Targeted Cross-Linking Mass Spectrometry on Single-Step Affinity Purified Molecular Complexes in the Yeast Saccharomyces cerevisiae.

Protein cross-linking mass spectrometry (XL-MS) has been developed into a powerful and robust tool that is now well implemented and routinely used by an increasing number of laboratories. While bulk cross-linking of complexes provides useful information on whole complexes, it is limiting for the probing of specific protein "neighbourhoods," or vicinity interactomes. For example, it is not unusual to find cross-linked peptide pairs that are disproportionately overrepresented compared to the surface areas of complexes, while very few or no cross-links are identified in other regions. When studying dynamic complexes along their pathways, some vicinity cross-links may be of too low abundance in the pool of heterogenous complexes of interest to be efficiently identified by standard XL-MS. In this chapter, we describe a targeted XL-MS approach from single-step affinity purified (ssAP) complexes that enables the investigation of specific protein "neighbourhoods" within molecular complexes in yeast, using a small cross-linker anchoring tag, the CH-tag. One advantage of this method over a general cross-linking strategy is the possibility to significantly enrich for localized anchored-cross-links within complexes, thus yielding a higher sensitivity to detect highly dynamic or low abundance protein interactions within a specific protein "neighbourhood" occurring along the pathway of a selected bait protein. Moreover, many variations of the method can be employed; the ssAP-tag and the CH-tag can either be fused to the same or different proteins in the complex, or the CH-tag can be fused to multiple protein components in the same cell line to explore dynamic vicinity interactions along a pathway.

Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway.

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.

DDX54 regulates transcriptome dynamics during DNA damage response.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A)+ RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.