UBA1-CDK16 : A Sex-Specific Chimeric RNA and Its Role in Immune Sexual Dimorphism.

RNA processing mechanisms, such as alternative splicing and RNA editing, have been recognized as critical means to expand the transcriptome. Chimeric RNAs formed by intergenic splicing provide another potential layer of RNA diversification. By analyzing a large set of RNA-Seq data and validating results in over 1,200 blood samples, we identified UBA1-CDK16 , a female-specific chimeric transcript. Intriguingly, both parental genes, are expressed in males and females. Mechanistically, UBA1-CDK16 is produced by cis-splicing between the two adjacent X-linked genes, originating from the inactive X chromosome. A female-specific chromatin loop, formed between the junction sites, facilitates the alternative splicing of its readthrough precursor. This unique chimeric transcript exhibits evolutionary conservation, evolving to be female-specific from non-human primates to humans. Furthermore, our investigation reveals that UBA1-CDK16 is enriched in the myeloid lineage and plays a regulatory role in myeloid differentiation. Notably, female COVID-19 patients who tested negative for this chimeric transcript displayed higher counts of neutrophils, highlighting its potential role in disease pathogenesis. These findings support the notion that chimeric RNAs represent a new repertoire of transcripts that can be regulated independently from the parental genes, and a new class of RNA variance with potential implications in sexual dimorphism and immune responses.

A protein-encoding CCDC7 circular RNA inhibits the progression of prostate cancer by up-regulating FLRT3.

Circular RNAs (circRNAs) are a family of endogenous RNAs that have become a focus of biological research in recent years. Emerging evidence has revealed that circRNAs exert biological functions by acting as transcriptional regulators, microRNA sponges, and binding partners with RNA-binding proteins. However, few studies have identified coding circRNAs, which may lead to a hidden repertoire of proteins. In this study, we unexpectedly discovered a protein-encoding circular RNA circCCDC7(15,16,17,18,19) while we were searching for prostate cancer related chimeric RNAs. circCCDC7(15,16,17,18,19) is derived from exon 19 back spliced to exon 15 of the CCDC7 gene. It is significantly downregulated in patients with high Gleason score. Prostate cancer patients with decreased circCCDC7(15,16,17,18,19) expression have a worse prognosis, while linear CCDC7 had no such association. Overexpressed circCCDC7(15,16,17,18,19) inhibited prostate cancer cell migration, invasion, and viability, supporting classification of circCCDC7(15,16,17,18,19) as a bona fide tumor suppressor gene. We provide evidence that its tumor suppressive activity is driven by the protein it encodes, and that circCCDC7(15,16,17,18,19) encodes a secretory protein. Consistently, conditioned media from circCCDC7(15,16,17,18,19) overexpressing cells has the same tumor suppressive activity. We further demonstrate that the tumor suppressive activity of circCCDC7(15,16,17,18,19) is at least partially mediated by FLRT3, whose expression also negatively correlates with Gleason score and clinical prognosis. In conclusion, circCCDC7(15,16,17,18,19) functions as a tumor suppressor in prostate cancer cells through the circCCDC7-180aa secretory protein it encodes, and is a promising therapeutic peptide for prostate cancer.

CTNNBIP1-CLSTN1 functions as a housekeeping chimeric RNA and regulates cell proliferation through SERPINE2.

The conventional understanding that chimeric RNAs are unique to carcinoma and are the products of chromosomal rearrangement is being challenged. However, experimental evidence supporting the function of chimeric RNAs in normal physiology is scarce. We decided to focus on one particular chimeric RNA, CTNNBIP1-CLSTN1. We examined its expression in various tissues and cell types and compared it quantitatively among cancer and noncancer cells. We further investigated its role in a panel of noncancer cells and investigated the functional mechanism. We found that this fusion transcript is expressed in almost all tissues and a wide range of cell types, including fibroblasts, epithelial cells, stem cells, vascular endothelial cells, and hepatocytes. In addition, the CTNNBIP1-CLSTN1 expression level in noncancerous cell lines was not evidently different from that in cancer cell lines. Furthermore, in at least three cell types, silencing CTNNBIP1-CLSTN1 significantly reduced the cell proliferation rate by inducing G2/M arrest and apoptosis. Importantly, rescue experiments confirmed that cell cycle arrest was restored by exogenous expression of the chimera but not the wild-type parental gene. Further evidence is provided that CTNNBIP1-CLSTN1 regulates cell proliferation through SERPINE2. Thus, CTNNBIP1-CLSTN1 is an example of a new class of fusion RNAs, dubbed "housekeeping chimeric RNAs".

CTNNBIP1-CLSTN1 Functions as a Housekeeping Chimeric RNA, and Regulates Cell Proliferation through SERPINE2.

The conventional wisdom that chimeric RNAs being peculiarity of carcinoma, and the products of chromosomal rearrangement is being challenged, However, experimental evidence supporting chimeric RNAs in normal physiology being functional is scarce. We decided to focus on one particular chimeric RNA, CTNNBIP1-CLSTN1 . We examined its expression among various tissues and cell types, and compared quantitatively among cancer and non-cancer cells. We further investigated its role in a panel of non-cancer cells and probed the functional mechanism. We found that this fusion transcript is expressed in almost all tissues, and a wide range of cell types including fibroblasts, epithelial, stem, vascular endothelial cells, and hepatocytes. The expression level in non-cancerous cell lines is also not evidently different from that in the cancer cell lines. Furthermore, silencing CTNNBIP1-CLSTN1 significantly reduces cell proliferation rate, by inducing G2/M arrest in cell cycle progress and apoptosis in at least three cell types. Importantly, rescue experiments confirmed that the cell cycle arrest can be regained by exogenous expression of the chimera, but not the wild type parental gene. Further evidence is provided that CTNNBIP1-CLSTN1 regulates cell proliferation through SERPINE2 . Thus, CTNNBIP1-CLSTN1 represents an example of a new class of fusion RNA, dubbed "housekeeping chimeric RNAs".

Characterization and Function of Glycans on the Spike Proteins of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.

Profile of chimeric RNAs and TMPRSS2-ERG e2e4 isoform in neuroendocrine prostate cancer.

PURPOSE: Specific gene fusions and their fusion products (chimeric RNA and protein) have served as ideal diagnostic markers and therapeutic targets for cancer. However, few systematic studies for chimeric RNAs have been conducted in neuroendocrine prostate cancer (NEPC). In this study, we explored the landscape of chimeric RNAs in different types of prostate cancer (PCa) cell lines and aimed to identify chimeric RNAs specifically expressed in NEPC. METHODS: To do so, we employed the RNA-seq data of eight prostate related cell lines from Cancer Cell Line Encyclopedia (CCLE) for chimeric RNA identification. Multiple filtering criteria were used and the candidate chimeric RNAs were characterized at multiple levels and from various angles. We then performed experimental validation on all 80 candidates, and focused on the ones that are specific to NEPC. Lastly, we studied the clinical relevance and effect of one chimera in neuroendocrine process. RESULTS: Out of 80 candidates, 15 were confirmed to be expressed preferentially in NEPC lines. Among them, 13 of the 15 were found to be specifically expressed in NEPC, and four were further validated in another NEPC cell line. Importantly, in silico analysis showed that tumor malignancy may be correlated to the level of these chimeric RNAs. Clinically, the expression of TMPRSS2-ERG (e2e4) was elevated in tumor tissues and indicated poor clinical prognosis, whereas the parental wild type transcripts had no such association. Furthermore, compared to the most frequently detected TMPRSS2-ERG form (e1e4), e2e4 encodes 31 more amino acids and accelerated neuroendocrine process of prostate cancer. CONCLUSIONS: In summary, these findings painted the landscape of chimeric RNA in NEPC and supported the idea that some chimeric RNAs may represent additional biomarkers and/or treatment targets independent of parental gene transcripts.

Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein-Protein Interactions and Drug Evaluation Under Physiological Conditions.

Identification of protein-protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2.

Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes.

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.

The intraviral protein-protein interaction of SARS-CoV-2 reveals the key role of N protein in virus-like particle assembly.

Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.

Loss of NEIL3 activates radiotherapy resistance in the progression of prostate cancer.

OBJECTIVE: To explore the genetic changes in the progression of castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) and the reason why these cancers resist existing therapies. METHODS: We employed our CRPC cell line microarray and other CRPC or NEPC datasets to screen the target gene NEIL3. Lentiviral transfection and RNA interference were used to construct overexpression and knockdown cell lines. Cell and animal models of radiotherapy were established by using a medical electron linear accelerator. Flow cytometry was used to detect apoptosis or cell cycle progression. Western blot and qPCR were used to detect changes in the protein and RNA levels. RESULTS: TCGA and clinical patient datasets indicated that NEIL3 was downregulated in CRPC and NEPC cell lines, and NEIL3 was correlated with a high Gleason score but a good prognosis. Further functional studies demonstrated that NEIL3 had no effect on the proliferation and migration of PCa cells. However, cell and animal radiotherapy models revealed that NEIL3 could facilitate the radiotherapy sensitivity of PCa cells, while loss of NEIL3 activated radiotherapy resistance. Mechanistically, we found that NEIL3 negatively regulated the expression of ATR, and higher NEIL3 expression repressed the ATR/CHK1 pathway, thus regulating the cell cycle. CONCLUSIONS: We demonstrated that NEIL3 may serve as a diagnostic or therapeutic target for therapy-resistant patients.

Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells.

The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.

Landscape of Chimeric RNAs in Non-Cancerous Cells.

Gene fusions and their products (RNA and protein) have been traditionally recognized as unique features of cancer cells and are used as ideal biomarkers and drug targets for multiple cancer types. However, recent studies have demonstrated that chimeric RNAs generated by intergenic alternative splicing can also be found in normal cells and tissues. In this study, we aim to identify chimeric RNAs in different non-neoplastic cell lines and investigate the landscape and expression of these novel candidate chimeric RNAs. To do so, we used HEK-293T, HUVEC, and LO2 cell lines as models, performed paired-end RNA sequencing, and conducted analyses for chimeric RNA profiles. Several filtering criteria were applied, and the landscape of chimeric RNAs was characterized at multiple levels and from various angles. Further, we experimentally validated 17 chimeric RNAs from different classifications. Finally, we examined a number of validated chimeric RNAs in different cancer and non-cancer cells, including blood from healthy donors, and demonstrated their ubiquitous expression pattern.

Autographa Californica Multiple Nucleopolyhedrovirus orf13 Is Required for Efficient Nuclear Egress of Nucleocapsids.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.

Association between anemia and outcome in patients hospitalized for acute heart failure syndromes: findings from Beijing Acute Heart Failure Registry (Beijing AHF Registry).

Whether the anemia increases the risk of mortality in patients with acute heart failure (AHF) remains unclear. This study aims to explore the relationship between anemia and outcomes in patients with AHF including subgroup analysis. This study included 3279 patients with hemoglobin available from the Beijing Acute Heart Failure Registry (Beijing AHF Registry) study. The primary endpoint was all-cause mortality in 1 year, and the secondary endpoint was 1-year all-cause events including all-cause death and readmission. Logistic regression models were applied to describe related variables of anemia in patients with AHF. Multivariate Cox proportional hazards models described associations of anemia with clinical outcomes in the overall cohort and subgroups. 45.4% of the patients were found anemic. They were older and had more comorbidities than non-anemic patients. Variables including older age, female, chronic kidney dysfunction (CKD), lower hematocrit, lower albumin, with loop diuretics applied, without beta-blockers, angiotensin-converting enzyme inhibitors /angiotensin receptor blockers (ACEIs/ARBs) and spironolactone applied in the emergency department (ED) were associated with anemia in AHF patients. Anemic patients had higher 1-year mortality (38.4% vs. 27.2%, p < 0.0001) and 1-year events rates (63.2% vs. 56.7%, p < 0.0001). After adjusted for covariates, anemia was associated with the increase of 1-year mortality (hazard ratio [HR] 1.278; 95% confidence interval [CI] 1.114-1.465; p = 0.0005) and 1-year events (HR 1.136; 95% CI 1.025-1.259; p = 0.0154). The severer anemia patients had higher risks both of 1-year mortality and events. In the subgroup analysis, the independent associations of anemia with 1-year mortality were shown in the subgroups including age < 75 years, male, body mass index < 25 kg/m2 and BMI ≥ 25 kg/m2, New York Heart Association (NYHA) functional class I-II and NYHA functional class III-IV, with and without cardiovascular ischemia, heart rate (HR) < 100 bpm and HR ≥ 100 bpm, systolic blood pressure (SBP) < 120 mmHg and SBP ≥ 120 mmHg, left ventricular ejection fraction (LVEF) < 40% and LVEF ≥ 40%, serum creatinine (Scr) < 133 umol/l, and with diuretics use, with and without beta-blockers use, without ACEIs/ARBs use in the ED. Anemia is associated with older age, female, CKD, volume overload, malnutrition, with loop diuretics, without beta-blockers, ACEIs/ARBs and spironolactone administration, and higher mortality and readmission in AHF. The risk associations are particular significantly obvious in younger, male, overweight, preserved LVEF, lower Scr, with diuretics and beta-blockers, without ACEIs/ARBs administration subgroups.Clinical trial No. ChiCTR-RIC-17014222.

Molecular Imaging of Prostate Cancer Targeting CD46 Using ImmunoPET.

PURPOSE: We recently identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted PET radiopharmaceutical, [89Zr]DFO-YS5, and evaluated its performance for immunoPET imaging in murine prostate cancer models. EXPERIMENTAL DESIGN: [89Zr]DFO-YS5 was prepared and its in vitro binding affinity for CD46 was measured. ImmunoPET imaging was conducted in male athymic nu/nu mice bearing DU145 [AR-, CD46+, prostate-specific membrane antigen-negative (PSMA-)] or 22Rv1 (AR+, CD46+, PSMA+) tumors, and in NOD/SCID gamma mice bearing patient-derived adenocarcinoma xenograft, LTL-331, and neuroendocrine prostate cancers, LTL-331R and LTL-545. RESULTS: [89Zr]DFO-YS5 binds specifically to the CD46-positive human prostate cancer DU145 and 22Rv1 xenografts. In biodistribution studies, the tumor uptake of [89Zr]DFO-YS5 was 13.3 ± 3.9 and 11.2 ± 2.5 %ID/g, respectively, in DU145 and 22Rv1 xenografts, 4 days postinjection. Notably, [89Zr]DFO-YS5 demonstrated specific uptake in the PSMA- and AR-negative DU145 model. [89Zr]DFO-YS5 also showed uptake in the patient-derived LTL-331 and -331R models, with particularly high uptake in the LTL-545 neuroendocrine prostate cancer tumors (18.8 ± 5.3, 12.5 ± 1.8, and 32 ± 5.3 %ID/g in LTL-331, LTL-331R, and LTL-545, respectively, at 4 days postinjection). CONCLUSIONS: [89Zr]DFO-YS5 is an excellent PET imaging agent across a panel of prostate cancer models, including in both adenocarcinoma and neuroendocrine prostate cancer, both cell line- and patient-derived xenografts, and both PSMA-positive and -negative tumors. It demonstrates potential for clinical translation as an imaging agent, theranostic platform, and companion biomarker in prostate cancer.

The iTRAQ-based chloroplast proteomic analysis of Triticum aestivum L. leaves subjected to drought stress and 5-aminolevulinic acid alleviation reveals several proteins involved in the protection of photosynthesis.

BACKGROUNDS: The perturbance of chloroplast proteins is a major cause of photosynthesis inhibition under drought stress. The exogenous application of 5-aminolevulinic acid (ALA) mitigates the damage caused by drought stress, protecting plant growth and development, but the regulatory mechanism behind this process remains obscure. RESULTS: Wheat seedlings were drought treated, and the iTRAQ-based proteomic approach was employed to assess the difference in chloroplast protein content caused by exogenous ALA. A total of 9499 peptides, which could be classified into 2442 protein groups, were identified with ≤0.01 FDR. Moreover, the contents of 87 chloroplast proteins was changed by drought stress alone compared to that of the drought-free control, while the contents of 469 was changed by exogenous ALA application under drought stress compared to that of drought stress alone. The Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results suggested that the ALA pretreatment adjusted some biological pathways, such as metabolic pathways and pathways involved in photosynthesis and ribosomes, to enhance the drought resistance of chloroplasts. Furthermore, the drought-promoted H2O2 accumulation and O2- production in chloroplasts were alleviated by the exogenous pretreatment of ALA, while peroxidase (POD) and glutathione peroxidase (GPX) activities were upregulated, which agreed with the chloroplast proteomic data. We suggested that ALA promoted reactive oxygen species (ROS) scavenging in chloroplasts by regulating enzymatic processes. CONCLUSIONS: Our results from chloroplast proteomics extend the understanding of the mechanisms employed by exogenous ALA to defend against drought stress in wheat.

The landscape of chimeric RNAs in non-diseased tissues and cells.

Chimeric RNAs and their encoded proteins have been traditionally viewed as unique features of neoplasia, and have been used as biomarkers and therapeutic targets for multiple cancers. Recent studies have demonstrated that chimeric RNAs also exist in non-cancerous cells and tissues, although large-scale, genome-wide studies of chimeric RNAs in non-diseased tissues have been scarce. Here, we explored the landscape of chimeric RNAs in 9495 non-diseased human tissue samples of 53 different tissues from the GTEx project. Further, we established means for classifying chimeric RNAs, and observed enrichment for particular classifications as more stringent filters are applied. We experimentally validated a subset of chimeric RNAs from each classification and demonstrated functional relevance of two chimeric RNAs in non-cancerous cells. Importantly, our list of chimeric RNAs in non-diseased tissues overlaps with some entries in several cancer fusion databases, raising concerns for some annotations. The data from this study provides a large repository of chimeric RNAs present in non-diseased tissues, which can be used as a control dataset to facilitate the identification of true cancer-specific chimeras.

Knockdown of Chimeric RNA by RNAi.

Knockdown assays are widely used to study the functions of a gene of interest. RNA interference (RNAi) describes a set of well-known methods used to reduce the expression of a target gene by degrading its mRNA with short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Knockdown of chimeric RNAs present different challenges than standard RNAi targeting for regular genes. Most specifically, sequence homology restricts the targeting region to the chimeric junction and can result in off-target effects on the parental genes. In this chapter, we provide guidelines and procedures for RNAi design of chimeric RNAs, knockdown of chimeric RNAs, downstream experiments for chimeric RNA functional studies and necessary controls to accompany each set of experiments.

Separation of Nuclear and Cytoplasmic Fractions for Chimeric RNA Characterization.

Cellular organelle fractionation, a basic technique in molecular biology, has been devised to separate various cell components, which can then be purified and analyzed biochemically. Isolation of proteins or RNAs from these fractions provides insight into fraction-specific or even organelle-specific expression, which may indicate potential modes of functionality or likelihood for a transcript to encode a protein. These findings can be further utilized to observe differences in expression between normal and diseased cell states, such as cancer. We utilize these techniques to observe expression of chimeric RNAs in these fractions. Within this chapter we describe the most frequently used cellular fractionation technique: the separation of the cytoplasmic fraction from the nuclear fraction in a cell.