Taxonomy of introns and the evolution of minor introns.

Classification of introns, which is crucial to understanding their evolution and splicing, has historically been binary and has resulted in the naming of major and minor introns that are spliced by their namesake spliceosome. However, a broad range of intron consensus sequences exist, leading us to here reclassify introns as minor, minor-like, hybrid, major-like, major and non-canonical introns in 263 species across six eukaryotic supergroups. Through intron orthology analysis, we discovered that minor-like introns are a transitory node for intron conversion across evolution. Despite close resemblance of their consensus sequences to minor introns, these introns possess an AG dinucleotide at the -1 and -2 position of the 5' splice site, a salient feature of major introns. Through combined analysis of CoLa-seq, CLIP-seq for major and minor spliceosome components, and RNAseq from samples in which the minor spliceosome is inhibited we found that minor-like introns are also an intermediate class from a splicing mechanism perspective. Importantly, this analysis has provided insight into the sequence elements that have evolved to make minor-like introns amenable to recognition by both minor and major spliceosome components. We hope that this revised intron classification provides a new framework to study intron evolution and splicing.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

Disruption of exon-bridging interactions between the minor and major spliceosomes results in alternative splicing around minor introns.

Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry-Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.

Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective Rnpc3-deficient mice.

Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by RNPC3, is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development. To pinpoint the impact of 65K loss during mammalian development and in adulthood, we generated germline and conditional Rnpc3-deficient mice. Homozygous Rnpc3-/- embryos died prior to blastocyst implantation, whereas Rnpc3+/- mice were born at the expected frequency, achieved sexual maturity, and exhibited a completely normal lifespan. Systemic recombination of conditional Rnpc3 alleles in adult (Rnpc3lox/lox ) mice caused rapid weight loss, leukopenia, and degeneration of the epithelial lining of the entire gastrointestinal tract, the latter due to increased cell death and a reduction in cell proliferation. Accompanying this, we observed a loss of both 65K and the pro-proliferative phospho-ERK1/2 proteins from the stem/progenitor cells at the base of intestinal crypts. RT-PCR analysis of RNA extracted from purified preparations of intestinal epithelial cells with recombined Rnpc3lox alleles revealed increased frequency of U12-type intron retention in all transcripts tested. Our study, using a novel conditional mouse model of Rnpc3 deficiency, establishes that U12-dependent splicing is not only important during development but is indispensable throughout life.

Eph-ephrin signaling modulated by polymerization and condensation of receptors.

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization-condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.

ahctf1 and kras mutations combine to amplify oncogenic stress and restrict liver overgrowth in a zebrafish model of hepatocellular carcinoma.

The nucleoporin (NUP) ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using both larval and adult zebrafish models of hepatocellular carcinoma (HCC), in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly decreases liver volume, while non-hyperplastic tissues are unaffected. We demonstrate that in the context of cancer, ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly, and chromosome segregation, leading to DNA damage and activation of a Tp53-dependent transcriptional programme that induces cell death and cell cycle arrest. Heterozygous expression of both ahctf1 and ranbp2 (encoding a second nucleoporin), or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocks krasG12V-driven hepatocyte hyperplasia. Gene expression analysis of patient samples in the liver hepatocellular carcinoma (LIHC) dataset in The Cancer Genome Atlas shows that high expression of one or more of the transcripts encoding the 10 components of the NUP107-160 subcomplex, which includes AHCTF1, is positively correlated with worse overall survival. These results provide a strong and feasible rationale for the development of novel cancer therapeutics that target ELYS function and suggest potential avenues for effective combinatorial treatments.

Delineation of the molecular mechanism for disulfide stress-induced aluminium toxicity.

Following our previous finding that the sulfhydryl-oxidising chemical diamide induced a marked elevation of cellular Al(3+) (Wu et al., Int J Mol Sci, 12:8119-8132, 2011), a further investigation into the underlying molecular mechanism was carried out, using the eukaryotic model organism Saccharomyces cerevisiae. The effects of non-toxic dose of diamide (0.8 mM) and a mild dose of aluminium sulphate (Al(3+)) (0.4 mM) were determined prior to the screening of gene deletion mutants. A total of 81 deletion mutants were selected for this study according to the available screening data against Al(3+) only (Kakimoto et al., BioMetals, 18: 467-474, 2005) and diamide only (Thorpe et al., Proc Natl Acad Sci USA, 101: 6564-6569, 2004). On the basis of our screening data and the cluster analysis, a cluster containing the gene deletions (rpe1∆, sec72∆, pdr5∆ and ric1∆) was found to be specifically sensitive to the mixture of diamide and Al(3+). However gnp1∆, mch5∆ and ccc1∆ mutants were resistant. Dithiothreitol (DTT) and ascorbate markedly reversed the diamide-induced Al(3+) toxicity. Inductively-coupled plasma optical emission spectrometry demonstrated that DTT reduced the intracellular Al(3+) content in diamide/Al(3+)-treated yeast cells six-fold compared to the non-DTT controls. These data together revealed that the pleiotropic drug resistance transporter (Pdr5p) and vacuolar/vesicular transport-related proteins (Ric1p and Sec72p) are the targets of diamide. A dysfunctional membrane-bound Pdr5p terminates the detoxification pathway for Al(3+) at the final step, leading to intracellular Al(3+) accumulation and hence toxicity. As Al(3+) toxicity has been a problem in agriculture and human health, this study has provided a significant step forward in understanding Al(3+) toxicity.

Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms.

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells.

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour.

Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy.

The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell.

Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.