RNA Helicases in 60S Ribosomal Subunit Biogenesis in Saccharomyces cerevisiae

Abstract

The ribosomes are the cellular organelles responsible for translating messenger RNA (mRNA) to proteins. In eukaryotic organisms, the ribosomes are composed by two subunits: the large subunit and the small subunit that come together to make a complete ribosome. The biogenesis of eukaryotic cytoplasmic ribosomes is a very complex process. The eukaryotic organism where this process has been best studied is the budding yeast Saccharomyces cerevisiae. This microorganism is considered a model organism of study due to its low complexity, rapid growth and dispersion of its cells. In S. cerevisiae, the large subunit or 60S is composed by three rRNAs called 25S, 5.8S and 5S and about 46 ribosomal proteins; the small subunit or 40S is composed by only one rRNA called 18S and about 33 ribosomal proteins. The ribosome biogenesis process is not only complex, but it is also compartmentalized. The precursors of rRNA (pre-rRNA) are transcribed in the nucleolus by the RNA polymerase I (25S, 5.8S y 18S) and by the RNA polymerase III (5S). Along the processing of the pre-rRNAs take place accurate endo- and exonucleolytic reactions, at the same time that some molecular building blocks of pre-rRNA are modified by methylases enzymes or by small nucleolar RNA complexes (snoRNPs). The processing and modification of the pre-rRNAs do not occur in naked RNAs, conversely, these reactions take place in pre-ribosomal particles, which are complexes where the ribosomal proteins are assembling hierarchically. The restructuration of the pre-ribosomal particles and its subsequent maturation to ribosomal subunits needs a large number of protein factors, more than 300, which are known as assembly factors or trans-acting factors. These factors are not present in the mature ribosomal particles. One part of these reactions occurs in the nucleoplasm or even in the cytoplasm. The ribosomal synthesis process has not only academic, but also biomedical interest, because a set of hereditary diseases with bad prognostic, called ribosomopathies are associated to loss of function mutation in some ribosomal proteins and/or assembly factors. Moreover, this process has been related with autoimmune pathologies and in a direct manner with large number of cancers and viral infections. The RNA dependent RNA helicases are among the assembly factors that facilitate the biogenesis of ribosomes. In a biochemical sense, these enzymes unwind RNA duplexes using the energy released by ATP hydrolysis. The RNA helicases are classified according to their sequence and structural properties in superfamilies (SF1-SF5). The superfamilies are formed at the same time by several subfamilies. Of special interest for our work are the RNA helicases belonging to the so-called DEAD-box protein subfamily, named as such due to the presence of the amino acids aspartic, glutamic, alanine and aspartic, in this order, in a specific place of its sequences. These amino acids are involved in ATP binding and hydrolysis. The DEAD-box proteins are composed by two structural domains, each one similar to the bacterial RecA protein, that contain a set of highly conserved sequence motifs. These motifs are involved in ATP binding and hydrolysis, nucleic acid binding, duplex unwinding or in the coordination between those reactions. Today, up to 19 helicases from the DEAD-box protein subfamily have been identified to be involved in the biogenesis of cytoplasmic ribosomes. However, the precise role of these enzymes in that process is clearly not fully understood. Therefore, the objective of this work has consisted in the biochemical and functional characterization of two functionally-related helicases during the biogenesis of the 60S ribosomal subunit. These helicases are Dbp7 and Dbp9. Both proteins are necessary for the 60S subunits biogenesis due to their roles during the processing of the early 27SA pre-rRNAs and therefore for the accumulation of the mature 25S and 5.8S rRNAs. However, the exact role of Dbp7 and Dbp9 in this process is still unknown. To accomplish these objectives, we have carried out a functional characterization of Dbp7 protein in vivo. The conserved motifs of this protein, and its two longs N- and C- terminal extensions were identified in the amino acid sequence. In the N-terminal extension, we could identify a putative nuclear localization signal (NLS). Several mutants were constructed for the study. The results of growth analysis and polysome profiles in point mutants in the motifs I and VI, involved in ATP binding and hydrolysis, demonstrated that these motifs play an important role in Dbp7 function, since they showed a delay in growth, possibly due to a decrease in the levels of 60S subunits. Similar results were obtained in mutants with deletions of specific regions in the N- and C- terminal extensions of the protein. The observed defects were not due to a delocalization of Dbp7, since in all the mutants with deletions in the N- and C- extensions, the protein was still associated to hight molecular weight complexes, probably pre-60S ribosomal particles. Moreover, by epifluorescence, we have checked that the putative NLS localized in the N-terminal end of Dbp7 is a bona fide nuclear localization signal. A dbp7 mutant lacking the NLS grew as an isogenic wild-type strain and the protein was able to reach the nucleus; therefore, Dbp7 must contain others redundant NLS not recognizable in a sequence analysis or is imported into the nucleus through binding to another protein that transfers it to that location. It has been demonstrated that Dbp7 is functionally associated to a protein complex called Npa1 complex and it is formed by the proteins Dbp6, Nop8, Rsa3, Npa1 and Npa2. Npa1 binds to specific positions in rRNA in the domains I and VI of rRNA 25S, so it has been proposed that the Npa1 complex is involved in the restructuration of these domains during the early nucleolar maturation of 60S subunits. This process involves the assembly of the ribosomal protein uL3. Dbp7 is genetically associated to this complex. Some snoRNAs participate in these restructuring reactions, among them snR190, whose target sequences of RNA are neighboring the region where Npa1 binds. Npa1 also binds directly to snR190. Our group has been identified mutations in the 25S rRNA that interfere with the binding of snR190 to the rRNA and partially suppress the growth defects of a dbp7 null mutant. In this work, we have generated a double dbp7Δ snR190 mutant strain, whose growth is improved compared to that of a single dbp7Δ mutant. Consistent with this result, snR190, among other snoRNAs, is retained in pre-ribosomal particles in the absence of Dbp7. This phenomenon does not occur in an isogenic wild-type strain. Thus, our work suggests that Dbp7, probably carrying out its function as a RNA helicase enzyme, participates in the releasing of the at least snR190 from the early 60S ribosomal particles in progression to maturation. In the second part of this Thesis work, we have studied the enzymatic characteristic of Dbp7 in vitro, more specifically its possible activities ATPase, binding to RNA and helicase. Making use of colorimetric enzymatic and radioactive assays, we have demonstrated that a recombinant protein of Dbp7 that was expressed and purified from Escherichia coli extracts presents an ATPase activity dependent of RNA and specific DNA oligonucleotides (i.e. poly dT of 30 nucleotides length). Simultaneously, we have demonstrated that Dbp7 binds to RNA and shows helicase activity in vitro. Dbp7 is able to unwind RNA duplexes in 5´-3´and 3´-5 directions. This activity is only dependent of ATP hydrolysis. Likewise, we have studied the previous activities in the Dbp7 point mutants in domains involved in the ATP binding and hydrolysis (Dbp7[K197A] and Dbp7[R553A]). However, surprisingly, both mutants maintain the activities of the wild-type protein. These results are discussed in the context of the functional and structural domains of Dbp7. Finally, in this work, we have started the preliminary characterization of the biochemical activities of the RNA helicase Dbp9. In a similar approach to that done with Dbp7, we have expressed and purified the recombinant Dbp9 protein from E. coli extracts and we have started it biochemical characterization. Our first assays suggest that the recombinant protein has ATPase activity dependent of an DNA oligonucleotide, but it neither has the ability to bind RNA nor helicase activity under the conditions tested.