Identification of the RNA polymerase I-RNA interactome.

Ribosome biogenesis is a complex process orchestrated by a host of ribosome assembly factors. Although it is known that many of the proteins involved in this process have RNA binding activity, the full repertoire of proteins that interact with the precursor ribosomal RNA is currently unknown. To gain a greater understanding of the extent to which RNA-protein interactions have the potential to control ribosome biogenesis, we used RNA affinity isolation coupled with proteomics to measure the changes in RNA-protein interactions that occur when rRNA transcription is blocked. Our analysis identified 211 out of 457 nuclear RNA binding proteins with a >3-fold decrease in RNA-protein interaction after inhibition of RNA polymerase I (RNAPI). We have designated these 211 RNA binding proteins as the RNAPI RNA interactome. As expected, the RNAPI RNA interactome is highly enriched for nucleolar proteins and proteins associated with ribosome biogenesis. Selected proteins from the interactome were shown to be nucleolar in location and to have RNA binding activity that was dependent on RNAPI activity. Furthermore, our data show that two proteins, which are required for rRNA maturation, AATF and NGDN, and which form part of the RNA interactome, both lack canonical RNA binding domains and yet are novel pre-rRNA binding proteins

Trans-acting translational regulatory RNA binding proteins.

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms

The cell stress response: extreme times call for post-transcriptional measures.

Following cell stress, a wide range of molecular pathways are initiated to orchestrate the stress response and enable adaptation to an environmental or intracellular perturbation. The post-transcriptional regulation strategies adopted during the stress response result in a substantial reorganization of gene expression, designed to prepare the cell for either acclimatization or programmed death, depending on the nature and intensity of the stress. Fundamental to the stress response is a rapid repression of global protein synthesis, commonly mediated by phosphorylation of translation initiation factor eIF2α. Recent structural and biochemical information have added unprecedented detail to our understanding of the molecular mechanisms underlying this regulation. During protein synthesis inhibition, the translation of stress-specific mRNAs is nonetheless enhanced, often through the interaction between RNA-binding proteins and specific RNA regulatory elements. Recent studies investigating the unfolded protein response (UPR) provide some important insights into how posttranscriptional events are spatially and temporally fine-tuned in order to elicit the most appropriate response and to coordinate the transition from an early, acute stage into the chronic state of adaptation. Importantly, cancer cells are known to hi-jack adaptive stress response pathways, particularly the UPR, to survive and proliferate in the unfavorable tumor environment. In this review, we consider the implications of recent research into stress-dependent post-transcriptional regulation and make the case for the exploration of the stress response as a strategy to identify novel targets in the development of cancer therapies. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Mechanisms > Translation Regulation

A cell-wide investigation of the RNA-binding proteins modulated by starvation and stimulation with insulin

The development of cutting-edge proteomic techniques has recently enabled to comprehensively catalogue more than a thousand of RBPs (RNA-binding proteins) across different cell types, tissues and whole organisms. Interestingly, these methods can be employed to interrogate the effect of either cell stress or stimulation on protein-RNA interactions and can be used to uncover novel RBP candidates or new functions of well-known RBPs.This study aimed at investigating RBPs dynamics in untransformed epithelial MCF10A cells subjected to growth factor deprivation and stimulation with insulin. Both treatments have been shown to modulate RNA metabolism and primarily mRNA translation through regulation of the mTOR signalling pathway.By performing OOPS (Orthogonal organic phase separation), it was possible to identify a large number of RBPs, the interaction of which was significantly affected by starvation and stimulation with insulin, including the tumour suppressor PDCD4 (Programmed cell death protein 4).Downregulation of PDCD4 has been observed to enhance tumour progression and invasion of many different cancers. The protein is primarily a translation repressor; it impedes the physical interaction between eIF4A and eIF4G and it compromises the expression of mRNAs with highly structured 5’UTR. However, PDCD4 was also found to directly bind specific region within the coding sequence of few mRNAs and slow down their translation elongation.Although the protein harbours an N-terminal disordered region with RNA-binding potential, its relevance and its direct RNA targets have not been fully investigated in a genome-wide study. With this in mind, the PDCD4-bound transcriptome was investigated by iCLIP (Individual-nucleotide resolution UV crosslinking and immunoprecipitation) in untransformed cells and it revealed more than 150 RNA partners, the regulation of which might play a significant role during cancer development.</div

Do entrepreneurial knowledge and innovative attitude overcome “imperfections” in the innovation process? Insights from SMEs in the UK and Italy

Purpose Entrepreneurial knowledge spurs innovation and, in turn, generates a competitive advantage. This paper aims to explore if entrepreneurial knowledge combined with the attitude to innovate can overcome the key “imperfections” of the innovation process generated by dynamic, current technological progress in the knowledge-intensive sector. The “imperfections” identified in risk management, asymmetric information in the knowledge management process and hold-up problems can all disrupt collaborative partnerships and limit opportunities for innovation. Design/methodology/approach A theory-building approach is applied which offers a case study analysis of two small- to medium-sized enterprises (SMEs). These two SMEs operate in Europe but in two different territories: the UK and Italy. The study explores three key imperfections, risk management, asymmetric information in the knowledge management process and hold-up problems, which occur in the innovation process. Findings The entrepreneurs face these imperfections by adopting an open innovation model. Notwithstanding, both entrepreneurs had to deal with all “imperfections”, and their skills, attributes, attitude and aptitude allowed them to grow their business and continually develop new products. Therefore, the imperfections do not limit the innovative capacity of an entrepreneur but rather enhance their challengeable attitude. In this regard, the case studies induce a further analysis on entrepreneurial knowledge intertwined with entrepreneurial risk management and networking skills. Research limitations/implications The empirical significance of the two cases does not allow theorisation. However, this research offers interesting results which can be strengthened by a comparative case study with other countries or deeper investigation by applying a quantitative approach. Originality/value By leveraging entrepreneurial knowledge, the imperfections noted in the innovation process can be overcome. Entrepreneurial knowledge is recognised as the main asset of an enterprise if it is combined with external talent or human resources. Entrepreneurs aim to develop innovative approaches and ideas through establishing both formal and informal collaborative partnership relationships which are used thanks to the entrepreneurs’ networking skills, knowledge and abilitie

System-wide analysis of RNA and protein subcellular localization dynamics.

Acknowledgements: We thank C. De Matos Ferraz Franco of the MRC-Toxicology Proteomics Facility for collection of all MS data and D. Scadden for kindly providing the G3BP1–GFP expression plasmid, B. Fisher for kindly sharing equipment and M. Marti-Solano for assistance with the manuscript writing. We also thank the Advanced Light Imaging facility at the MRC Toxicology Unit and the Light Imaging Facility at the Human Technopole for their support in the acquisition of light microscopy data. E.V., T.S., R.M.L.Q., M.P. and M.E. are supported by Wellcome Trust, grant numbers 110170/Z/15/Z and 110071/Z/15/Z awarded to A.E.W. and K.S.L.; R.F.H, V.D. and M.M. are supported by the Medical Research Council, grant number 5TR00. L.M.B. is supported by EU Horizon 2020 programme INFRAIA project EPIC-XS (project 823839). O.M.C. was supported by a Wellcome Trust Mathematical Genomics and Medicine studentship funded by the Cambridge School of Clinical Medicine and by the Todd-Bird Junior Research Fellowship from New College, Oxford.Funder: Mathematical Genomics and Medicine studentship funded by the Cambridge School of Clinical MedicineAlthough the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics

Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism