A MYC-GCN2-eIF2α negative feedback loop limits protein synthesis to prevent MYC-dependent apoptosis in colorectal cancer.

Tumours depend on altered rates of protein synthesis for growth and survival, which suggests that mechanisms controlling mRNA translation may be exploitable for therapy. Here, we show that loss of APC, which occurs almost universally in colorectal tumours, strongly enhances the dependence on the translation initiation factor eIF2B5. Depletion of eIF2B5 induces an integrated stress response and enhances translation of MYC via an internal ribosomal entry site. This perturbs cellular amino acid and nucleotide pools, strains energy resources and causes MYC-dependent apoptosis. eIF2B5 limits MYC expression and prevents apoptosis in APC-deficient murine and patient-derived organoids and in APC-deficient murine intestinal epithelia in vivo. Conversely, the high MYC levels present in APC-deficient cells induce phosphorylation of eIF2α via the kinases GCN2 and PKR. Pharmacological inhibition of GCN2 phenocopies eIF2B5 depletion and has therapeutic efficacy in tumour organoids, which demonstrates that a negative MYC-eIF2α feedback loop constitutes a targetable vulnerability of colorectal tumours.

Serine threonine kinase receptor associated protein regulates early follicle development in the mouse ovary.

The molecular mechanisms involved in regulating the development of small, gonadotrophin-independent follicles are poorly understood; however, many studies have highlighted an essential role for TGFB ligands. Canonical TGFB signalling is dependent upon intracellular SMAD proteins that regulate transcription. STRAP has been identified in other tissues as an inhibitor of the TGFB-SMAD signalling pathway. Therefore, in this study we aimed to determine the expression and role of STRAP in the context of early follicle development. Using qPCR, Strap, Smad3 and Smad7 revealed similar expression profiles in immature ovaries from mice aged 4-16 days containing different populations of early growing follicles. STRAP and SMAD2/3 proteins co-localised in granulosa cells of small follicles using immunofluorescence. Using an established culture model, neonatal mouse ovary fragments with a high density of small non-growing follicles were used to examine the effects of Strap knockdown using siRNA and STRAP protein inhibition by immuno-neutralisation. Both interventions caused a reduction in the proportion of small, non-growing follicles and an increase in the proportion and size of growing follicles in comparison to untreated controls, suggesting inhibition of STRAP facilitates follicle activation. Recombinant STRAP protein had no effect on small, non-growing follicles, but increased the mean oocyte size of growing follicles in the neonatal ovary model and also promoted the growth of isolated preantral follicles in vitro Overall findings indicate STRAP is expressed in the mouse ovary and is capable of regulating development of small follicles in a stage-dependent manner.

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

lin28 proteins promote expression of 17∼92 family miRNAs during amphibian development.

BACKGROUND: Lin28 proteins are post-transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let-7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let-7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let-7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure. RESULTS: Here, we show that mir-17∼92 and mir-106∼363 cluster miRNAs are down-regulated in response to lin28 knockdown, and RNAs from these clusters are co-expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre-mir-363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre-mir-363 with an affinity similar to that of let-7, and that this high affinity interaction requires to conserved a GGAG motif. CONCLUSIONS: Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir-17∼92 family miRNAs.

Lin28 proteins are required for germ layer specification in Xenopus.

Lin28 family proteins share a unique structure, with both zinc knuckle and cold shock RNA-binding domains, and were originally identified as regulators of developmental timing in Caenorhabditis elegans. They have since been implicated as regulators of pluripotency in mammalian stem cells in culture. Using Xenopus tropicalis, we have undertaken the first analysis of the effects on the early development of a vertebrate embryo resulting from global inhibition of the Lin28 family. The Xenopus genome contains two Lin28-related genes, lin28a and lin28b. lin28a is expressed zygotically, whereas lin28b is expressed both zygotically and maternally. Both lin28a and lin28b are expressed in pluripotent cells of the Xenopus embryo and are enriched in cells that respond to mesoderm-inducing signals. The development of axial and paraxial mesoderm is severely abnormal in lin28 knockdown (morphant) embryos. In culture, the ability of pluripotent cells from the embryo to respond to the FGF and activin/nodal-like mesoderm-inducing pathways is compromised following inhibition of lin28 function. Furthermore, there are complex effects on the temporal regulation of, and the responses to, mesoderm-inducing signals in lin28 morphant embryos. We provide evidence that Xenopus lin28 proteins play a key role in choreographing the responses of pluripotent cells in the early embryo to the signals that regulate germ layer specification, and that this early function is probably independent of the recognised role of Lin28 proteins in negatively regulating let-7 miRNA biogenesis.

Identification of novel Notch target genes in T cell leukaemia.

BACKGROUND: Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. RESULTS: RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1DeltaE or N3DeltaE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1). CONCLUSION: The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.