Splice Forms Research Articles

Sequence characterization of extracellular HBV RNA in patient plasma.

Antiviral nucleos(t)ide analogue therapies inhibit HBV replication and suppress the HBV DNA levels in patients with chronic HBV infection. Since HBV RNAs are expressed from cccDNA or HBV integrated sequences, independently of viral genome replication, levels of HBV RNAs in plasma may remain high following treatment with nucleos(t)ide analogue. Thus, HBV RNAs have been proposed to be used as a viral biomarker for treatment outcome and disease progression. Recent investigations of plasma HBV RNAs described the presence of full length as well as subgenomic forms of RNA. To support the usage of plasma HBV RNAs as a viral biomarker, further understanding of HBV RNA composition in clinical samples is needed. Here, sequence of extracellular HBV RNAs was characterized in plasma samples of patients with chronic HBV infection using two independent RNA amplification methods that do not use HBV-specific primers for amplification: total RNA (NuGEN RNAseq) and mRNA (TruSeq RNAseq). Sequencing coverage was obtained across the full length of HBV genome for both methods, confirming the presence of full-length HBV RNA in plasma. The sequence of HBV RNA was nearly identical to plasma HBV DNA sequence in each sample with only 0-14 (median 4) mismatches over 3kb. Thus, sequence of HBV RNA plasma reflects the intrahepatic viral reservoir and can be used for monitoring of sequence variants such as resistance in clinical trials. Additionally, RNA splice forms, different polyA tails start positions and presence of HBV-human chimeric transcript were identified.

Inhibition on XBP1s-driven lipogenesis by Qushi Huayu Decoction contributes to amelioration of hepatic steatosis induced by fructose

Ethnopharmacological relevanceQushi Huayu Decoction (QHD) is a traditional Chinese medicine formula consisting of five herbs, which has been used for non-alcoholic fatty liver disease (NAFLD) treatment in clinic for decades in China and validated in several NAFLD animal models. The hepatic de novo lipogenesis (DNL) is enhanced greatly to contribute to steatosis in NAFLD. The spliced form of X-box binding protein 1 (XBP1s) initiates DNL independently of sterol regulatory element-binding protein (SREBP) and carbohydrate-responsive element-binding protein (ChREBP). Aim of the studyTo disclose the mechanism of inhibition on hepatic DNL by QHD and the responsible compounds. MethodsThe effects of QHD on hepatic DNL were evaluated in mice induced by high-fructose diet (HFru). The effects of the serum-absorbed compounds of QHD on XBP1s were evaluated in HepG2 cells induced by tunicamycin. Hepatic histology, triglyceride (TG) and nonesterified fatty acids were observed. Hepatic apolipoprotein B100 and very low-density lipoprotein were measured to reflect lipid out-transport. The mRNA expression of XBP1s and its target genes were detected by real-time polymerase chain reaction. The protein expression of TG synthetases and DNL enzymes, and inositol requirement enzyme 1 alpha (IRE1α), phosphorylated IRE1α and XBP1s were detected in liver tissue and HepG2 cells by western-blot. The binding activity of SREBP1, protein expression of ChREBP and XBP1s were detected in the nuclear extracts of liver tissue. ResultsDynamical observing suggested feeding with HFru for 2 weeks was sufficient to induce hepatic lipogenesis and XBP1s. QHD ameliorated liver steatosis without enhancing out-transport of lipids, accompanied with more inhibitory effects on DNL enzymes than TG synthetases. QHD inhibits the nuclear XBP1s without affecting ChREBP and SREBP1. In QHD, chlorogenic acid, geniposide and polydatin inhibit lipogenesis initiated by XPB1s. ConclusionQHD probably decreases hepatic DNL by inhibiting XBP1s independent of SREBP1 and ChREBP. Chlorogenic acid, geniposide and polydatin are the potential responsible compounds.

A common intronic single nucleotide variant modifies PKD1 expression level.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in PKD1 and PKD2 (PKD1/2), has unexplained phenotypic variability likely affected by environmental and other genetic factors. Approximately 10% of individuals with ADPKD phenotype have no causal mutation detected, possibly due to unrecognized risk variants of PKD1/2. This study was designed to identify risk variants of PKD genes through population genetic analyses. We used Wright's F-statistics (Fst) to evaluate common single nucleotide variants (SNVs) potentially favored by positive natural selection in PKD1 from 1000 Genomes Project (1KG) and genotyped 388 subjects from the Rogosin Institute ADPKD Data Repository. The variants with >90th percentile Fst scores underwent further investigation by in silico analysis and molecular genetics analyses. We identified a deep intronic SNV, rs3874648G> A, located in a conserved binding site of the splicing regulator Tra2-β in PKD1 intron 30. Reverse-transcription PCR (RT-PCR) of peripheral blood leukocytes (PBL) from an ADPKD patient homozygous for rs3874648-A identified an atypical PKD1 splice form. Functional analyses demonstrated that rs3874648-A allele increased Tra2-β binding affinity and activated a cryptic acceptor splice-site, causing a frameshift that introduced a premature stop codon in mRNA, thereby decreasing PKD1 full-length transcript level. PKD1 transcript levels were lower in PBL from rs3874648-G/A carriers than in rs3874648-G/G homozygotes in a small cohort of normal individuals and patients with PKD2 inactivating mutations. Our findings indicate that rs3874648G > A is a PKD1 expression modifier attenuating PKD1 expression through Tra2-β, while the derived G allele advantageously maintains PKD1 expression and is predominant in all subpopulations.

Carm1 and the Epigenetic Control of Stem Cell Function.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a methyltransferase whose function has been highly studied in the context of nuclear receptor signaling. However, CARM1 is known to epigenetically regulate expression of several myogenic genes involved in differentiation such as Myog and MEF2C. CARM1 also acts to regulate myogenesis through its influence on various cellular processes from embryonic to adult myogenesis. First, CARM1 has a crucial role in establishing polarity-regulated gene expression during an asymmetric satellite cell division by methylating PAX7, leading to the expression of Myf5. Second, satellite cells express the CARM1-FL and CARM1-ΔE15 isoforms. The former has been shown to promote pre-mRNA splicing through its interaction with CA150 and U1C, leading to their methylation and increased activity, while the latter displays a reduction in both metrics, thus, modulating alternative pre-mRNA splice forms in muscle cells. Third, CARM1 is a regulator of autophagy through its positive reinforcement of AMPK activity and gene expression. Autophagy already has known implications in ageing and disease, and CARM1 could follow suite. Thus, CARM1 is a central regulator of several important processes impacting muscle stem cell function and myogenesis.

A pathogenic proteolysis–resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function

Huntington’s disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTTΔ12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTTΔ12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTTΔ12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTTΔ12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.

Alternative splicing of Arabidopsis G6PD5 recruits NADPH-producing OPPP reactions to the endoplasmic reticulum.

Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the oxidative pentose-phosphate pathway (OPPP). The OPPP mainly provides NADPH and sugar-phosphate building blocks for anabolic pathways and is present in all eukaryotes. In plant cells, the irreversible part of the OPPP is found in several compartments. Among the isoforms catalyzing the first OPPP step in Arabidopsis, G6PD1 to G6PD4 target plastids (with G6PD1 being also directed to peroxisomes), whereas G6PD5 and G6PD6 operate in the cytosol. We noticed that alternative splice forms G6PD5.4 and G6PD5.5 encode N-terminally extended proteoforms. Compared to G6PD5.1, RT-PCR signals differed and fluorescent reporter fusions expressed in Arabidopsis protoplasts accumulated in distinct intracellular sites. Co-expression with organelle-specific markers revealed that the G6PD5.4 and G6PD5.5 proteoforms label different subdomains of the endoplasmic reticulum (ER), and analysis of C-terminal roGFP fusions showed that their catalytic domains face the cytosol. In g6pd5-1 g6pd6-2 mutant protoplasts lacking cytosolic G6PDH activity, the ER-bound proteoforms were both active and thus able to form homomers. Among the Arabidopsis 6-phosphogluconolactonases (catalyzing the second OPPP step), we noticed that isoform PGL2 carries a C-terminal CaaX motif that may be prenylated for membrane attachment. Reporter-PGL2 fusions co-localized with G6PD5.4 in ER subdomains, which was abolished by Cys-to-Ser exchange in the 256CSIL motif. Among the Arabidopsis 6-phosphogluconate dehydrogenases (catalyzing the third OPPP step), S-acylated peptides were detected for all three isoforms in a recent palmitoylome, with dual cytosolic/peroxisomal PGD2 displaying three sites. Co-expression of GFP-PGD2 diminished crowding of OFP-G6PD5.4 at the ER, independent of PGL2's presence. Upon pull-down of GFP-G6PD5.4, not only unlabeled PGD2 and PGL2 were enriched, but also enzymes that depend on NADPH provision at the ER, indicative of physical interaction with the OPPP enzymes. When membrane-bound G6PD5.5 and 5.4 variants were co-expressed with KCR1 (ketoacyl-CoA reductase, involved in fatty acid elongation), ATR1 (NADPH:cytochrome-P450 oxidoreductase), or pulled C4H/CYP73A5 (cinnamate 4-hydroxylase) as indirectly (via ATR) NADPH-dependent cytochrome P450 enzyme, co-localization in ER subdomains was observed. Thus, alternative splicing of G6PD5 can direct the NADPH-producing OPPP reactions to the cytosolic face of the ER, where they may operate as membrane-bound metabolon to support several important biosynthetic pathways of plant cells.

Functions of G protein-coupled receptor 56 in health and disease.

Human G protein-coupled receptor 56 (GPR56) is encoded by gene ADGRG1 from chromosome 16q21 and is homologously encoded in mice, at chromosome 8. Both 687 and 693 splice forms are present in humans and mice. GPR56 has a 381 amino acid-long N-terminal extracellular segment and a GPCR proteolysis site upstream from the first transmembrane domain. GPR56 is mainly expressed in the heart, brain, thyroid, platelets, and peripheral blood mononuclear cells. Accumulating evidence indicates that GPR56 promotes the formation of myelin sheaths and the development of oligodendrocytes in the cerebral cortex of the central nervous system. Moreover, GPR56 contributes to the development and differentiation of hematopoietic stem cells, induces adipogenesis, and regulates the function of immune cells. The lack of GPR56 leads to nervous system dysfunction, platelet disorders, and infertility. Abnormal expression of GPR56 is related to the malignant transformation and tumor metastasis of several cancers including melanoma, neuroglioma, and gastrointestinal cancer. Metabolic disorders and cardiovascular diseases are also associated with dysregulation of GPR56 expression, and GPR56 is involved in the pharmacological resistance to some antidepressant and cancer drug treatments. In this review, the molecular structure, expression profile, and signal transduction of GPR56 are introduced, and physiological and pathological functions of GRP56 are comprehensively summarized. Attributing to its significant biological functions and its long N-terminal extracellular region that interacts with multiple ligands, GPR56 is becoming an attractive therapeutic target in treating neurological and hematopoietic diseases.

Activation of the canonical ER stress IRE1–XBP1 pathway by insulin regulates glucose and lipid metabolism

Knockout of the transcription factor X-box binding protein (XBP1) is known to decrease liver glucose production and lipogenesis. However, whether insulin can regulate gluconeogenesis and lipogenesis through XBP1 and how insulin activates the inositol-requiring enzyme-XBP1 ER stress pathway remains unexplored. Here, we report that in the fed state, insulin-activated kinase AKT directly phosphorylates inositol-requiring enzyme 1 at S724, which in turn mediates the splicing of XBP1u mRNA, thus favoring the generation of the spliced form, XBP1s, in the liver of mice. Subsequently, XBP1s stimulate the expression of lipogenic genes and upregulates liver lipogenesis as previously reported. Intriguingly, we find that fasting leads to an increase in XBP1u along with a drastic decrease in XBP1s in the liver of mice, and XBP1u, not XBP1s, significantly increases PKA-stimulated CRE reporter activity in cultured hepatocytes. Furthermore, we demonstrate that overexpression of XBP1u significantly increases cAMP-stimulated expression of rate-limiting gluconeogenic genes, G6pc and Pck1, and glucose production in primary hepatocytes. Reexpression of XBP1u in the liver of mice with XBP1 depletion significantly increases fasting blood glucose levels and gluconeogenic gene expression. These data support an important role of XBP1u in upregulating gluconeogenesis in the fasted state. Taken together, we reveal that insulin signaling via AKT controls the expression of XBP1 isoforms and that XBP1u and XBP1s function in different nutritional states to regulate liver gluconeogenesis and lipogenesis, respectively.

Study on Vertical Joint Performance of Single-Faced Superposed Shear Wall Based on Finite Element Analysis and Calculation.

In this paper, a new type of single-faced superposed shear wall system is proposed based on the single-faced superposed shear wall structure. The nonlinear finite element analysis of a new splicing form of single-faced superposed shear wall components is carried out by using the method of combining experimental research and ABAQUS finite element software analysis. The impact of noncolumn on the connection performance of vertical seam is studied. The analysis shows no significant difference in the effect of eliminating concealed column on the hysteretic performance and bearing capacity of the single-faced superposed shear wall with the joint. The single-faced composite shear wall with two different splicing forms presents bending-shear failure. The single-faced composite shear wall model without concealed column design can also maintain good overall performance and seismic behavior.

Abstract 6339: Deep plasma proteomics at scale enabling proteogenomic analyses in a lung cancer (NSCLC) study

Abstract Our ~20,000 genes encode over one million protein variants, given alternative splice forms, allelic variation, and protein modification. Though large-scale genomics studies have expanded our understanding of biology, similarly, scaled deep and untargeted proteomics studies of biofluids have remained impractical due to complex workflows. To address this need, we have previously described Proteograph࣪, a novel platform that leverages the protein-corona interactions of nanoparticles for deep and untargeted proteomic sampling at scale. Using Proteograph࣪ in a non-small cell lung cancer (NSCLC) cohort, we previously conducted a deep interrogation of plasma from 141 subjects: 61 early-stage NSCLC subjects and 80 non-cancer controls. We identified 2,499 plasma proteins, with 1,992 present in ≥ 25% of the samples. Leveraging this data, we created a biomarker classifier distinguishing NSCLC from controls with area under the receiver operating characteristic curve of 0.911. In this study, we now re-analyze the data with the more sensitive DIA-NN software to enhance protein depth while preserving the accuracy of the classifier. In addition, to show the added value of our platform in combination with genomic data, we integrate previously sequenced exome data with our proteomic data to build a multi-modal proteogenomic deep learning classifier. Our results outline proteogenomic workflows for robust biomarker discovery and cohort subtyping. The Proteograph࣪ platform interrogates the plasma proteome at previously impractical combinations of scale, depth and coverage, and enables the development of improved classification models and the study of proteogenomics. 1Blume et al. Nat. Comm. (2020) Citation Format: Mahdi Zamanighomi, Harendra Guturu, Jian Wang, Amir Alavi, Tristan Brown, Daniel Hornburg, Moaraj Hasan, Shadi Ferdosi, Khatereh Motamedchaboki, Margaret Donovan, Theodore Platt, Ryan Benz, Asim Siddiqui, Serafim Batzoglou. Deep plasma proteomics at scale enabling proteogenomic analyses in a lung cancer (NSCLC) study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6339.

Abstract 1961: Discovery and validation of prostate cancer biomarkers of biochemical recurrence in low-risk prostate cancer patients

Abstract Introduction: Prostate cancer (CaP) is predominantly indolent disease with a small fraction of patients undergoing metastatic progression. Therefore, it is critical to identify prognostic markers for the early detection of an aggressive subtype. We hypothesized that tissue specimens from early-stage, low risk CaP may harbor predictive prognostic signatures for disease progression after radical prostatectomy. Previously, we had systematically evaluated and optimized the NanoString platform for CaP gene expression analysis in formalin fixed paraffin embedded (FFPE) whole mounted prostate specimens. The goal of the study is to validate the CPDR findings on the differential mRNA expression of candidate markers from patients with BCR (biochemical recurrence) and non-BCR in an independent cohort. Methods: This is a retrospective case cohort study based on 78 tumor-normal paired (N=156) archived whole mounted FFPE prostate samples received from biobank at UTHSCSA. We performed an optimized NanoString analysis of 203-CaP probe set to evaluate the association of markers with BCR outcome in a racially diverse patient population comprising of 21.6% AA and 78.38% CA men. The cohort comprised of 28 patients with BCR, and 50 patients with no BCR (based on a minimum of 5 years of follow-up). ERG gene expression was validated by immunohistochemistry (IHC) assay using CPDR ERG-MAb (9FY). Normalized nCounter gene expression data was analyzed using the NanoString nSolver (v 4.0) platform. Fisher exact and Wilcoxon-Mann-Whitney tests were used for categorical and continuous variables respectively. Results: We found 96.1% (73/76) concordance between ERG IHC and NanoString datasets, which provides strong QC and validation for NanoString data. ERG positive tumors had strong expression of both TMPRSS2-ERG fusion and several other ERG splice forms. Additionally, the prostate epithelial cell markers PSA, MSMB and PAP had the highest signals in all samples reflecting the prostate epithelial origin of the specimens. Differential gene expression analysis showed that a total of 37 genes were found to be significantly (P adj <0.05) different across tumors and normal. Established CaP genes like AMACR, PCA3, OR51E2, ERG, HOXC6, DLX1, CACNA1D and PSGR were most highly over-expressed in tumors compared to matched normal while GSTP1, CAV1, PAP and MSMB were significantly downregulated in tumors compared to adjacent normal prostate epithelia. Race stratified analysis in this randomly selected patient cohort showed that AA men were found to be significantly younger (median age: 56 years AA vs. 63 years CA; p 0.01) and had higher frequency of BCR events (p 0.004) than CA men. Data analysis for disease outcome across AA and CA men is ongoing. Conclusions: We confirmed that NanoString approach is useful for evaluation of prognostic biomarker candidates in prostate cancer using FFPE specimens. Citation Format: Indu Kohaar, Kaitlyn Bejar, Denise Young, Yingjie Song, Jiji Jiang, Jacob Kagan, Sudhir Srivastava, Javier Hernandez, Gregory Chesnut, Isabell A. Sesterhenn, Robin J. Leach, Gyorgy Petrovics. Discovery and validation of prostate cancer biomarkers of biochemical recurrence in low-risk prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1961.

Abstract 5125: Biological and clinical value of OCT4A/SPP1C axis in endometrial and lung adenocarcinoma

Abstract Background: It is largely unclear whether octamer-binding transcription factor 4A (OCT4A, OCT3/4A) functions physiologically or pathologically in adult somatic tissues and related cancers. We have recently identified OCT4A translation in a human cancer cell subpopulation that might exacerbate cell migration and invasion. Here, we focus on biological and clinical features of OCT4A and its relationship with secreted phosphoprotein 1(SPP1), which is strongly related to the malignant progression of numerous tumors. Methods: RT-PCR or qPCR analysis was used to identify full-length spliced forms of OCT4A and SPP1 transcripts in non-tumor and tumor human cells. An OCT4A-FLAG-tagged genomic transgene identified OCT4A-positive cancer cells. Cell-ablation of OCT4A-positive cells using promoter-driven diphtheria toxin A was used to examine the potential role of OCT4A in lung and endometrial adenocarcinoma cells. OCT4A and SPP1 transcripts in early-stage lung adenocarcinoma (stage I, fifty-eight cases) were analyzed and compared with pathohistological and prognostic features. Results: We found that OCT4A and SPP1C are co-expressed in highly aggressive human lung and endometrial adenocarcinoma cell lines but not in mesothelioma or non-tumor cell lines. Ablation of OCT4A-positive cells significantly decreased cancer-cell migration and SPP1C mRNA levels. The OCT4A/SPP1C axis was found in primary, early-stage, lung adenocarcinomas, and all four cases of recurrence and/or metastasis out of 58 cases were included in the OCT4A and SPP1C positive group. Conclusions: Co-expression of OCT4A and SPP1C may correlate with human cancer cell migration and the OCT4A/SPP1C axis may have a prognostic value in high-risk, early-stage lung adenocarcinomas.

Structural basis of the IL-1 receptor TIR domain-mediated IL-1 signaling

SummaryThe cytoplasmic Toll/interleukin-1 receptor (TIR) domains of IL-1 receptors (IL-1Rs) are evolutionally conserved and essential for transmitting signals. IL-1RAcP is a shared co-receptor in the IL-1R family for signaling. Its splicing form IL-1RAcPb contains a different TIR domain and is unable to transduce NF-κB signaling. Here, we determined crystal structures of TIR domains of IL-1RAcPb and other IL-1Rs including IL-18Rβ, IL-1RAPL2, and zebrafish SIGIRR (zSIGIRR). Structurally variant regions in the TIR domain important for signaling were revealed by structural comparisons. Taking advantage of the IL-1RAcP/IL-1RAcPb pair, we demonstrated that differential TIR domain determines signaling discrepancies between IL-1RAcP and IL-1RAcPb. We also proved the functional importance of two helices (αC and αD) in the structurally variable regions and pinpointed critical residues in αC and αD for signaling. These results collectively provide additional and important knowledge for fully understanding the molecular basis of IL-1R TIR domain in mediating signaling.

A cell-free RNA-based next-generation sequencing (NGS) assay for the detection of actionable gene fusions in patients with non–small cell lung cancer (NSCLC).

3040 Background: Gene fusions and alternate RNA splice forms represent clinically actionable driver and resistance-conferring alterations in NSCLC and other cancers. Where tissue samples are inadequate for molecular testing, liquid biopsies could address the gap in detection of clinically relevant gene fusions. Targeted cell-free (cf) DNA-based NGS methods are typically used for non-invasive blood-based testing of gene fusions, but have limited sensitivity if fusion breakpoints involve long intronic regions or repetitive sequences (e.g. NTRKs and NRG1). RNA-based approaches are not affected by introns, can identify expressed fusion genes and can discriminate splicing. We developed an NGS assay optimized for cfRNA analyte for the detection of actionable gene fusions and exon deletion/skipping events in liquid biopsies. Methods: A highly multiplexed molecular-barcoded primer panel was designed for cfRNA-based detection of actionable fusion genes in NSCLC ( ALK, BRAF, FGFR2, FGFR3, MET (including exon 14 skipping), NRG1, NTRK1/2/3, RET and ROS1) covering > 90% of reported driver and partner gene exons in COSMIC database. The panel also targets housekeeping genes as endogenous sample controls. We developed a custom bioinformatics pipeline to call fusions and exon skipping based on split and spanning reads. Results: In initial analytical validation using fragmented RNA from pre-characterized reference material (representing 32 unique fusions at known copy numbers), the assay could detect as low as 10 fusion copies with a sensitivity of 97.6% and a specificity of 100%. For clinical testing, cfRNA co-eluted with cfDNA in nucleic acid extracts from plasma was used. In plasma samples (n = 103) from advanced NSCLC, 76% (15/21) of fusions (5 ALK, 3 RET, 2 ROS1, 5 MET ex14 skipping, 1 FGFR3-TACC3) detected in a clinically validated cfDNA assay (LiquidHALLMARK) were also detected in corresponding cfRNA. In a subset of samples that were driver-negative untreated or tyrosine kinase-inhibitor treated, the cfRNA assay yielded 9 gene fusions or breakpoints (1 CD74-NRG1, 4 BRAF, 2 MET, 2 FGFR3-TACC3 fusions) that could not be detected by the cfDNA assay. This represents an increase of 8.7% (9/103) of actionable alterations (driver or resistance) identified using cfRNA. Together cfRNA and cfDNA resulted in 30 fusion events to be detected compared to 21 by the cfDNA assay alone, representing a 42.8% (9/21) increase in fusion-specific detection of the combined assay. Conclusions: This novel cfRNA assay can detect actionable gene fusions and exon skipping events in liquid biopsies with high sensitivity. Combining cfRNA with more routine cfDNA testing can increase the total actionable diagnostic information from non-invasive testing in NSCLC patients where tissue samples are lacking, especially for gene fusions not amenable to detection in cfDNA.

PRECOGx: exploring GPCR signaling mechanisms with deep protein representations.

In this study we show that protein language models can encode structural and functional information of GPCR sequences that can be used to predict their signaling and functional repertoire. We used the ESM1b protein embeddings as features and the binding information known from publicly available studies to develop PRECOGx, a machine learning predictor to explore GPCR interactions with G protein and β-arrestin, which we made available through a new webserver (https://precogx.bioinfolab.sns.it/). PRECOGx outperformed its predecessor (e.g. PRECOG) in predicting GPCR-transducer couplings, being also able to consider all GPCR classes. The webserver also provides new functionalities, such as the projection of input sequences on a low-dimensional space describing essential features of the human GPCRome, which is used as a reference to track GPCR variants. Additionally, it allows inspection of the sequence and structural determinants responsible for coupling via the analysis of the most important attention maps used by the models as well as through predicted intramolecular contacts. We demonstrate applications of PRECOGx by predicting the impact of disease variants (ClinVar) and alternative splice forms from healthy tissues (GTEX) of human GPCRs, revealing the power to dissect system biasing mechanisms in both health and disease.

WNT1 expression influences the development of dysplasia of the hip via regulating RBPMS2/NOG-BMP2/4-GDF5- WISP2 pathway

Objective: To explore the role of WNT family member 1 (WNT1) in the development of dysplasia of the hip (DDH) and the molecular mechanism involved in this process. Methods: Si-WNT1, pcDNA3.1-WNT1 or corresponding negative controls were transfected into human osteoblast hFOB1.19 and human chondrocyte C28/I2, respectively. The proliferation of cells was measured by EdU assay. The relative expressions of human noggin gene (NOG), growth differentiating factor 5 (GDF5), WNT1, and WNT1-inducible-signaling pathway protein 2 (WISP2) were determined by immunofluorescence analysis. The protein expressions of RNA-binding protein of multiple splice forms 2 (RBPMS2), NOG, bone morphogenetic protein 2 (BMP2), BMP4, WNT1 and WISP2 were determined by western blot. Animal experiment was also performed and the morphological development of hip joint was observed. Results: Overexpression of WNT1 promoted osteoblast proliferation and inhibited chondrocyte proliferation, while knockdown of WNT1 inhibited osteoblast proliferation. In chondrocytes, knockdown of WNT1 upregulated NOG expression, while overexpression of WNT1 downregulated its expression. In osteoblasts and chondrocytes, overexpression of WNT1 increased BMP2, BMP4, WNT1, and WISP2 expression. RBPMS2 and NOG were slightly expressed in each group. Conclusion: Overexpression of WNT1 promoted osteoblast proliferation, inhibited chondrocyte proliferation, and increased the expressions of BMP2, BMP4, WNT1, and WISP2. Therefore, WNT1 may be a new therapeutic target for DDH.

IRE1α Inhibitors as a Promising Therapeutic Strategy in Blood Malignancies.

Simple SummaryBlood malignancies account for 6.9% of all cancer deaths. Inositol-requiring enzyme 1 alpha (IRE1α), a part of the unfolded protein response (UPR), has been shown to be pivotal for cancer cell development and progression, including blood cancers. Furthermore, IRE1α levels are often elevated in blood cancer cells, and they correspond with cell survival, response to treatment, and prognosis. The aim of our study is to summarize the current knowledge on IRE1α in blood cancers and to evaluate the potential utility of IRE1α inhibitors in the treatment of blood malignancies. The introduction of new therapies based on IRE1α inhibition may increase treatment efficacy and reduce the side effects of blood cancer therapy.Synthesis, folding, and structural maturation of proteins occur in the endoplasmic reticulum (ER). Accumulation of misfolded or unfolded proteins in the ER lumen contributes to the induction of ER stress and activation of the unfolded protein response (UPR) signaling pathway. Under ER stress, the UPR tries to maintain cellular homeostasis through different pathways, including the inositol-requiring enzyme 1 alpha (IRE1α)-dependent ones. IRE1α is located in an ER membrane, and it is evolutionarily the oldest UPR sensor. Activation of IRE1α via ER stress triggers the formation of the spliced form of XBP1 (XBP1s), which has been linked to a pro-survival effect in cancer cells. The role of IRE1α is critical for blood cancer cells, and it was found that the levels of IRE1α and XBP1s are elevated in various hematological malignancies. This review paper is focused on summarizing the latest knowledge about the role of IRE1α and on the assessment of the potential utility of IRE1α inhibitors in blood cancers.

Upregulation of the serine palmitoyltransferase subunit SPTLC2 by endoplasmic reticulum stress inhibits the hepatic insulin response

Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor β (IRβ). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRβ in hepatocytes.

Oxidative Stress-Induced Growth Inhibitor (OSGIN1), a Target of X-Box-Binding Protein 1, Protects Palmitic Acid-Induced Vascular Lipotoxicity through Maintaining Autophagy.

Saturated free fatty acids (FFAs) strongly correlate with metabolic syndromes and are well-known risk factors for cardiovascular diseases (CVDs). The mechanism of palmitic acid (PA)-induced vascular lipotoxicity under endoplasmic reticulum (ER) stress is unknown. In the present paper, we investigate the roles of spliced form of X-box-binding protein 1 (XBP1s) target gene oxidative stress-induced growth inhibitor 1 (OSGIN1) in PA-induced vascular dysfunction. PA inhibited the tube formation assay of primary human umbilical vein endothelial cells (HUVECs). Simultaneously, PA treatment induced the XBP1s expression in HUVECs. Attenuate the induction of XBP1s by silencing the XBP1s retarded cell migration and diminished endothelial nitric oxide synthase (eNOS) expression. OSGIN1 is a target gene of XBP1s under PA treatment. The silencing of OSGIN1 inhibits cell migration by decreasing phospho-eNOS expression. PA activated autophagy in endothelial cells, inhibiting autophagy by 3-methyladenine (3-MA) decreased endothelial cell migration. Silencing XBP1s and OSGIN1 would reduce the induction of LC3 II; therefore, OSGIN1 could maintain autophagy to preserve endothelial cell migration. In conclusion, PA treatment induced ER stress and activated the inositol-requiring enzyme 1 alpha–spliced XBP1 (IRE1α–XBP1s) pathway. OSGIN1, a target gene of XBP1s, could protect endothelial cells from vascular lipotoxicity by regulating autophagy.

Regulation of alternative splicing of PaFT and PaFDL1, the FT and FD homologs in Platanus acerifolia

Alternative splicing (AS) selects different alternative splice sites and produces a variety of transcripts with different exon/intron combinations, which may result in multiple protein isoforms. The splicing signals include cis-elements and RNA structures; however, the mechanisms of AS regulation in plants have yet to be elucidated. Previous studies have shown that in Platanus acerifolia, the FLOWERING LOCUS T (FT) homolog PaFT has a unique and complex AS pattern, in which most of the splice forms of PaFT involve the first and/or second intron, and the FD homolog PaFDL1 produces two transcripts via AS, whereas the other FT homolog PaFTL is not regulated by AS. In this study, the regulatory mechanism of the AS of PaFT was demonstrated to be conserved in different plant species. To define the distribution of the AS regulatory signals, the intron-swap, site-directed mutagenesis of alternative splice sites, and deletion experiment were performed. For the PaFT gene, all the signals that regulate the AS of the first intron were located within this intron, while the usage of the first alternative splice site in the second intron was determined by the first intron. Meanwhile, the AS of PaFDL1 might be co-regulated by exons and the first intron. Additionally, the first alternative splice site and adjacent region in PaFT intron 1 might contain cis-elements and/or RNA structures that affect the use of the other sites. This study had provided a deeper insight into the distribution of AS signals in plants, namely the AS signals of different splice sites might exist in the intron where the sites were present, and might also be distributed in exons or other introns.