BaseScope™ Approach to Visualize Alternative Splice Variants in Tissue

Methods in molecular biology (Clifton, N.J.)

2022 Jul 27

Bunda, A;Andrade, A;
PMID: 35895265 | DOI: 10.1007/978-1-0716-2521-7_11

Defining the cell-specific alternative splicing landscape in complex tissues is an important goal to gain functional insights. Deep-sequencing techniques coupled to genetic strategies for cell identification has provided important cues on cell-specific exon usage in complex tissues like the nervous system. BaseScope has emerged as a powerful and highly sensitive alternative to in situ hybridization to determine exon composition in tissue with spatial and morphological context. In this protocol, we will review how BaseScope was utilized to detect the e37a-Cacna1b splice variant of the presynaptic calcium channel CaV2.2 or N-type. This splice variant arises from a pair of mutually exclusive exons (e37a and e37b). E37a-Cacna1b is heavily underrepresented relative to e37b-Cacna1b and both exons share 60% of their sequence. By using BaseScope , we were able to discover that e37a-Cacna1b is expressed in excitatory pyramidal neurons of hippocampus and cortex, as well as motor neurons of the ventral horn of the spinal cord.

Minor intron splicing is critical for survival of lethal prostate cancer

Molecular cell

2023 Jun 15

Augspach, A;Drake, KD;Roma, L;Qian, E;Lee, SR;Clarke, D;Kumar, S;Jaquet, M;Gallon, J;Bolis, M;Triscott, J;Galván, JA;Chen, Y;Thalmann, GN;Kruithof-de Julio, M;Theurillat, JP;Wuchty, S;Gerstein, M;Piscuoglio, S;Kanadia, RN;Rubin, MA;
PMID: 37295433 | DOI: 10.1016/j.molcel.2023.05.017

The evolutionarily conserved minor spliceosome (MiS) is required for protein expression of ∼714 minor intron-containing genes (MIGs) crucial for cell-cycle regulation, DNA repair, and MAP-kinase signaling. We explored the role of MIGs and MiS in cancer, taking prostate cancer (PCa) as an exemplar. Both androgen receptor signaling and elevated levels of U6atac, a MiS small nuclear RNA, regulate MiS activity, which is highest in advanced metastatic PCa. siU6atac-mediated MiS inhibition in PCa in vitro model systems resulted in aberrant minor intron splicing leading to cell-cycle G1 arrest. Small interfering RNA knocking down U6atac was ∼50% more efficient in lowering tumor burden in models of advanced therapy-resistant PCa compared with standard antiandrogen therapy. In lethal PCa, siU6atac disrupted the splicing of a crucial lineage dependency factor, the RE1-silencing factor (REST). Taken together, we have nominated MiS as a vulnerability for lethal PCa and potentially other cancers.

ebv-circRPMS1 promotes the progression of EBV-associated gastric carcinoma via Sam68-dependent activation of METTL3

Cancer letters

2022 Jun 01

Zhang, JY;Du, Y;Gong, LP;Shao, YT;Pan, LJ;Feng, ZY;Pan, YH;Huang, JT;Wen, JY;Sun, LP;Chen, GF;Chen, JN;Shao, CK;
PMID: 35304258 | DOI: 10.1016/j.canlet.2022.215646

Epstein-Barr virus (EBV) is a tumor virus that is associated with a variety of neoplasms, including EBV-associated gastric carcinoma (EBVaGC). Recently, EBV was reported to generate various circular RNAs (circRNAs). CircRNAs are important regulators of tumorigenesis by modulating the malignant behaviors of tumor cells. However, to date, the functions of ebv-circRNAs in EBVaGC remain poorly understood. In the present study, we observed high ebv-circRPMS1 expression in EBVaGC and showed that ebv-circRPMS1 promoted the proliferation, migration, and invasion and inhibited the apoptosis of EBVaGC cells. In addition, METTL3 was upregulated in GC cells overexpressing ebv-circRPMS1. Mechanistically, ebv-circRPMS1 bound to Sam68 to facilitate its physical interaction with the METTL3 promotor, resulting in the transactivation of METTL3 and cancer progression. In clinical EBVaGC samples, ebv-circRPMS1 was associated with distant metastasis and a poor prognosis. Based on these findings, ebv-circRPMS1 contributed to EBVaGC progression by recruiting Sam68 to the METTL3 promoter to induce METTL3 expression. ebv-circRPMS1, Sam68, and METTL3 might serve as therapeutic targets for EBVaGC.

A KO mouse model for the lncRNA Lhx1os produces motor neuron alterations and locomotor impairment

iScience

2022 Dec 01

Pellegrini, F;Padovano, V;Biscarini, S;Santini, T;Setti, A;Galfrè, S;Silenzi, V;Vitiello, E;Mariani, D;Nicoletti, C;Torromino, G;De Leonibus, E;Martone, J;Bozzoni, I;
| DOI: 10.1016/j.isci.2022.105891

Here we describe a conserved motor neuron specific long non-coding RNA, Lhx1os, whose knock-out in mice produces motor impairment and post-natal reduction of mature motor neurons (MNs). The endoplasmic reticulum (ER)-stress response pathway resulted specifically altered with the downregulation of factors involved in the Unfolded Protein Response (UPR). Lhx1os was found to bind the ER-associated PDIA3 disulfide isomerase and to affect the expression of the same set of genes controlled by this protein, indicating that the two factors act in conjunction to modulate the UPR. Altogether, the observed phenotype and function of Lhx1os indicate its important role in the control of MN homeostasis and function.

Alternative Splicing

Methods in Molecular Biology

2022 Jul 27

Scheiffele, P;Mauger, O;
| DOI: 10.1007/978-1-0716-2521-7

This detailed volume collects commonly used and cutting-edge methods to analyze alternative splicing, a key step in gene regulation. After an introduction of the alternative splicing mechanism and its targeting for therapeutic strategies, the book continues with techniques for analyzing alternative splicing profiles in complex biological systems, visualizing and localizing alternative spliced transcripts with cellular and sub-cellular resolution, probing regulators of alternative splicing, as well as assessing the functional consequences of alternative splicing. Written for the highly successful _Methods in Molecular Biology_ series, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, reproducible protocols, and tips on troubleshooting and avoiding known pitfalls.

Nanoparticle-mediated transgene expression of insulin-like growth factor 1 in the growth restricted guinea pig placenta increases placenta nutrient transporter expression and fetal glucose concentrations

Molecular reproduction and development

2022 Sep 12

Wilson, RL;Lampe, K;Gupta, MK;Duvall, CL;Jones, HN;
PMID: 36094907 | DOI: 10.1002/mrd.23644

Fetal growth restriction (FGR) significantly contributes to neonatal and perinatal morbidity and mortality. Currently, there are no effective treatment options for FGR during pregnancy. We have developed a nanoparticle gene therapy targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF1) to correct fetal growth trajectories. Using the maternal nutrient restriction guinea pig model of FGR, an ultrasound-guided, intraplacental injection of nonviral, polymer-based hIGF1 nanoparticle containing plasmid with the hIGF1 gene and placenta-specific Cyp19a1 promotor was administered at mid-pregnancy. Sustained hIGF1 expression was confirmed in the placenta 5 days after treatment. Whilst increased hIGF1 did not change fetal weight, circulating fetal glucose concentration were 33%-67% higher. This was associated with increased expression of glucose and amino acid transporters in the placenta. Additionally, hIGF1 nanoparticle treatment increased the fetal capillary volume density in the placenta, and reduced interhaemal distance between maternal and fetal circulation. Overall, our findings, that trophoblast-specific increased expression of hIGF1 results in changes to glucose transporter expression and increases fetal glucose concentrations within a short time period, highlights the translational potential this treatment could have in correcting impaired placental nutrient transport in human pregnancies complicated by FGR.

MALAT1-dependent hsa_circ_0076611 regulates translation rate in triple-negative breast cancer

Communications biology

2022 Jun 16

Turco, C;Esposito, G;Iaiza, A;Goeman, F;Benedetti, A;Gallo, E;Daralioti, T;Perracchio, L;Sacconi, A;Pasanisi, P;Muti, P;Pulito, C;Strano, S;Ianniello, Z;Fatica, A;Forcato, M;Fazi, F;Blandino, G;Fontemaggi, G;
PMID: 35710947 | DOI: 10.1038/s42003-022-03539-x

Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA, induce back-splicing of VEGFA exon 7, producing circular RNA circ_0076611. Circ_0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ_0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ_0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells.

Clinical and Pathological Characteristics of Hyaline-Vascular Type Unicentric Castleman Disease: A 20-Year Retrospective Analysis

Diagnostics (Basel, Switzerland)

2021 Oct 28

Nishimura, MF;Nishimura, Y;Nishikori, A;Maekawa, Y;Maehama, K;Yoshino, T;Sato, Y;
PMID: 34829355 | DOI: 10.3390/diagnostics11112008

The first case of hyaline vascular type of unicentric Castleman disease (HV-UCD) was reported more than six decades ago. Since patients with HV-UCD are often asymptomatic and this condition is generally discovered incidentally on imaging tests, most of the previous reports were of mediastinal origin detected by chest radiography. In recent years, improved access to imaging modalities has provided new insights in the diagnosis of this condition. In this study, we reviewed the detailed clinical and pathological findings of 38 HV-UCD cases (20 males and 18 females, mean age: 42.8 years). The most common site involved was the abdominal cavity (34.2%), followed by mediastinum (23.7%) and retroperitoneum (15.8%). In the abdominal cavity, mesenteric origin was the most common. The mean size of masses was 4.8 cm. Pathologically, thick hyalinized collagen fibers surrounding large blood vessels and calcification were observed (81.6% and 23.7%, respectively). Multinucleated giant cells resembling Warthin-Finkeldey cell were also observed in occasional cases (23.7%). This is a unique paper that summarizes detailed clinical and pathological findings of a large series of a rare disease. The clinical information presented in this paper is more plausible than previous views and is useful for accurate diagnosis and understanding of the disease.

hsa_circ_0005358 suppresses cervical cancer metastasis by interacting with PTBP1 protein to destabilize CDCP1 mRNA

Molecular therapy. Nucleic acids

2022 Mar 08

Cen, Y;Zhu, T;Zhang, Y;Zhao, L;Zhu, J;Wang, L;Xu, J;Ding, T;Xie, X;Wang, X;Lu, W;
PMID: 34976440 | DOI: 10.1016/j.omtn.2021.11.020

Metastasis is the main cause of cervical cancer lethality, but to date, no effective treatment has been developed to block metastasis. Circular RNAs (circRNAs) were recently found to be involved in cancer metastasis. In this study, we identified a downregulated circRNA derived from the host gene Gli1 (hsa_circ_0005358) in cervical cancer tissues, which was expressed at lower levels in tissues with extracervical metastasis than in those without extracervical metastasis. Upregulation of hsa_circ_0005358 significantly suppressed the migration and invasion of cervical cancer cells in vitro, and downregulation of hsa_circ_0005358 had the opposite effect. A mouse model revealed that cervical cancer cells overexpressing hsa_circ_0005358 possessed weaker metastatic potential in vivo. RNA-pull-down assay, mass spectrometry, and RNA immunoprecipitation validated the findings that hsa_circ_0005358 functions via its 215-224 sequence, which interacts with polypyrimidine tract-binding protein 1 (PTBP1). RNA-sequencing profiling revealed that CUB-domain-containing protein 1 (CDCP1) is a common target for hsa_circ_0005358 and PTBP1. We further confirmed that hsa_circ_0005358 sequestered PTBP1, preventing it from stabilizing CDCP1 mRNA, reducing CDCP1 protein translation and ultimately suppressing cancer metastasis. Our findings reveal the function of hsa_circ_0005358 in tumor metastasis, which may be applied to a potential therapeutic approach for patients with metastatic cervical cancer.

Circular RNA circ-TNPO3 suppresses metastasis of gastric cancer by acting as a protein decoy for IGF2BP3 to regulate the expression of MYC and SNAIL

Molecular Therapy - Nucleic Acids

2021 Aug 01

Yu, T;Ran, L;Zhao, H;Yin, P;Li, W;Lin, J;Mao, H;Cai, D;Ma, Q;Pan, X;Wang, X;Wu, J;Zeng, H;Zhang, W;Lu, D;Luo, P;Zou, Q;Xiao, B;
| DOI: 10.1016/j.omtn.2021.08.029

Gastric cancer (GC) continues to be the most common gastrointestinal malignancy in China, and tumor metastases are a major reason for poor prognosis. Circular RNAs (circRNAs) are an intriguing type of noncoding RNAs with important regulatory roles. However, the roles of circRNAs in GC metastasis have not been fully elucidated. Here, we reported that circ-TNPO3 was significantly downregulated in 103 pairs of GC tissues compared with matched noncancerous tissues. The level of circ-TNPO3 expression correlated with differentiation of GC, and plasma circ-TNPO3 could serve as a potential diagnostic biomarker. Functionally, circ-TNPO3 inhibited proliferation and migration of GC in vitro and in vivo. We further verified that circ-TNPO3 competitively interacted with IGF2BP3 protein, thus, the role of IGF2BP3 in stabilizing MYC mRNA was weakened, which inhibited the expression of MYC and its target SNAIL. Taken together, circ-TNPO3 acts as a protein decoy for IGF2BP3 to regulate the MYC-SNAIL axis, thereby suppressing the proliferation and metastasis of GC. Therefore, circ-TNPO3 has the potential to serve as a therapeutic target for GC.

Enhanced detection of expanded repeat mRNA foci with hybridization chain reaction

Acta neuropathologica communications

2021 Apr 23

Glineburg, MR;Zhang, Y;Krans, A;Tank, EM;Barmada, SJ;Todd, PK;
PMID: 33892814 | DOI: 10.1186/s40478-021-01169-8

Transcribed nucleotide repeat expansions form detectable RNA foci in patient cells that contribute to disease pathogenesis. The most widely used method for detecting RNA foci, fluorescence in situ hybridization (FISH), is powerful but can suffer from issues related to signal above background. Here we developed a repeat-specific form of hybridization chain reaction (R-HCR) as an alternative method for detection of repeat RNA foci in two neurodegenerative disorders: C9orf72 associated ALS and frontotemporal dementia (C9 ALS/FTD) and Fragile X-associated tremor/ataxia syndrome. R-HCR to both G4C2 and CGG repeats exhibited comparable specificity but > 40 × sensitivity compared to FISH, with better detection of both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. Using R-HCR, we observed that integrated stress response (ISR) activation significantly increased the number of endogenous G4C2 repeat RNA foci and triggered their selective nuclear accumulation without evidence of stress granule co-localization in patient fibroblasts and patient derived neurons. These data suggest that R-HCR can be a useful tool for tracking the behavior of repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.

Targeted RNA editing in brainstem alleviates respiratory dysfunction in a mouse model of Rett syndrome

Proceedings of the National Academy of Sciences of the United States of America

2022 Aug 16

Sinnamon, JR;Jacobson, ME;Yung, JF;Fisk, JR;Jeng, S;McWeeney, SK;Parmelee, LK;Chan, CN;Yee, SP;Mandel, G;
PMID: 35939700 | DOI: 10.1073/pnas.2206053119

Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editasewt) and either Mecp2-targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2-targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2-targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editasewt. Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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