hMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors.

Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments.

Proteomic insight into the effects of the Salmonella ubiquitin ligase SlrP on host cells.

The virulence of the human and animal pathogen Salmonella enterica serovar Typhimurium is dependent on two type III secretion systems. These systems translocate proteins called effectors into eukaryotic host cells. SlrP is a Salmonella type III secretion effector with ubiquitin ligase activity. Here, we used two complementary proteomic approaches, two-dimensional gel electrophoresis and iTRAQ (isobaric tags for relative and absolute quantification) to study the consequences of the presence of SlrP in human epithelial cells. We identified 37 proteins that were differentially expressed in HeLa cells expressing slrP compared to control cells. Microarray analysis revealed that more than a half of differentially expressed proteins did not show changes in the transcriptome, suggesting post-transcriptional regulation. A gene ontology overrepresentation test carried out on the differentially expressed proteins revealed enrichment of ontology terms related to several types of junctions mediating adhesion in epithelial cells. Consistently, slrP-transfected cells showed defects in migration and adhesion. Our results suggest that the modification of cell-cell interaction ability of the host could be one of the final consequences of the action of SlrP during an infection.

Dynamics of the Spb4 interactome monitored by affinity purification.

RNA helicases constitute the largest class of NTPases involved in ribosome biogenesis, a fundamental process that has been best characterized in the eukaryotic model organism Saccharomyces cerevisiae. In yeast, genetic and biochemical analyses indicate that these RNA helicases are energy-consuming modulators of local structures inside pre-ribosomal particles that actively promote the establishment or dissociation of snoRNA:pre-rRNA base pairings, the activity of certain pre-rRNA nucleases, and/or the acquisition of pre-rRNA folds required for the recruitment or release of ribosome assembly factors and the stable assembly of ribosomal proteins. Despite significant recent advances, the precise molecular functions of RNA helicases involved in ribosome biogenesis remain largely elusive. In recent years, the purification and compositional analysis of distinct pre-ribosomal particles via affinity purification methods has been established as one of the most useful techniques to explore the yeast ribosome biogenesis pathway. In this chapter, we describe the use of different affinity purification methods to study the physical environment of RNA helicases involved in ribosome biogenesis, using as an example the putative RNA helicase Spb4 required for 60S ribosomal subunit biogenesis.

Yeast and human RNA helicases involved in ribosome biogenesis: current status and perspectives.

Ribosome biogenesis is a fundamental process that is conserved in eukaryotes. Although spectacular progress has been made in understanding mammalian ribosome synthesis in recent years, by far, this process has still been best characterised in the yeast Saccharomyces cerevisiae. In yeast, besides the rRNAs, the ribosomal proteins and the 75 small nucleolar RNAs, more than 250 non-ribosomal proteins, generally referred to as trans-acting factors, are involved in ribosome biogenesis. These factors include nucleases, RNA modifying enzymes, ATPases, GTPases, kinases and RNA helicases. Altogether, they likely confer speed, accuracy and directionality to the ribosome synthesis process, however, the precise functions for most of them are still largely unknown. This review summarises our current knowledge on eukaryotic RNA helicases involved in ribosome biogenesis, particularly focusing on the most recent advances with respect to the molecular roles of these enzymes and their co-factors in yeast and human cells. This article is part of a Special Issue entitled: The Biology of RNA helicases-Modulation for life.

Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae.

Spb4 is a putative ATP-dependent RNA helicase that is required for proper processing of 27SB pre-rRNAs and therefore for 60S ribosomal subunit biogenesis. To define the timing of association of this protein with preribosomal particles, we have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, some protein factors of 90S preribosomal particles and the 35S and 27SA pre-rRNAs also copurify with TAP-tagged Spb4. Moreover, we have obtained by site-directed mutagenesis an allele that results in the R360A substitution in the conserved motif VI of the Spb4 helicase domain. This allele causes a dominant-negative phenotype when overexpressed in the wild-type strain. Cells expressing Spb4(R360A) display an accumulation of 35S and 27SB pre-rRNAs and a net 40S ribosomal subunit defect. TAP-tagged Spb4(R360A) displays a greater steady-state association with 90S preribosomal particles than TAP-tagged wild-type Spb4. Together, our data indicate that Spb4 is a component of early nucle(ol)ar pre-60S ribosomal particles containing 27SB pre-rRNA. Apparently, Spb4 binds 90S preribosomal particles and dissociates from pre-60S ribosomal particles after processing of 27SB pre-rRNA.

Nop6, a component of 90S pre-ribosomal particles, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, ribosome biogenesis requires, in addition to rRNA and ribosomal proteins, a myriad of small nucleolar RNAs (snoRNAs) and over two hundred protein trans-acting factors. There are protein trans-acting factors predicted to participate in ribosome biogenesis that have not been so far characterized. Here, we report the functional analysis of the Nucleolar protein 6 (Nop6) in ribosome biogenesis. Our results show that Nop6 is needed for optimal 40S ribosomal subunit biogenesis. Deletion of NOP6 leads to an appropriate 20% reduction in 18S rRNA levels and therefore in 40S ribosomal subunits. This is due to mild inhibition of pre-rRNA processing at cleavage site A 2. Tandem affinity purification followed by mass spectrometry and northern blot analyses indicate that Nop6 is a component of 90S pre-ribosomal particles. rDNA chromatin immunoprecipitation experiments and analysis of the intracellular localisation of Nop6-eGFP after in vivo shut down of pre-rRNA transcription strongly suggest that Nop6 binds to the pre-rRNA early during transcription. Genetic data suggest that Nop6 and the snoRNA snR57 both interact similarly with the protein trans-acting factor Nep1. It has been proposed that snR57 and Nep1 participate in a pre-rRNA conformational switch that allows the proper assembly of 40S ribosomal protein S19. Our results strongly suggest that the role Nop6 might have in this conformational switch is independent of snR57.