snRNA Research Articles

Differential levels of circulating RNAs prior to endometrial cancer diagnosis.

Endometrial cancer (EC) is one of the most common female cancers and there is currently no routine screening strategy for early detection. An altered abundance of circulating microRNAs (miRNAs) and other RNA classes have the potential as early cancer biomarkers. We analyzed circulating RNA levels using small RNA sequencing, targeting RNAs in the size range of 17-47 nucleotides, in EC patients with samples collected prior to diagnosis compared to cancer-free controls. The analysis included 316 cases with samples collected 1-11 years prior to EC diagnosis, and 316 matched controls, both from the Janus Serum Bank cohort in Norway. We identified differentially abundant (DA) miRNAs, isomiRs, and small nuclear RNAs between EC cases and controls. The top EC DA miRNAs were miR-155-5p, miR-200b-3p, miR-589-5p, miR-151a-5p, miR-543, miR-485-5p, miR-625-p, and miR-671-3p. miR-200b-3p was previously reported to be among one of the top miRNAs with higher abundance in EC cases. We observed 47, 41, and 32 DA miRNAs for EC interacting with BMI, smoking status, and physical activity, respectively, including two miRNAs (miR-223-3p and miR-29b-3p) interacting with all three factors. The circulating RNAs are altered and show temporal dynamics prior to EC diagnosis. Notably, DA miRNAs for EC had the lowest q-value 4.39-6.66 years before diagnosis. Enrichment analysis of miRNAs showed that signaling pathways Fc epsilon RI, prolactin, toll-like receptor, and VEGF had the strongest associations.

The transcriptional landscape of coding and non-coding RNAs in recurrent and non-recurrent colon cancer

A considerable number of colon cancer patients with local or local advanced disease suffer from recurrence and there is an urgent need for better prognostic biomarkers in this setting. Here, the transcriptomic landscape of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs (scaRNAs), pseudogenes and circular RNAs (circRNAs), as well as RNAs denoted as miscellaneous RNAs, was profiled by total RNA sequencing. In addition to well-known coding and non-coding RNAs, differential expression analysis also uncovered transcripts, which have not previously been implicated in colon cancer, such as RNA5SP149, RNU4-2 and SNORD3A. Moreover, there was a profound global upregulation of snRNA pseudogenes, snoRNAs and rRNA pseudogenes in more advanced tumours. A global downregulation of circRNAs in tumours relative to normal tissues was observed, while only few were differentially expressed between tumour stages. Many previously undescribed transcripts, including RNU6-620P, RNU2-20P, VTRNA1-3 and RNA5SP60 indicated strong prognostic biomarker potential in ROC analyses. In summary, this study unveiled numerous differentially expressed RNAs across various classes between recurrent and non-recurrent colon cancer. Notably, there was a significant global upregulation of snRNA pseudogenes, snoRNAs and rRNA pseudogenes in advanced tumours. Many of these newly discovered candidates demonstrated a strong prognostic potential for stage II colon cancer.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

Role of TOE1 variants at the nuclear localization motif in pontocerebellar hypoplasia 7.

Biallelic TOE1 variants can cause pontocerebellar hypoplasia type 7 (PCH7), a condition characterized by pontocerebellar hypoplasia with genital abnormality. TOE1 is a 3'-exonuclese for 3'-end maturation in small nuclear RNA. TOE1 pathogenic variants have been reported at the DEDD catalytic domain and zinc finger motif. Here, we describe a PCH7 patient with novel compound heterozygous TOE1 variants and a detailed clinical course. The patient was a 3-year-old female and showed developmental delay without cerebellar ataxic behavior. Head MRI revealed delayed myelination without pontocerebellar hypoplasia at 9 months of age. Progressive pontocerebellar atrophy was prominent at follow-up MRI. Cerebral abnormalities are characteristic features of PCH7 before pontocerebellar atrophy is observed. One variant, p.Arg331*, was located at the nuclear localization motif (NLM) and partially escaped from nonsense-mediated decay. This variant affected nuclear localization in mutant expressing cells, thus, the TOE1 variant at NLM leads to TOE1 dysfunction associated with nuclear mis-localization.

Small Non-Coding RNAs and Their Role in Locoregional Metastasis and Outcomes in Early-Stage Breast Cancer Patients.

Deregulation of small non-coding RNAs (sncRNAs) has been associated with the onset of metastasis. We evaluated the expression of sncRNAs in patients with early-stage breast cancer, performing RNA sequencing in 60 patients for whom tumor and sentinel lymph node (SLN) samples were available, and conducting differential expression, gene ontology, enrichment and survival analyses. Sequencing annotation classified most of the sncRNAs into small nucleolar RNA (snoRNAs, 70%) and small nuclear RNA (snRNA, 13%). Our results showed no significant differences in sncRNA expression between tumor or SLNs obtained from the same patient. Differential expression analysis showed down-regulation (n = 21) sncRNAs and up-regulation (n = 2) sncRNAs in patients with locoregional metastasis. The expression of SNHG5, SNORD90, SCARNA2 and SNORD78 differentiated luminal A from luminal B tumors, whereas SNORD124 up-regulation was associated with luminal B HER2+ tumors. Discriminating analysis and receiver-operating curve analysis revealed a signature of six snoRNAs (SNORD93, SNORA16A, SNORD113-6, SNORA7A, SNORA57 and SNORA18A) that distinguished patients with locoregional metastasis and predicted patient outcome. Gene ontology and Reactome pathway analysis showed an enrichment of biological processes associated with translation initiation, protein targeting to specific cell locations, and positive regulation of Wnt and NOTCH signaling pathways, commonly involved in the promotion of metastases. Our results point to the potential of several sncRNAs as surrogate markers of lymph node metastases and patient outcome in early-stage breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant sncRNAs and to validate our results in a larger cohort of patients.

Circulating microRNA-21-3p in patients with arterial hypertension

Objective — to study changes in the blood plasma levels of microRNA‑21‑3p in patients with arterial hypertension (AH) and their relationship with the characteristics of the course of the disease.
 Materials and methods. Examinations involved 24 patients with hypertension stage II and degree 2—3 (11 men, 13 women) aged from 42 to 60 years. The control group consisted of 12 practically healthy individuals, age and gender matching. The examinations were carried out in the Department of Hypertension and Kidney Diseases of the GI «L. T. Mala National Therapy Institute of NAMS of Ukraine». The degree, stage of hypertension and the presence of risk factors for cardiovascular diseases (CVD), established CVD and kidney disease were assessed in accordance with current recommendations. All patients underwent general clinical laboratory and instrumental examinations. The blood plasma level of circulating microRNA‑21‑3p was determined by quantitative reverse transcriptase‑polymerase chain reaction (qRT‑PCR) using reagent kits for monitoring and analyzing microRNA expression «TaqMan microRNA Assay» (ThermoFisher Scientific, USA). Small nuclear RNA U6 was used as a reference. Relative expression was calculated in relative units (r.u.) using the ΔΔCt method. Statistical analysis of the obtained data was carried out using standard methods with application packages Microsoft Excel and SPSS 19.0.
 Results. A significant decrease in the plasma levels of microRNA‑21‑3p in patients with hypertension was established in comparison with practically healthy individuals. The most pronounced decrease in plasma microRNA‑21‑3p levels was observed in patients with the most severe hypertension (stage 3 AH) and in subjects, in whom antihypertensive therapy did not result in the achieving target levels of «office» blood pressure (BP). Significant negative correlations were found between the plasma microRNA‑21‑3p levels and hemodynamic parameters (BP levels) and the left ventricular myocardial mass index — an indicator of the severity of left ventricular hypertrophy, one of the key lesions of target organs. The obtained results indicate the presence in patients with hypertension of a significant deficiency in the production of microRNA‑21‑3p, which, according to the literature data, has pronounced antihypertensive and cardiovascular‑ and renoprotective properties.
 Conclusions. A significant decrease in the blood plasma levels of microRNA‑21‑3p, the endogenous regulator of gene expression with antihypertensive and organoprotective properties, was established in patients with hypertension in comparison with practically healthy subjects. The revealed decrease in the blood plasma levels of this microRNA was enhanced at severe and uncontrolled hypertension and associated with the degree of hypertensive heart damage. The data obtained indicate the important role of microRNA‑21‑3p in the hypertension pathogenesis and the prospects of finding methods for correcting its production.

LUC7L3 is a downstream factor of SRSF1 and prevents genomic instability

The RNA-binding protein LUC7L3 is the human homolog of yeast U1 small nuclear RNA (snRNA)-related splicing factor Luc7p. While the primary function of LUC7L3 as an RNA-binding protein is believed to be involved in RNA metabolism, particularly in the splicing process, its exact role and other functions are still not fully understood. In this study, we aimed to elucidate the role of LUC7L3 and its impact on cell proliferation. Our study revealed that LUC7L3 depletion impairs cell proliferation compared to the other Luc7p paralogs, resulting in cell apoptosis and senescence. We explored the underlying mechanisms and found that LUC7L3 depletion leads to R-loop accumulation, DNA replication stress, and genome instability. Furthermore, we discovered that LUC7L3 depletion caused abnormalities in spindle assembly, leading to the formation of multinuclear cells. This was attributed to the dysregulation of protein translation of spindle-associated proteins. Additionally, we investigated the interplay between LUC7L3 and SRSF1 and identified SRSF1 as an upper stream regulator of LUC7L3, promoting the translation of LUC7L3 protein. These findings highlight the importance of LUC7L3 in maintaining genome stability and its relationship with SRSF1 in this regulatory pathway.

Abstract 2350: Comparison of normalization methods for RT-qPCR microRNA expression in cancer datasets

Abstract Real-time quantitative PCR (RT-qPCR) is a widely used method for quantifying microRNA (miRNA) expression, but its accuracy depends on appropriate normalization. RT-qPCR is susceptible to technical variation from several sources, including sample collection and storage, miRNA quantity/quality, and extraction and amplification efficiencies. Normalization corrects for these factors, theoretically leaving behind only biological variation. With growing interest in miRNAs as biomarkers for the diagnosis and prognosis of various cancers, analyses must be accurate and consistent. Primarily, the comparative Ct method is used, which compares the expression of miRNAs to a reference value. Unfortunately, there is a lack of consistency on what to use as a reference. A standard recommendation is the average of multiple, stable miRNAs referred to as endogenous controls (ECs). Currently, there are no known universally stable miRNAs. Therefore, ECs should be selected on a per-disease and per-sample type basis. Multiple algorithms exist to identify stable ECs, but few studies indicate what, if any, steps were taken to implement them correctly. Also, many studies have used non-ideal reference values such as small nuclear RNAs or a single miRNA. This lack of agreement on protocol often leads to difficulty comparing studies and may produce incorrect results. This work addresses these inconsistencies by making recommendations for the normalization of RT-qPCR miRNA expression. We compared 2 widely used methods for EC selection, NormFinder and GeNorm, and to show how normalization influences results we also used an unstable miRNA. Only miRNAs with expression in all samples were considered as potential ECs. GeNorm is sensitive to correlated genes. Therefore, for any pair or trio of correlated miRNAs 1 was kept. NormFinder performs optimally with 5-10 candidate genes; therefore, we tested the 10 miRNAs with the lowest coefficients of variation (CV). The top 3 stable miRNAs were selected from each algorithm to serve as ECs, and we used their average expression to normalize each sample. For the single miRNA, samples were scaled based on its value. We applied these methods to 3 independent datasets: First, plasma samples from a study on canine osteosarcoma (OSA), including pre-amputation (n=45), post-amputation (n=27), and healthy controls (n=21). Second, tissue samples from a study on canine OSA, including primary tumour (n=42) and lung metastases (n=12). Third, serum samples from a study on canine lymphoma included B-cell (n=24), T-cell (n=16), and healthy controls (n=14). For datasets 1 and 3, NormFinder provided a better reduction in gene-specific CV and a more favourable cumulative distribution of the CV. For dataset 2, there was less initial variability, but NormFinder still had slightly better results. Both algorithms had pros and cons, but NormFinder consistently provides a better reduction in sample variation across datasets. Citation Format: Heather Treleaven, Latasha Ludwig, Alicia Viloria-Petit, R. Darren Wood, Ayesha Ali, Geoffrey A. Wood. Comparison of normalization methods for RT-qPCR microRNA expression in cancer datasets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2350.

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

Abstract 6939: Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program

Abstract Background: In HER2+ breast cancer (BC), residual disease (RD) after neoadjuvant HER2 inhibition is associated with increased risk of recurrence. However, the genetic and non-genetic resistance mechanisms underlying RD remain incompletely characterized. Here we used single nuclei (sn) RNA and DNA sequencing to profile genetic and transcriptional changes in growing, residual, and recurrent tumors from an inducible mouse tumor model of HER2+ breast cancer. Methods: We used mammary tumors/tumor bed (n=2-3/time point) from female MMTV-rtTA; TetO-ERBB2 mice at 9 weeks DoxOn (HER2on), 7 days DoxOff (HER2off) and 12-21 weeks Doxoff (recurrent). Samples were snap frozen and single nuclei isolation was performed. snRNAseq was performed using 10 × 5’ v2 kits for library preparation and sequencing. snDNAseq was performed using a custom amplicon panel targeting the mouse genome and human ERBB2 locus via the MissionBio Tapestri platform. Results: For snRNAseq analysis, tumor cell clusters were identified by Epcam and ERBB2 gene expression. Tumor cells were further subclustered into 18 unique clusters that separated by ERBB2 expression. Seven subclusters of HER2on tumor cells were characterized by low ERRB2 expression: 1.38 vs 2.94 (median normalized log counts) in HER2on-ERBB2high cells. Top 5 differentially expressed genes (DEG) per condition (log2 fold change >1.5, adjusted p value < 0.05) were: HER2on: Erbb2, Csn3, Lcn2, Csn2, Wfdc18; HER2on-ERBB2high: Csn3, Igfbp5, Csn2, Lcn2, Trf; HER2on-ERBB2low: Lars2, Gm47283, Cmss1, Gm42418, Neat1; HER2off: Lars2, Hexb, Cmss1, Gphn, Gm42418; Recurrent: Capza2, Vim, Lgals1, Crabp1, S100a6. HER2on-ERBB2low cells were more closely related in transcriptional space to HER2off residual tumor cells than HER2on-ERBB2high tumor cells. Trajectory analysis suggested that HER2off RD evolved from HER2on-ERRB2low cells and formed the basis of recurrent disease. To determine clonal evolution over time, we used common top 10 DEGs between HER2on vs HER2off vs HER2low snRNAseq data and intersected that set with significantly enriched single nucleotide variants (SNVs) from our snDNAseq data. We identified 16 clones containing at least 1 SNV at >1% frequency across all samples. We observed clonal persistence, relative enrichment and extinguishment between HER2on, HER2off and recurrent samples but without clonal dominance. Conclusion: Time-series analysis of genetic and transcriptional evolution in a mouse model of HER2+ BC RD suggests that a tumor cell subpopulation characterized by an ERBB2low-associated transcriptional program drives RD after HER2 inhibition. Citation Format: Sheheryar K. Kabraji, Weihua Wang, Yali Zhang, Johann Bergholz, Tao Jiang, Vittal Kurisetty, Maia Homsi, Jianmin Wang, Jean Zhao. Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6939.

Activation of ERβ hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor β (ERβ) in TNBC, but the detailed molecular mechanisms downstream ERβ activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERβ agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERβ were critical for the binding between U2AF1 and ERβ. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERβ-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERβ activation in TNBC, which provides rationale for ERβ activation-based single or combined therapy for patients with TNBC.

Structural basis of U12-type intron engagement by the fully assembled human minor spliceosome.

The minor spliceosome, which is responsible for the splicing of U12-type introns, comprises five small nuclear RNAs (snRNAs), of which only one is shared with the major spliceosome. In this work, we report the 3.3-angstrom cryo-electron microscopy structure of the fully assembled human minor spliceosome pre-B complex. The atomic model includes U11 small nuclear ribonucleoprotein (snRNP), U12 snRNP, and U4atac/U6atac.U5 tri-snRNP. U11 snRNA is recognized by five U11-specific proteins (20K, 25K, 35K, 48K, and 59K) and the heptameric Sm ring. The 3' half of the 5'-splice site forms a duplex with U11 snRNA; the 5' half is recognized by U11-35K, U11-48K, and U11 snRNA. Two proteins, CENATAC and DIM2/TXNL4B, specifically associate with the minor tri-snRNP. A structural analysis uncovered how two conformationally similar tri-snRNPs are differentiated by the minor and major prespliceosomes for assembly.

POLR3A-mutated Wiedemann-Rautenstrauch fibroblasts display differential profile of intron retention and expression of TP53 isoforms

BACKGROUND: Wiedemann-Rautenstrauch Syndrome (WRS) is a neonatal progeroid syndrome for which biallelic pathogenic variants in RNA polymerase III subunit A (POLR3A) have recently been described. POLR3 is a 17 subunits protein complex responsible for the transcription of short RNAs including all the transfer RNAs (tRNAs), the 5 S subunit of ribosomal RNA, the short nuclear RNA U6, among other regulatory RNAs. OBJECTIVE: We aim to evaluate the impact of POLR3A pathogenic variants on the relative expression of the short nuclear RNA U6 and on the differential profile of intron retention RNA U6, p53 isoforms and in fibroblasts derived from patients with WRS and control fibroblasts. METHODS: RNA was extracted by the TRIzol method; intron retention analysis was performed by using IRFinder from an mRNA sequencing (RNA-Seq) platform; P53 isoforms, short nuclear RNA U6 and additional genes related to cell senescence were measured by RT-PCR. RESULTS: No significant differences were found in the percentage of intron retention (control: 7.8%, WRS1 : 6.3%and WRS2 : 8.14%). Genes showing higher intron retention profile in both groups were mainly related to RNA binding pathways, cell cycle regulation, positive regulation of transcription, positive regulation of inflammatory pathways, negative regulation of apoptosis, RNA transcription, mitochondria, and regulation of translation initiation. However, in WRS fibroblasts the genes with more intron retention were those related to the immune response and mitochondrial function; while in control those related to the response to oxidative stress had the most introns retained. WRS1 showed higher expression of short nuclear RNA U6 compared to control and WRS2; while both WRS cells showed higher expression of p53β and lower percentage of Δ133p63α, consistent with a higher expression of the cellular senescence markers p16 and p21. CONCLUSIONS: These results demonstrated the important role of POLR3A in the maintenance of cellular homeostasis and highlight its potential role in cell senescence in WRS.

Abstract P26: Altered RNA Export Sensitizes to Nuclear Export Inhibition in SF3B1 Mutant MDS

Abstract SF3B1 mutations, the most frequent spliceosomal alterations across cancers, occur in 25% of myelodysplastic syndromes (MDS) patients yet lack effective therapies. Two phase 2 clinical trials with the XPO1 inhibitors in high-risk MDS revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs) out of the nucleus. Given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome, we hypothesized that XPO1 inhibition may preferentially affect SF3B1-mutant cells and that SF3B1-mutant high-risk MDS patients would have a better response to rational targeted drug combinations with XPO1 inhibitors. To evaluate the mechanism underlying the preferential sensitivity of SF3B1-mutant cells to XPO1 inhibition, we conducted subcellular RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and mutant cells. Transcriptomic analysis revealed increased global retention of RNA transcripts and elevated snRNAs in the nucleus after XPO1 inhibition in the SF3B1 mutant cell line. Global RNA expression and splicing analysis revealed increased alternative splicing in SF3B1 mutant cells after XPO1 inhibition. These results suggest that the preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through increased nuclear retention of spliceosomal snRNAs and select mRNAs that results in perturbation of apoptotic pathways. Despite the promising efficacy of XPO1 inhibition in SF3B1-mutated MDS, dose escalation is limited by toxicity. In order to identify novel drug combination targets with lower XPO1 inhibitor doses, we employed whole genome CRISPR screens in two acute myeloid leukemia (AML) cell lines treated with XPO1 inhibitor. We identified two drug targets that greatly synergized with eltanexor specifically in the SF3B1 mutant cell lines: venetoclax (a BCL2 inhibitor), and A1331852 (a BCL-XL inhibitor). In addition, BH3 profiling demonstrated increased priming of the BCL2 and BCL-XL in SF3B1 mutant cells. We further validated these combinations in vivo using competitive transplant assays in Sf3b1 K700E conditional knock-in mice. The combination of eltanexor with venetoclax and eltanexor with A1331852 showed a significant decrease in the Sf3b1-mutant burden in the peripheral blood and bone marrow progenitor compartments, suggesting a preferential sensitivity of the combinations for SF3B1 mutant cells. Although A1331852 exhibited similar results to venetoclax, there was increased weight loss and decrease in hemoglobin associated with BCLXL inhibition. In conclusion, our study provides insight on the mechanisms underlying the heightened sensitivity of XPO1 inhibition in SF3B1-mutant MDS/AML. The identification of BCL2 and BCL-XL as synergistic targets with XPO1 inhibitors, validated by in vivo testing, BH3 profiling, and RNA sequencing, highlights the potential for therapeutic combinations. Specifically, the combination of eltanexor and venetoclax could be a promising SF3B1-specific therapy. Citation Format: Sana Chaudhry, Felipe Beckedorff, Shaista Shabbir Jasdanwala, Tulasigeri M Totiger, Maurizio Affer, Nidhi Hariramani, Olivia Tonini, Stephan Noudali, Alexandra Chirino, Alyssa Cornista, Skye Montoya, Daniel Bilbao, Jumana Afaghani, Jose Antonio Rodríguez, Shruti Bhatt, Eric Wang, Justin Taylor. Altered RNA Export Sensitizes to Nuclear Export Inhibition in SF3B1 Mutant MDS [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P26.

Cell-mediated exon skipping normalizes dystrophin expression and muscle function in a new mouse model ofDuchenne Muscular Dystrophy.

Cell therapy for muscular dystrophy has met with limited success, mainly due to the poor engraftment of donor cells, especially in fibrotic muscle at an advanced stage of the disease. We developed a cell-mediated exon skipping that exploits the multinucleated nature of myofibers to achieve cross-correction of resident, dystrophic nuclei by the U7 small nuclear RNA engineered to skip exon 51 of the dystrophin gene. We observed that co-culture of genetically corrected human DMD myogenic cells (but not of WT cells) with their dystrophic counterparts at a ratio of either 1:10 or 1:30 leads to dystrophin production at a level several folds higher than what predicted by simple dilution. This is due to diffusion of U7 snRNA to neighbouring dystrophic resident nuclei. When transplanted into NSG-mdx-Δ51mice carrying a mutation of exon 51, genetically corrected human myogenic cells produce dystrophin at much higher level than WT cells, well in the therapeutic range, and lead to force recovery even with an engraftment of only 3-5%. This level of dystrophin production is an important step towards clinical efficacy for cell therapy.

Unconventional features in the transcription and processing of spliceosomal small nuclear RNAs in the protozoan parasite Trichomonas vaginalis

Trichomonas vaginalis is a medically important protozoan parasite, and a deep-branching, evolutionarily divergent unicellular eukaryote that has conserved several key features of eukaryotic gene expression. Trichomonas vaginalis possesses a metazoan/plant-like capping apparatus, mRNAs with a cap 1 structure and spliceosomes containing the five small nuclear RNAs (snRNAs). However, in contrast to metazoan and plant snRNAs, the structurally conserved T. vaginalis snRNAs were initially identified as lacking the canonical guanosine cap nucleotide. To explain this unusual condition, we sought to investigate transcriptional and processing features of the spliceosomal snRNAs in this protist. Here, we show that T. vaginalis spliceosomal snRNA genes mostly lack typical eukaryotic promoters. In contrast to other eukaryotes, the putative TATA box in the T. vaginalis U6 snRNA gene was found to be dispensable for transcription or RNA polymerase selectivity. Moreover, U6 transcription in T. vaginalis was virtually insensitive to tagetitoxin compared with other cellular transcripts produced by the same RNA polymerase III. Most important and unexpected, snRNA transcription in T. vaginalis appears to bypass capping as we show that these transcripts retain their original 5′-triphosphate groups. In conclusion, transcription and processing of spliceosomal snRNAs in T. vaginalis deviate considerably from the conventional rules of other eukaryotes.

Housekeeping U1 snRNA facilitates antiviral innate immunity by promoting TRIM25-mediated RIG-I activation

U1 small nuclear RNA (snRNA) is an abundant and evolutionarily conserved 164-nucleotide RNA species that functions in pre-mRNA splicing, and it is considered to be a housekeeping non-coding RNA. However, the role of U1 snRNA in regulating host antiviral immunity remains largely unexplored. Here, we find that RNVU1-18, a U1 pseudogene, is significantly upregulated in the host infected with RNA viruses, including influenza and respiratory syncytial virus. Overexpression of U1 snRNA protects cells against RNA viruses, while knockdown of U1 snRNA leads to more viral burden invitro and invivo. Knockout of RNVU1-18 is sufficient to impair the type I interferon-dependent antiviral innate immunity. U1 snRNA is required to fully activate the retinoic acid-inducible gene I (RIG-I)-dependent antiviral signaling, since it interacts with tripartite motif 25 (TRIM25) and enhances the RIG-I-TRIM25 interaction to trigger K63-linked ubiquitination of RIG-I. Our study reveals the important role of housekeeping U1 snRNA in regulating host antiviral innate immunity and restricting RNA virus infection.

Chromosome-level genome assembly and annotation of rare and endangered tropical bivalve, Tridacna crocea

Tridacna crocea is an ecologically important marine bivalve inhabiting tropical coral reef waters. High quality and available genomic resources will help us understand the population structure and genetic diversity of giant clams. This study reports a high-quality chromosome-scale T. crocea genome sequence of 1.30 Gb, with a scaffold N50 and contig N50 of 56.38 Mb and 1.29 Mb, respectively, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences cover 71.60% of the total length, and a total of 25,440 protein-coding genes were annotated. A total of 1,963 non-coding RNA (ncRNA) were determined in the T. crocea genome, including 62 micro RNA (miRNA), 58 small nuclear RNA (snRNA), 83 ribosomal RNA (rRNA), and 1,760 transfer RNA (tRNA). Phylogenetic analysis revealed that giant clams diverged from oyster about 505.7 Mya during the evolution of bivalves. The genome assembly presented here provides valuable genomic resources to enhance our understanding of the genetic diversity and population structure of giant clams.

Circulating serum profile of small non-coding RNAs in patients with anaphylaxis beyond microRNAs.

Anaphylaxis is the most severe manifestation of allergic disorders. Currently, an increasing number of cells, pathways and molecules involved in the etiopathogenesis of anaphylaxis are being discovered. However, there are no conclusive biomarkers to confirm its diagnosis. Small non-coding RNAs (sncRNAs) are 18-200 nucleotide molecules that can be divided into: microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transference RNA derived fragments (tRFs) and YRNA derived fragments (YRFs). These molecules participate in cell-cell communication modulating various physiological processes and have been postulated as non-invasive biomarkers of several pathologies. Therefore, in this study we characterized the serum circulating profile of other sncRNA beyond miRNAs in two populations of 5 adults and 5 children with drug- and food-mediated anaphylaxis, respectively. Samples were obtained from each patient under two different conditions: during anaphylaxis and 14 days after the reaction (control). The sncRNA analysis was carried out by Next Generation Sequencing (NGS). A total of 671 sncRNAs (3 piRNAs, 74 snoRNAs, 54 snRNAs, 348 tRFs and 192 YRFs) were identified in adults with drug-induced anaphylaxis, while 612 sncRNAs (2 piRNAs, 73 snoRNAs, 52 snRNAs, 321 tRFs and 164 YRFs) were characterized in children with food-mediated anaphylaxis. However, only 33 (1 piRNA, 4 snoRNAs, 1 snRNAs, 7 tRFs and 20 YRFs) and 80 (4 snoRNAs, 6 snRNAs, 54 tRFs and 16 YRFs) of them were statistically different between both conditions, respectively. Among them, only three (Y_RNA.394, Y_RNA.781 and SCARNA2) were common to both adults and children analysis. This study provides a differential profile of circulating serum sncRNAs beyond miRNAs in patients with anaphylaxis, postulating them as candidate biomarkers for this pathological event and as novel mediators of the reaction.

USP4 regulates TUT1 ubiquitination status in concert with SART3

The ubiquitin system plays pivotal roles in diverse cellular processes, including signal transduction, transcription and translation, organelle quality control, and protein degradation. Recent investigations have revealed the regulatory influence of ubiquitin systems on RNA metabolism. Previously, we reported that the deubiquitinating enzyme, ubiquitin specific peptidase 15 (USP15), promotes deubiquitination of terminal uridylyl transferase 1 (TUT1), a key regulator within the U4/U6 spliceosome, thereby instigating significant alterations in global RNA splicing [1]. In this study, we report that ubiquitin specific peptidase 4 (USP4), a homologous protein to USP15, also exerts control over the ubiquitination status of TUT1. Analogous to USP15, the expression of USP4 results in a reduction of TUT1 ubiquitination. Furthermore, squamous cell carcinoma antigen recognized by T-cells 3 (SART3) collaborates in enhancing the deubiquitinating activity of USP4 towards TUT1. A crucial revelation is that USP4 orchestrates the subnuclear relocation of TUT1 from the nucleolus to the nucleoplasm and facilitates the stability of U6 small nuclear RNA (snRNA). Notably, USP4 has a more profound effect on TUT1 redistribution compared to USP15. Our findings suggest that USP4 intricately modulates the ubiquitination status of TUT1, thereby exerting pronounced effects on the spliceosome functions.