S rRNA type and many variable ones (Vierna et al., 2013). In eukaryotic cells, 5S rRNA is synthesized by RNA polymerase III, is transported from the nucleus to the cytosol in a complex with TFIIIA (transcription factor), and then re-enters the nucleus in the complex with protein L5. It is the only reported RNA species that creates complexes with ribosomal proteins before incorporation into the ribosomes in prokaryotes and eukaryotes (Szymański et al., 2003). Furthermore, it has been already reported that 5S rRNA plays roles outside the ribosome. The most recent publication, shows this small RNA particle in a complex with L5 and L11 protein implicated in p53 level regulation (Donati et al., 2013). This pre-ribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition, which activates p53, as a result of defective ribosome biogenesis. These intriguing data implicate 5S rRNA in very complicated and still elusive processes of carcinogenesis, as p53 is known as a tumour suppressor, which is lost or mutated in over 50% of all human tumours (Cairns and White, 1998). The role, if any, of 5S rRNA in mitoribosomes is even more mysterious. There are very limited data available

50 concerning the function of this molecule in mammalian mitochondria. As already

mentioned it was not detected in a 13.5 Å resolution structure of bovine 55S obtained by cryoelectron microscopy (Sharma et al., 2003). However, this observation was challenged in a recent publication, showing its presence in mitoribosomes

immunoprecipitated via either overexpressed FLAG tagged ICT1 or MRPS27 (Smirnov et al., 2011). The ratio of mitoribosomes to bound 5S rRNAs was reported as 1:1. This astonishing result needs further investigation as it is the first reported finding, standing against very strongly established dogma in mitochondrial research. There are no more publications supporting it directly, but there are at least a few that strongly suggest the presence of 5S rRNA in mammalian mitochondria. Two of the first reports shows the presence of this particle in stringently purified mitochondria and mitoplasts of bovine, chicken, rabbit, rat and human cells (Yoshionari et al., 1994, Magalhães et al., 1998). However, since the cytosolic ribosomes co-purify with mitochondria it is very difficult to eliminate contamination. It is important to underline, that the new mammalian 39S structure has an RNA fragment present that is not the 16S but has not been identified (Greber et al., 2013).

Structural analysis of 5S rRNA suggests two distinct regions, α- and γ- domains as critical for its mitochondrial targeting in vitro, confirmed by decline of import in vivo (Smirnov et al., 2008). The same research group, point out two proteins, mitochondrial enzyme rhodanese and MRPL18, as involved in 5S rRNA transport into mammalian mitochondria (Smirnov et al., 2010, Smirnov et al., 2011). There are also two

independent publications showing involvement of PNPase (polyribonucleotide nucleotidyltransferase) in mitochondrial RNA import. This protein has a 3’ to 5’ exoribonuclease and poly(A) polymerase activity. As mentioned before it has a dual location in mitochondria; inner membrane and matrix. PNPase has also been shown to enhance transport of RNase P RNA, 5S rRNA and MRP RNAs into the mammalian mitochondrial matrix (Wang et al., 2010). These results were reinforced with a recent report, describing two patients with homozygous missense mutation in the PNPase gene who presented with severe encephalomyopathy, choreoathetotic movements (irregular involuntary movements that may involve the face, neck, trunk, limbs or respiratory muscles) and respiratory chain deficiencies (Vedrenne et al., 2012). The analysis of patient 1 fibroblasts showed a reduction in 5S rRNA and MRP RNA import into mitochondria, and a decrease in translation. Overexpression of the wild type PNPase

51 cDNA enhanced 5S rRNA transfer to mitochondria and rescued the translation

deficiency.

Overall, the significance and functions of 5S rRNA in human cells is far from defined. Presented in this subsection publications clearly show how incomplete is our knowledge about this 120 nt RNA species especially in regard to its mitochondrial appearance. Although, few published findings suggest that 5S rRNA is a part of mitochondrial ribosome in human cells with the most recent cryo-EM structure of the porcine 39S at 4.9 Å resolution presenting additional density of unidentified short RNA fragment positioned in the CP, the only strong conclusion which can be made at this stage is that the 5S rRNA is present in human mitochondria. Beyond this conclusion there is not enough experimental evidence deciphering its exact location or functions.