Trim25 is an RNA-specific activator of Lin28a/TuT4-mediated uridylation

SummaryRNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity

Towards an understanding of the role the culture of designers can play in organisations

This thesis contributes to the debate about the role of design and designers in a modern organisation. It aims to engage in academic dialogue through an exploration of the way designers perceive themselves and their methods (and also how they are being perceived); providing an outline of their professional culture and its interactions in an organisation. Its rationale is based on the assumption that various occupational and professional cultures can have significant influence on the way organisations are run. Thus, by examining the dimensions and sense-making within the professional culture of designers, this PhD aims to establish the analytical grounds for understanding the role design and designers in an organisational setting. By conducting a number of in-depth interviews with general managers, senior designers, management consultants and design managers from IDEO, Wolff Olins, Nissan Design and Philips Design this thesis looks at constructs attributed to designers that emerge at the core of their culture. Grounded theory methodology helped to establish a picture of the cultural arrangements in design-intensive organisations. 'Consolidating multidimensional meanings', `Creating bringing to 4/, Embracing discontinuityand gbenendedness', Embracing persona/ and commercial empathy', Engaging porensorial aesthetics', are the five theoretical categories that have been recognised as important to describing designers' professional culture. The analysis suggests that designers, by focusing on adding fundamental rake through unconfined exploration can play the part of 0/J-temological change agetas'in an organisation. Additionally, the thesis develops a model of design's influence on organisations. According to this model there are three 'channels' through which design affects organisations: (i) frameniarhs such as ontologies, epistemologies, theories, methods, tools, techniques etc., (ii) outcomes including physical artefacts, environments, brands and experiences, (iii) a'esigwers and their professional culture with certain values, assumptions, skills, preferences, behaviours etc. Designers, as a professional group, might have a more or less active roles within those groups of influences, modifying them in accordance with their culture

RNA-targeted Therapies and High-throughput Screening Methods

RNA-binding proteins (RBPs) are involved in regulating all aspects of RNA metabolism, including processing, transport, translation, and degradation. Dysregulation of RNA metabolism is linked to a plethora of diseases, such as cancer, neurodegenerative diseases, and neuromuscular disorders. Recent years have seen a dramatic shift in the knowledge base, with RNA increasingly being recognised as an attractive target for precision medicine therapies. In this article, we are going to review current RNA-targeted therapies. Furthermore, we will scrutinise a range of drug discoveries targeting protein-RNA interactions. In particular, we will focus on the interplay between Lin28 and let-7, splicing regulatory proteins and survival motor neuron (SMN) pre-mRNA, as well as HuR, Musashi, proteins and their RNA targets. We will highlight the mechanisms RBPs utilise to modulate RNA metabolism and discuss current high-throughput screening strategies. This review provides evidence that we are entering a new era of RNA-targeted medicine

Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain-of-function toxicity mechanism contributing to the pathogenesis of fragile X-associated premature ovarian insufficiency

Fragile X-associated premature ovarian insufficiency (FXPOI) is among a family of disorders caused by expansion of a CGG trinucleotide repeat sequence located in the 5’ untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene on the X chromosome. Women with FXPOI have a depleted ovarian follicle population, resulting in amenorrhea, hypoestrogenism, and loss of fertility before the age of 40. FXPOI is caused by expansions of the CGG sequence to lengths between 55 and 200 repeats, known as a FMRI premutation, however the mechanism by which the premutation drives disease pathogenesis remains unclear. Two main hypotheses exist, which describe an mRNA toxic gain-of-function mechanism or a protein-based mechanism, where repeat-associated non-AUG (RAN) translation results in the production of an abnormal protein, called FMRpolyG. Here, we have developed an in vitro granulosa cell model of the FMR1 premutation by ectopically expressing CGG-repeat RNA and FMRpolyG protein. We show that expanded CGG-repeat RNA accumulated in intranuclear RNA structures, and these aggregates were able to cause significant granulosa cell death independent of FMRpolyG expression. Using an innovative RNA pulldown, mass spectrometry-based approach we have identified proteins that are specifically sequestered by CGG RNA aggregates in granulosa cells in vitro, and thus may be deregulated as consequence of this interaction. Furthermore, we have demonstrated reduced expression of three proteins identified via our RNA pulldown (FUS, PA2G4 and TRA2β) in ovarian follicles in a FMR1 premutation mouse model. Collectively, these data provide evidence for the contribution of an mRNA gain-of-function mechanism to FXPOI disease biology

Tissue-specific control of brain-enriched miR-7 biogenesis

MicroRNA (miRNA) biogenesis is a highly regulated process in eukaryotic cells. Several mature miRNAs exhibit a tissue-specific pattern of expression without an apparent tissue-specific pattern for their corresponding primary transcripts. This discrepancy is suggestive of post-transcriptional regulation of miRNA abundance. Here, we demonstrate that the brain-enriched expression of miR-7, which is processed from the ubiquitous hnRNP K pre-mRNA transcript, is achieved by inhibition of its biogenesis in nonbrain cells in both human and mouse systems. Using stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry combined with RNase-assisted RNA pull-down, we identified Musashi homolog 2 (MSI2) and Hu antigen R (HuR) proteins as inhibitors of miR-7 processing in nonneural cells. This is achieved through HuR-mediated binding of MSI2 to the conserved terminal loop of pri-miR-7. Footprinting and electrophoretic gel mobility shift analysis (EMSA) provide further evidence for a direct interaction between pri-miR-7-1 and the HuR/MSI2 complex, resulting in stabilization of the pri-miR-7-1 structure. We also confirmed the physiological relevance of this inhibitory mechanism in a neuronal differentiation system using human SH-SY5Y cells. Finally, we show elevated levels of miR-7 in selected tissues from MSI2 knockout (KO) mice without apparent changes in the abundance of the pri-miR-7 transcript. Altogether, our data provide the first insight into the regulation of brain-enriched miRNA processing by defined tissue-specific factors

Posttranscriptional Regulation of 14q32 microRNAs by RNA Binding Proteins CIRBP and HADHB during Vascular Regeneration after Ischemia:Posttranscriptional regulation of 14q32 microRNAs

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB−/− 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs. Keywords: microRNA, 14q32, microRNA cluster, miR-329, miR-495, HADHB, CIRBP, RNA binding proteins, ischemia, hindlimb ischemia mode

Loss of 5-methylcytosine alters the biogenesis of vault-derived small RNAs to coordinate epidermal differentiation.

The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.We thank everybody who provided us with reagents, in particular we thank James Stevenin for sending us recombinant SRSF2. We gratefully acknowledge the support of all the WT-MRC Stem Cell Institute core facility managers. This work was funded by Cancer Research UK (CR-630 UK) and the European Research Council (ERC). Parts of this research in Michaela Frye's laboratory was supported by core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute. Juri Rappsilber’s laboratory was supported by Wellcome Trust Senior Research Fellowship (084229). Gracjan Michlewski’s laboratory was supported by the MRC Career Development Award (G10000564), Wellcome Trust Seed Award (210144/Z/18/Z) and Wellcome Trust Centre for Cell Biology Core Grants (077707 and 092076). Abdulrahim Sajini was supported by a scholarship from the University of Tabuk and Khalifa University of Science and Technology Faculty start-up award number FSU-2018-01. Rebecca Wagner was supported by the Wellcome Trust PhD Programme in Stem Cell Biology & Medicine