In eukaryotes, the synthesis of ribosomal subunits, which involves the maturation of the ribosomal (r)RNAs and assembly of ribosomal proteins, requires the co-ordinated action of a plethora of ribosome biogenesis factors. Many of these cofactors remain to be characterized in human cells. Here, we demonstrate that the human G-patch protein NF-κB-repressing factor (NKRF) forms a pre-ribosomal subcomplex with the DEAH-box RNA helicase DHX15 and the 5΄-3΄ exonuclease XRN2. Using UV crosslinking and analysis of cDNA (CRAC), we reveal that NKRF binds to the transcribed spacer regions of the pre-rRNA transcript. Consistent with this, we find that depletion of NKRF, XRN2 or DHX15 impairs an early pre-rRNA cleavage step (A’). The catalytic activity of DHX15, which we demonstrate is stimulated by NKRF functioning as a cofactor, is required for efficient A’ cleavage, suggesting that a structural remodelling event may facilitate processing at this site. In addition, we show that depletion of NKRF or XRN2 also leads to the accumulation of excised pre-rRNA spacer fragments and that NKRF is essential for recruitment of the exonuclease to nucleolar pre-ribosomal complexes. Our findings therefore reveal a novel pre-ribosomal subcomplex that plays distinct roles in the processing of pre-rRNAs and the turnover of excised spacer fragments.

Ribosome synthesis is an essential cellular process, and perturbation of human ribosome production is linked to cancer and genetic diseases termed ribosomopathies. During their assembly, pre-ribosomal particles undergo numerous structural rearrangements, which establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome biogenesis. RNA helicases are essential mediators of such remodelling events and here, we demonstrate that the DEAH-box RNA helicase DHX37 is required for maturation of the small ribosomal subunit in human cells. Our data reveal that the presence of DHX37 in early pre-ribosomal particles is monitored by a quality control pathway and that failure to recruit DHX37 leads to pre-rRNA degradation. Using an in vivo crosslinking approach, we show that DHX37 binds directly to the U3 small nucleolar RNA (snoRNA) and demonstrate that the catalytic activity of the helicase is required for dissociation of the U3 snoRNA from pre-ribosomal complexes. This is an important event during ribosome assembly as it enables formation of the central pseudoknot structure of the small ribosomal subunit. We identify UTP14A as a direct interaction partner of DHX37 and our data suggest that UTP14A can act as a cofactor that stimulates the activity of the helicase in the context of U3 snoRNA release.

A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

No abstract

One of the major research interests of nanomedicine is the designing of harmless and biocompatible medical devices. To improve the features of Ti surface, TiO2 based nanotube (TNT) films (50 nm diameter) achieved by anodic oxidation and thermal treatment were grown on titanium and on Ti6Al4V and Ti6Al7Nb alloys. Their in vitro toxicity and biocompatibility were investigated using G292 osteoblast cell line. The LDH release after 24 and 48 h of exposure demonstrated that TNT layers were not cytotoxic. The cell growth on TNT films deposited on titanium and Ti6Al4V was significantly increased compared with Ti6Al7Nb. F-actin staining showed a better organized actin cytoskeleton in osteoblasts grown on these two samples, which provide the best conditions for osteoblast attachment and spreading. Analysis of GSH distribution revealed a higher nuclear level in the samples with TNTs compared with Ti plate without nanotubes, indicating an active proliferation. Thus, nuclear glutathione levels can be used as a useful biomarker for biocompatibility assessment. Our results suggest that the substrate for TNTs can have a significant impact on cell morphology and fate. In conclusion, the TNT/Ti and TNT/Ti6Al4V were toxicity-free and can provide a proper nanostructure for a positive cell response.

Herewith I declare that I prepared the Ph.D. dissertation "Insights into the regulation of RNA helicases by protein cofactors" on my own and with no other sources and aids than quoted.