Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro.

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.

Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis.

HP1α/CBX5 is an epigenetic regulator with a suspected role in multiple sclerosis (MS). Here, using high-depth RNA sequencing on monocytes, we identified a subset of MS patients with reduced CBX5 expression, correlating with progressive stages of the disease and extensive transcriptomic alterations. Examination of rare non-coding RNA species in these patients revealed impaired maturation/degradation of U snRNAs and enhancer RNAs, indicative of reduced activity of the Integrator, a complex with suspected links to increased MS risk. At protein-coding genes, compromised Integrator activity manifested in reduced pre-mRNA splicing efficiency and altered expression of genes regulated by RNA polymerase II pause-release. Inactivation of Cbx5 in the mouse mirrored most of these transcriptional defects and resulted in hypersensitivity to experimental autoimmune encephalomyelitis. Collectively, our observations suggested a major contribution of the Integrator complex in safeguarding against transcriptional anomalies characteristic of MS, with HP1α/CBX5 emerging as an unexpected regulator of this complex's activity. These findings bring novel insights into the transcriptional aspects of MS and provide potential new criteria for patient stratification.

Molecular basis for the activation of human spliceosome.

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.

Therapy-induced secretion of spliceosomal components mediates pro-survival crosstalk between ovarian cancer cells.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.

Mechanism for the initiation of spliceosome disassembly.

Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome1. Recent studies have shed light on spliceosome assembly and remodelling for catalysis2-6, but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes.

A role for the C. elegans Argonaute protein CSR-1 in small nuclear RNA 3' processing.

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing.

Single-cell and single-nucleus RNA-sequencing from paired normal-adenocarcinoma lung samples provide both common and discordant biological insights.

Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be context-dependent. Herein, a head-to-head comparison was performed in matched normal-adenocarcinoma human lung samples to assess biological insights derived from scRNA-seq versus snRNA-seq and better understand the cellular transition that occurs from normal to tumoral tissue. Here, the transcriptome of 160,621 cells/nuclei was obtained. In non-tumor lung, cell type proportions varied widely between scRNA-seq and snRNA-seq with a predominance of immune cells in the former (81.5%) and epithelial cells (69.9%) in the later. Similar results were observed in adenocarcinomas, in addition to an overall increase in cell type heterogeneity and a greater prevalence of copy number variants in cells of epithelial origin, which suggests malignant assignment. The cell type transition that occurs from normal lung tissue to adenocarcinoma was not always concordant whether cells or nuclei were examined. As expected, large differential expression of the whole-cell and nuclear transcriptome was observed, but cell-type specific changes of paired normal and tumor lung samples revealed a set of common genes in the cells and nuclei involved in cancer-related pathways. In addition, we showed that the ligand-receptor interactome landscape of lung adenocarcinoma was largely different whether cells or nuclei were evaluated. Immune cell depletion in fresh specimens partly mitigated the difference in cell type composition observed between cells and nuclei. However, the extra manipulations affected cell viability and amplified the transcriptional signatures associated with stress responses. In conclusion, research applications focussing on mapping the immune landscape of lung adenocarcinoma benefit from scRNA-seq in fresh samples, whereas snRNA-seq of frozen samples provide a low-cost alternative to profile more epithelial and cancer cells, and yield cell type proportions that more closely match tissue content.

HIV-1 Infection Reduces NAD Capping of Host Cell snRNA and snoRNA.

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

Functional analysis of the zinc finger modules of the Saccharomyces cerevisiae splicing factor Luc7.

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.

Spliceosomal snRNAs, the Essential Players in pre-mRNA Processing in Eukaryotic Nucleus: From Biogenesis to Functions and Spatiotemporal Characteristics.

Spliceosomal small nuclear RNAs (snRNAs) are a fundamental class of non-coding small RNAs abundant in the nucleoplasm of eukaryotic cells, playing a crucial role in splicing precursor messenger RNAs (pre-mRNAs). They are transcribed by DNA-dependent RNA polymerase II (Pol II) or III (Pol III), and undergo subsequent processing and 3' end cleavage to become mature snRNAs. Numerous protein factors are involved in the transcription initiation, elongation, termination, splicing, cellular localization, and terminal modification processes of snRNAs. The transcription and processing of snRNAs are regulated spatiotemporally by various mechanisms, and the homeostatic balance of snRNAs within cells is of great significance for the growth and development of organisms. snRNAs assemble with specific accessory proteins to form small nuclear ribonucleoprotein particles (snRNPs) that are the basal components of spliceosomes responsible for pre-mRNA maturation. This article provides an overview of the biological functions, biosynthesis, terminal structure, and tissue-specific regulation of snRNAs.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

ARDS and aging: TYMS emerges as a promising biomarker and therapeutic target.

Background: Acute Respiratory Distress Syndrome (ARDS) is a common condition in the intensive care unit (ICU) with a high mortality rate, yet the diagnosis rate remains low. Recent studies have increasingly highlighted the role of aging in the occurrence and progression of ARDS. This study is committed to investigating the pathogenic mechanisms of cellular and genetic changes in elderly ARDS patients, providing theoretical support for the precise treatment of ARDS. Methods: Gene expression profiles for control and ARDS samples were obtained from the Gene Expression Omnibus (GEO) database, while aging-related genes (ARGs) were sourced from the Human Aging Genomic Resources (HAGR) database. Differentially expressed genes (DEGs) were subjected to functional enrichment analysis to understand their roles in ARDS and aging. The Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning pinpointed key modules and marker genes, with ROC curves illustrating their significance. The expression of four ARDS-ARDEGs was validated in lung samples from aged mice with ARDS using qRT-PCR. Gene set enrichment analysis (GSEA) investigated the signaling pathways and immune cell infiltration associated with TYMS expression. Single-nucleus RNA sequencing (snRNA-Seq) explored gene-level differences among cells to investigate intercellular communication during ARDS onset and progression. Results: ARDEGs are involved in cellular responses to DNA damage stimuli, inflammatory reactions, and cellular senescence pathways. The MEmagenta module exhibited a significant correlation with elderly ARDS patients. The LASSO, RRF, and XGBoost algorithms were employed to screen for signature genes, including CKAP2, P2RY14, RBP2, and TYMS. Further validation emphasized the potential role of TYMS in the onset and progression of ARDS. Immune cell infiltration indicated differential proportion and correlations with TYMS expression. SnRNA-Seq and cell-cell communication analysis revealed that TYMS is highly expressed in endothelial cells, and the SEMA3 signaling pathway primarily mediates cell communication between endothelial cells and other cells. Conclusion: Endothelial cell damage associated with aging could contribute to ARDS progression by triggering inflammation. TYMS emerges as a promising diagnostic biomarker and potential therapeutic target for ARDS.

Structural insights into human exon-defined spliceosome prior to activation.

Spliceosome is often assembled across an exon and undergoes rearrangement to span a neighboring intron. Most states of the intron-defined spliceosome have been structurally characterized. However, the structure of a fully assembled exon-defined spliceosome remains at large. During spliceosome assembly, the pre-catalytic state (B complex) is converted from its precursor (pre-B complex). Here we report atomic structures of the exon-defined human spliceosome in four sequential states: mature pre-B, late pre-B, early B, and mature B. In the previously unknown late pre-B state, U1 snRNP is already released but the remaining proteins are still in the pre-B state; unexpectedly, the RNAs are in the B state, with U6 snRNA forming a duplex with 5'-splice site and U5 snRNA recognizing the 3'-end of the exon. In the early and mature B complexes, the B-specific factors are stepwise recruited and specifically recognize the exon 3'-region. Our study reveals key insights into the assembly of the exon-defined spliceosomes and identifies mechanistic steps of the pre-B-to-B transition.

Locus-specific detection of pseudouridine with CRISPR-Cas13a.

We combined the CRISPR-Cas13a system with CMC chemical labeling, developing an approach that enables precise identification of pseudouridine (Ψ) sites at specific loci within ribosomal RNA (rRNA), messenger RNA (mRNA) and small nuclear RNAs (snRNA). This method, with good efficiency and simplicity, detects Ψ sites through fluorescence measurement, providing a straightforward and fast validation for targeted Ψ sites of interest.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion.

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates ß-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of ß-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy.

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 in vitro and in vivo. 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.

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

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.

2'-O-methylation (Nm) in RNA: progress, challenges, and future directions.

RNA 2'-O-methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs has been demonstrated to impact cellular processes, including translation and splicing. In addition, Nm modifications at the 5' cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self-mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field.

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs.

Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.