The Impact of ETV6-NTRK3 Oncogenic Gene Fusions on Molecular and Signaling Pathway Alterations.

Chromosomal translocations creating fusion genes are common cancer drivers. The oncogenic ETV6-NTRK3 (EN) gene fusion joins the sterile alpha domain of the ETV6 transcription factor with the tyrosine kinase domain of the neurotrophin-3 receptor NTRK3. Four EN variants with alternating break points have since been detected in a wide range of human cancers. To provide molecular level insight into EN oncogenesis, we employed a proximity labeling mass spectrometry approach to define the molecular context of the fusions. We identify in total 237 high-confidence interactors, which link EN fusions to several key signaling pathways, including ERBB, insulin and JAK/STAT. We then assessed the effects of EN variants on these pathways, and showed that the pan NTRK inhibitor Selitrectinib (LOXO-195) inhibits the oncogenic activity of EN2, the most common variant. This systems-level analysis defines the molecular framework in which EN oncofusions operate to promote cancer and provides some mechanisms for therapeutics.

A novel partial de novo duplication of JARID2 gene causing a neurodevelopmental phenotype.

BACKGROUND: Deletions covering the entire or partial JARID2 gene as well as pathogenic single nucleotide variants leading to haploinsufficiency of JARID2 have recently been shown to cause a clinically distinct neurodevelopmental syndrome. Here, we present a previously undescribed partial de novo duplication of the JARID2 gene in a patient displaying features similar to those of patients with JARID2 loss-of-function variants. CASE REPORT: The index patient presents with abnormalities in gross motor skills and speech development as well as neuropsychiatric disorders. The patient has markedly dark infraorbital circles and slightly prominent supraorbital ridges.Whole-genome sequencing and array comparative genomic hybridization revealed a novel disease-causing variant type, a partial tandem duplication of JARID2, covering the exons 1-7. Furthermore, RNA sequencing validated the increased expression of these exons. Expression alterations were also detected in target genes of the PRC2 complex, in which JARID2 acts as an essential member. CONCLUSION: Our data add to the variety of different pathogenic variants associated with JARID2 neurodevelopmental syndrome.

Depletion of Mediator Kinase Module Subunits Represses Superenhancer-Associated Genes in Colon Cancer Cells.

In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by ß-catenin, which has previously been shown to associate with MED12. Importantly, ß-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.

Stromal Lkb1 deficiency leads to gastrointestinal tumorigenesis involving the IL-11-JAK/STAT3 pathway.

Germline mutations in the gene encoding tumor suppressor kinase LKB1 lead to gastrointestinal tumorigenesis in Peutz-Jeghers syndrome (PJS) patients and mouse models; however, the cell types and signaling pathways underlying tumor formation are unknown. Here, we demonstrated that mesenchymal progenitor- or stromal fibroblast-specific deletion of Lkb1 results in fully penetrant polyposis in mice. Lineage tracing and immunohistochemical analyses revealed clonal expansion of Lkb1-deficient myofibroblast-like cell foci in the tumor stroma. Loss of Lkb1 in stromal cells was associated with induction of an inflammatory program including IL-11 production and activation of the JAK/STAT3 pathway in tumor epithelia concomitant with proliferation. Importantly, treatment of LKB1-defcient mice with the JAK1/2 inhibitor ruxolitinib dramatically decreased polyposis. These data indicate that IL-11-mediated induction of JAK/STAT3 is critical in gastrointestinal tumorigenesis following Lkb1 mutations and suggest that targeting this pathway has therapeutic potential in Peutz-Jeghers syndrome.

Protein Coating of DNA Nanostructures for Enhanced Stability and Immunocompatibility.

Fully addressable DNA nanostructures, especially DNA origami, possess huge potential to serve as inherently biocompatible and versatile molecular platforms. However, their use as delivery vehicles in therapeutics is compromised by their low stability and poor transfection rates. This study shows that DNA origami can be coated by precisely defined one-to-one protein-dendron conjugates to tackle the aforementioned issues. The dendron part of the conjugate serves as a cationic binding domain that attaches to the negatively charged DNA origami surface via electrostatic interactions. The protein is attached to dendron through cysteine-maleimide bond, making the modular approach highly versatile. This work demonstrates the coating using two different proteins: bovine serum albumin (BSA) and class II hydrophobin (HFBI). The results reveal that BSA-coating significantly improves the origami stability against endonucleases (DNase I) and enhances the transfection into human embryonic kidney (HEK293) cells. Importantly, it is observed that BSA-coating attenuates the activation of immune response in mouse primary splenocytes. Serum albumin is the most abundant protein in the blood with a long circulation half-life and has already found clinically approved applications in drug delivery. It is therefore envisioned that the proposed system can open up further opportunities to tune the properties of DNA nanostructures in biological environment, and enable their use in various delivery applications.

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5' splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11-48K and U11/U12-65K genes.

Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5' splice sites (5'ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50 nt downstream from the target 3' splice site (3'ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3'ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5'ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5'ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.

Global analysis of the nuclear processing of transcripts with unspliced U12-type introns by the exosome.

U12-type introns are a rare class of introns in the genomes of diverse eukaryotes. In the human genome, they number over 700. A subset of these introns has been shown to be spliced at a slower rate compared to the major U2-type introns. This suggests a rate-limiting regulatory function for the minor spliceosome in the processing of transcripts containing U12-type introns. However, both the generality of slower splicing and the subsequent fate of partially processed pre-mRNAs remained unknown. Here, we present a global analysis of the nuclear retention of transcripts containing U12-type introns and provide evidence for the nuclear decay of such transcripts in human cells. Using SOLiD RNA sequencing technology, we find that, in normal cells, U12-type introns are on average 2-fold more retained than the surrounding U2-type introns. Furthermore, we find that knockdown of RRP41 and DIS3 subunits of the exosome stabilizes an overlapping set of U12-type introns. RRP41 knockdown leads to slower decay kinetics of U12-type introns and globally upregulates the retention of U12-type, but not U2-type, introns. Our results indicate that U12-type introns are spliced less efficiently and are targeted by the exosome. These characteristics support their role in the regulation of cellular mRNA levels.

Virus-encapsulated DNA origami nanostructures for cellular delivery.

DNA origami structures can be programmed into arbitrary shapes with nanometer scale precision, which opens up numerous attractive opportunities to engineer novel functional materials. One intriguing possibility is to use DNA origamis for fully tunable, targeted, and triggered drug delivery. In this work, we demonstrate the coating of DNA origami nanostructures with virus capsid proteins for enhancing cellular delivery. Our approach utilizes purified cowpea chlorotic mottle virus capsid proteins that can bind and self-assemble on the origami surface through electrostatic interactions and further pack the origami nanostructures inside the viral capsid. Confocal microscopy imaging and transfection studies with a human HEK293 cell line indicate that protein coating improves cellular attachment and delivery of origamis into the cells by 13-fold compared to bare DNA origamis. The presented method could readily find applications not only in sophisticated drug delivery applications but also in organizing intracellular reactions by origami-based templates.

Regulation of gene expression through inefficient splicing of U12-type introns.

U12-type introns are a rare class of nuclear introns that are removed by a dedicated U12-dependent spliceosome and are thought to regulate the expression of their target genes owing through their slower splicing reaction. Recent genome-wide studies on the splicing of U12-type introns are now providing new insights on the biological significance of this parallel splicing machinery. The new studies cover multiple different organisms and experimental systems, including human patient cells with mutations in the components of the minor spliceosome, zebrafish with similar mutations and various experimentally manipulated human cells and Arabidopsis plants. Here, we will discuss the potential implications of these studies on the understanding of the mechanism and regulation of the minor spliceosome, as well as their medical implications.

The significant other: splicing by the minor spliceosome.

The removal of non-coding sequences, introns, from the mRNA precursors is an essential step in eukaryotic gene expression. U12-type introns are a minor subgroup of introns, distinct from the major or U2-type introns. U12-type introns are present in most eukaryotes but only account for less than 0.5% of all introns in any given genome. They are processed by a specific U12-dependent spliceosome, which is similar to, but distinct from, the major spliceosome. U12-type introns are spliced somewhat less efficiently than the major introns, and it is believed that this limits the expression of the genes containing such introns. Recent findings on the role of U12-dependent splicing in development and human disease have shown that it can also affect multiple cellular processes not directly related to the functions of the host genes of U12-type introns. At the same time, advances in understanding the regulation and phylogenetic distribution of the minor spliceosome are starting to shed light on how the U12-type introns and the minor spliceosome may have evolved.

An ancient mechanism for splicing control: U11 snRNP as an activator of alternative splicing.

Alternative pre-mRNA splicing is typically regulated by specific protein factors that recognize unique sequence elements in pre-mRNA and affect, directly or indirectly, nearby splice site usage. We show that 5' splice site sequences (5'ss) of U12-type introns, when repeated in tandem, form a U11 snRNP-binding splicing enhancer, USSE. Binding of U11 to the USSE regulates alternative splicing of U2-type introns by activating an upstream 3'ss. The U12-type 5'ss-like sequences within the USSE have a regulatory role and do not function as splicing donors. USSEs, present both in animal and plant genes encoding the U11/U12 di-snRNP-specific 48K and 65K proteins, create sensitive switches that respond to intracellular levels of functional U11 snRNP and alter the stability of 48K and 65K mRNAs. We conclude that U11 functions not only in 5'ss recognition in constitutive splicing, but also as an activator of U2-dependent alternative splicing and as a regulator of the U12-dependent spliceosome.

Neural and mechanical responses of the triceps surae muscle group after 1 h of repeated fast passive stretches.

Experiments were carried out to examine interaction between mechanical changes of the muscle-tendon unit and reduced reflex sensitivity after repeated and prolonged passive muscle stretching (RPS). There is some evidence that this interaction might be relevant also during active stretch-shortening cycle type of fatigue tasks. The results demonstrated a clear deterioration of voluntarily and electrically induced muscle contractions after RPS. Maximal voluntary contraction (MVC), average electromyographic activities of the gastrocnemius and soleus muscles, and maximal twitch contraction decreased on average by 13.8, 10.4, 7.6, and 16.8%, respectively. In addition, there was a 14% lengthening in the total duration of the twitch. MVCs measured at different ankle joint angles revealed a downward and rightward shift in the torque-fascicle length curve after RPS. Interestingly, there was a crossing in the torque-fascicle length curves while measured at different activation levels but at the same joint angle before and after RPS. Even though no changes were observed in the activation level during MVCs, all the reflex parameters showed a clear reduction after RPS. This study presents evidence that repeated and prolonged passive muscle stretching can lead to some modification of material behavior of the aponeurosis-tendon system, such as stress relaxation and/or plastic deformation. In addition, altered material properties seem to affect proprioceptive feedback and, therefore, the motor unit activation in proportion to the contractile failure.