Silencing of SRRM4 suppresses microexon inclusion and promotes tumor growth across cancers.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.

Engineering of weak helper interactions for high-efficiency FRET probes.

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

Mammary Microvessels are Sensitive to Menstrual Cycle Sex Hormones.

The mammary gland is a highly vascularized organ influenced by sex hormones including estrogen (E2) and progesterone (P4). Beyond whole-organism studies in rodents or cell monocultures, hormonal effects on the breast microvasculature remain largely understudied. Recent methods to generate 3D microvessels on-chip have enabled direct observation of complex vascular processes; however, these models often use non-tissue-specific cell types, such as human umbilical vein endothelial cells (HUVECs) and fibroblasts from various sources. Here, novel mammary-specific microvessels are generated by coculturing primary breast endothelial cells and fibroblasts under optimized culture conditions. These microvessels are mechanosensitive (to interstitial flow) and require endothelial-stromal interactions to develop fully perfusable vessels. These mammary-specific microvessels are also responsive to exogenous stimulation by sex hormones. When treated with combined E2 and P4, corresponding to the four phases of the menstrual cycle (period, follicular, ovular, and luteal), vascular remodeling and barrier function are altered in a phase-dependent manner. The presence of high E2 (ovulation) promotes vascular growth and remodeling, corresponding to high depletion of proangiogenic factors, whereas high P4 concentrations (luteal) promote vascular regression. The effects of combined E2 and P4 hormones are not only dose-dependent but also tissue-specific, as are shown by similarly treating non-tissue-specific HUVEC microvessels.

Flow in fetoplacental-like microvessels in vitro enhances perfusion, barrier function, and matrix stability.

Proper placental vascularization is vital for pregnancy outcomes, but assessing it with animal models and human explants has limitations. We introduce a 3D in vitro model of human placenta terminal villi including fetal mesenchyme and vascular endothelium. By coculturing HUVEC, placental fibroblasts, and pericytes in a macrofluidic chip with a flow reservoir, we generate fully perfusable fetal microvessels. Pressure-driven flow facilitates microvessel growth and remodeling, resulting in early formation of interconnected and lasting placental-like vascular networks. Computational fluid dynamics simulations predict shear forces, which increase microtissue stiffness, decrease diffusivity, and enhance barrier function as shear stress rises. Mass spectrometry analysis reveals enhanced protein expression with flow, including matrix stability regulators, proteins associated with actin dynamics, and cytoskeleton organization. Our model provides a powerful tool for deducing complex in vivo parameters, such as shear stress on developing vascularized placental tissue, and holds promise for unraveling gestational disorders related to the vasculature.

Classical and Non-classical Fibrosis Phenotypes Are Revealed by Lung and Cardiac Like Microvascular Tissues On-Chip.

Fibrosis, a hallmark of many cardiac and pulmonary diseases, is characterized by excess deposition of extracellular matrix proteins and increased tissue stiffness. This serious pathologic condition is thought to stem majorly from local stromal cell activation. Most studies have focused on the role of fibroblasts; however, the endothelium has been implicated in fibrosis through direct and indirect contributions. Here, we present a 3D vascular model to investigate vessel-stroma crosstalk in normal conditions and following induced fibrosis. Human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are co-cultured with (and without) primary human cardiac and lung fibroblasts (LFs) in a microfluidic device to generate perfusable microvasculature in cardiac- and pulmonary-like microenvironments. Endothelial barrier function, vascular morphology, and matrix properties (stiffness and diffusivity) are differentially impacted by the presence of stromal cells. These vessels (with and without stromal cells) express inflammatory cytokines, which could induce a wound-healing state. Further treatment with transforming growth factor-ß (TGF-ß) induced varied fibrotic phenotypes on-chip, with LFs resulting in increased stiffness, lower MMP activity, and increased smooth muscle actin expression. Taken together, our work demonstrates the strong impact of stromal-endothelial interactions on vessel formation and extravascular matrix regulation. The role of TGF-ß is shown to affect co-cultured microvessels differentially and has a severe negative impact on the endothelium without stromal cell support. Our human 3D in vitro model has the potential to examine anti-fibrotic therapies on patient-specific hiPSCs in the future.

rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions.

Knowing which proteins and RNAs directly interact is essential for understanding cellular mechanisms. Unfortunately, discovering such interactions is costly and often unreliable. To overcome these limitations, we developed rec-YnH, a new yeast two and three-hybrid-based screening pipeline capable of detecting interactions within protein libraries or between protein libraries and RNA fragment pools. rec-YnH combines batch cloning and transformation with intracellular homologous recombination to generate bait-prey fusion libraries. By developing interaction selection in liquid-gels and using an ORF sequence-based readout of interactions via next-generation sequencing, we eliminate laborious plating and barcoding steps required by existing methods. We use rec-Y2H to simultaneously map interactions of protein domains and reveal novel putative interactors of PAR proteins. We further employ rec-Y2H to predict the architecture of published coprecipitated complexes. Finally, we use rec-Y3H to map interactions between multiple RNA-binding proteins and RNAs-the first time interactions between protein and RNA pools are simultaneously detected.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

BACKGROUND: Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). METHODS: The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. RESULTS: Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. CONCLUSIONS: Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

Measuring activity of endocytosis-regulating factors in T-lymphocytes by flow cytometry.

Elucidation of the mechanisms regulating membrane traffic of lymphocyte receptors is of great interest to manipulate the immune response, as well as for accurately delivering drugs and nanoprobes to cells. Aiming to detect and characterize regulators of endocytosis and intracellular traffic, we have modified the FACS-based endocytosis assay to measure and quantify the activity of putative endocytic regulators as EGFP chimeras. To study the activity of putative endocytosis regulators, we transfected Jurkat T-lymphocytes with EGFP-tagged constructs of the regulators to be tested. Cells were then incubated with a αCD3(APC) antibody, and were allowed to internalize the label. After acid-washing the cells, APC fluorescence was measured by flow cytometry in cells gated for EGFP(+), as well as in their EGFP(-) (transfection-resistant) counterparts that were taken as internal controls. This approach facilitated intra- and inter-assay normalization of endocytic rates/loads by comparison with the internal control. We have used this assay to test the regulatory activity of polarity kinase EMK1, and here we substantiate a role for EMK1 in the control of receptor internalization in T-lymphocytes. The method here presented gives quantitative measures of internalization, and will facilitate the development of tools to modulate endocytic rates or the intracellular fate of internalized materials.

Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering.

Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce 'TEMTAC', a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2.

Angiotensin converting enzyme genotype and chronic allograft nephropathy in protocol biopsies.

Genotype DD of the angiotensin-converting enzyme (ACE) is not associated with an increased incidence of native renal diseases, although it could modulate progression to renal failure in patients who already display chronic lesions. Because its role in renal allograft degeneration is not well characterized, whether ACE genotype was associated with the prevalence of chronic allograft nephropathy (CAN) was studied, in a group of protocol biopsies from 180 patients, or with the incidence of CAN in 152 patients with at least two sequential biopsies. As a control group, ACE genotype was also studied in 41 donors and 72 healthy subjects. For analyzing the influence of ACE genotype in graft survival, patients were grouped into six categories (II-normal biopsy, ID-normal, DD-normal, II-CAN, ID-CAN and DD-CAN). Finally, relative renal ACE mRNA levels were measured in 67 cases by real-time PCR using the delta threshold cycle method. ACE-DD genotype was more frequent in patients who received a transplant than in control subjects (43.3% versus 30.1%, P = 0.026), but prevalence (DD = 42.7% versus non-DD = 42.2%) or incidence (DD = 24.6% versus non-DD = 29.9%) of CAN was not different regarding recipient ACE genotype. Furthermore, patients with the ACE-DD genotype and CAN had the poorest graft survival (II-normal = 100%, ID-normal = 91%, DD-normal = 84%, II-CAN = 100%, ID-CAN = 66%, and DD-CAN = 36%; P = 0.034) and higher ACE mRNA levels than non-DD and CAN (DD = -3.36 +/- 2.35 versus non-DD = -5.65 +/- 1.72-fold in ACE copies; P = 0.012). It is concluded that ACE-DD genotype is not associated with an increased prevalence or incidence of CAN but is actually associated with higher ACE mRNA levels and poorer graft survival in patients who already display CAN.