snRNA Research Articles

RNA Methyltransferase METTL16's Protein Domains Have Differential Functional Effects on Cell Processes.

METTL16, a human m6A RNA methyltransferase, is currently known for its modification of U6 and MAT2A RNAs. Several studies have identified additional RNAs to which METTL16 binds, however whether METTL16 modifies these RNAs is still in question. Moreover, a recent study determined that METTL16 contains more than one RNA-binding domain, leaving the importance of each individual RNA-binding domain unknown. Here we examined the effects of mutating the METTL16 protein in certain domains on overall cell processes. We chose to mutate the N-terminal RNA-binding domain, the methyltransferase domain, and the C-terminal RNA-binding domain. With these mutants, we identified changes in RNA-binding ability, protein and RNA expression, cell cycle phase occupancy, and proliferation. From the resulting changes in RNA and protein expression, we saw effects on cell cycle, metabolism, intracellular transport, and RNA processing pathways, which varied between the METTL16 mutant lines. We also saw significant effects on the G1 and S phase occupancy times and proliferative ability with some but not all the mutants. We have therefore concluded that while METTL16 may or may not m6A-modify all RNAs it binds, its binding (or lack of) has a significant outcome on a variety of cell processes.

Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XYGONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Identifying small non-coding RNAs as biomarkers for cervical cancer.

e17507 Background: Small non-coding RNAs (sncRNAs) that control post-transcriptional gene expression are attractive candidate molecular targets for cancer screening tests. We evaluated whether sncRNAs expression are markedly different in benign, precancerous, and cancerous cervical tissue. Methods: We examined the expression of six sncRNA species: circular RNAs (circRNAs) microRNAs (miRNAs), piwi-interacting RNA (piRNAs), small nuclear RNA (snRNAs), small nucleolar RNA (snoRNAs), and transfer RNA (tRNA), from archival formalin-fixed paraffin-embedded cervical specimen obtained from 172 women (74 benign, 58 high-grade squamous intraepithelial lesion (HSIL) and 40 invasive cervical cancer). We used DESeq2 to identify differentially expressed sncRNA molecules with adjusted p-value < 0.05 and at least 2-fold up- or down-regulation. Results: Comparing benign to HSIL samples, we foundseveral statistically significant, differentially expressed sncRNAs: 906 circRNAs (*circ_0066810, p=3.76x10-16), 87 miRNAs (*miR-149-5p, p=1.30x10-14), 11 piRNAs (*piR_016945, p=8.32x10-13), 32 snRNAs (*RNU1-17P, p=1.90x10-10), 5 snoRNAs (*SCARNA18, p=1.35x10-5), and 24 tRNA (*tRNA-Glu-TTC-8-1, p=6.45x10-17). Comparing benign to invasive cervical cancer samples, we also foundseveral statistically significant, differentially expressed sncRNAs: 3,192 circRNAs (*circ_0123115, p=7.46x10-50), 325 miRNAs (*miR-7-5p, p=6.72x10-36), 82 piRNAs (*piR-hsa-27513, p=1.44x10-15), 71 snRNAs (*RNU1-20P, p=1.37x10-45), 52 snoRNAs (*SNORD95, p=1.68x10-23), and 68 tRNA (*tRNA-Cys-GCA-9-3, p=1.01x10-21). * Indicates the sncRNA with the smallest p-value, for each specie. Conclusions: Our results identify sncRNAs with potential utility of as diagnostic and prognostic biomarkers, and therapeutic targets for cervical precancer and cancer.

Minor intron splicing is critical for survival of lethal prostate cancer

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

7SK methylation by METTL3 promotes transcriptional activity.

A fundamental feature of cell signaling is the conversion of extracellular signals into adaptive transcriptional responses. The role of RNA modifications in this process is poorly understood. The small nuclear RNA 7SK prevents transcriptional elongation by sequestering the cyclin dependent kinase 9/cyclin T1 (CDK9/CCNT1) positive transcription elongation factor (P-TEFb) complex. We found that epidermal growth factor signaling induces phosphorylation of the enzyme methyltransferase 3 (METTL3), leading to METTL3-mediated methylation of 7SK. 7SK methylation enhanced its binding to heterogeneous nuclear ribonucleoproteins, causing the release of the HEXIM1 P-TEFb complex subunit1 (HEXIM1)/P-TEFb complex and inducing transcriptional elongation. Our findings establish the mechanism underlying 7SK activation and uncover a previously unknown function for the m6A modification in converting growth factor signaling events into a regulatory transcriptional response via an RNA methylation-dependent switch.

Small RNA shuffling between murine sperm and their cytoplasmic droplets during epididymal maturation.

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

A Patient With Hypogammaglobulinemia and Bi-allelic Variants in the RNU4ATAC Gene

IntroductionThe RNU4ATAC gene codes for a small nuclear RNA involved in U12 minor intron splicing. Bi-allelic compound heterozygous pathogenic variants in the RNU4ATAC gene have been associated with Roifman Syndrome, which is an autosomal recessive disease manifesting with immunodeficiency, retinal dystrophy, developmental delay, poor growth, spondyloepiphyseal dysplasia and dysmorphic facial features. We present a case of a patient with similar characteristics seen in Roifman syndrome including impaired humoral immunity, short stature, developmental delay, osseous abnormalities and characteristic facial features found to have one likely pathogenic variant and one variant of uncertain significance (VUS) on the RNU4ATAC gene, raising the suspicion for Roifman syndrome. Case PresentationA 9-year-old male patient with a history of congenital hydrocephalus, seizure disorder, developmental delay, skeletal dysplasia, short stature and alopecia presented with headaches in the setting of EBV-meningitis requiring craniotomy and external ventricular drain placement. Prior infectious history was remarkable for four bacterial pneumonias, one sinusitis and recurrent otitis media infections in childhood. Initial evaluation of his immune system revealed decreased levels of IgG at 453 mg/dL (698–1560 mg/dL), IgM at 19 mg/dL (31–179 mg/dL), IgA at 36 mg/dL (63–236 mg/dL), and decreased CD19+ B-cells at 59/uL (200–600/uL), with suboptimal response to tetanus, pneumococcal-13, haemophilus influenzae and diphtheria vaccines. Although after receiving booster vaccinations, specific antibodies rose to protective levels, they began to drop within one year and IgG levels remained low, so he was started on immunoglobulin replacement, and has remained free of infections. Genetic evaluation revealed one de novo VUS and one maternally inherited likely pathogenic variant on the RNU4ATAC gene. Further VUS were detected in various genes with mode of inheritance and disease associations not consistent with our patient’s phenotype. DiscussionDisease-causing variants in compound heterozygosity have been associated with Roifman syndrome. The clinical similarities shared between this condition and our patient, including epiphyseal dysplasia, unique facial features, developmental delay and immunodeficiency characterized by hypogammaglobulinemia and impaired specific antibody response, raise the suspicion for Roifman syndrome. As broad testing failed to reveal any other possible etiologies, confirmation of clinical diagnosis may lead to future reclassification of the VUS into presumably pathogenic.

Hexanucleotide repeat expansions in C9orf72 alter microglial responses and prevent a coordinated glial reaction in ALS (P1-6.002)

<h3>Objective:</h3> Neuroinflammation is an important hallmark in amyotrophic lateral sclerosis (ALS). Experimental evidence has highlighted a role of microglia in the modulation of motor neuron degeneration. However, the exact contribution of microglia to both sporadic and genetic forms of ALS is still unclear. <h3>Background:</h3> Single nuclei (sn) RNA sequencing is an interesting approach to study transcriptional changes at the cellular level in human tissues from patients with neurodegenerative disorders. For this study we included motor cortex and spinal cord samples from 5 patients with ALS caused by a C9orf72 hexanucleotide repeat expansion (HRE), from 5 patients with sporadic ALS (mutations in C9orf72, SOD1, TARDBP and FUS excluded) and from 5 control individuals. <h3>Design/Methods:</h3> We generated single nuclei profiles of spinal cord and motor cortex from sporadic and <i>C9orf72</i> ALS patients, as well as controls. We particularly focused on the transcriptomic responses of both microglia and astrocytes. <h3>Results:</h3> We confirmed that <i>C9orf72</i> is highly expressed in microglia and shows a diminished expression as a result of the HRE. This resulted in an impaired response to disease, with specific deficits in phagocytic and lysosomal transcriptional pathways. Astrocytes also displayed a dysregulated response in <i>C9orf72</i> ALS patients, remaining in a homeostatic state. This suggesting that <i>C9orf72</i> HRE alters a coordinated glial response, which ultimately would increase the risk for developing ALS. <h3>Conclusions:</h3> Our results indicate that <i>C9orf72</i> HRE results in a selective microglial loss-of-function, likely impairing microglial-astrocyte communication and preventing a global glial response. This is relevant as it indicates that sporadic and familial forms of ALS may present a different cellular substrate, which is of great importance for patient stratification and selecting treatments. <b>Disclosure:</b> Dr. Masrori has received research support from European Academy of Neurology. An immediate family member of Miss Bijnens has received personal compensation in the range of $0-$499 for serving as a Consultant for Lundbeck . The institution of Miss Bijnens has received research support from University of Antwerp . Dr. Davie has nothing to disclose. Suresh Kumar Poovathingal has nothing to disclose. Miss Storm has nothing to disclose. Nicole Hersmus has nothing to disclose. Laura Fumagalli has nothing to disclose. The institution of Ludo Van Den Bosch has received research support from FWO-Vlaanderen. The institution of Ludo Van Den Bosch has received research support from Muscular Dystrophy Association (MDA). The institution of Ludo Van Den Bosch has received research support from American ALS Association (ALSA). The institution of Ludo Van Den Bosch has received research support from Thierry Latran Foundation. The institution of Ludo Van Den Bosch has received research support from Target ALS. The institution of Ludo Van Den Bosch has received research support from ABMM. Mark Fiers has nothing to disclose. Dietmar Thal has nothing to disclose. Prof. Mancuso has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sanofi. The institution of Dr. Van Damme has received research support from CSL Behring.

Functional investigation of SCN1A deep-intronic variants activating poison exons inclusion.

Dravet syndrome is a devastating epileptic syndrome characterized by intractable epilepsy with an early age of onset, regression of developmental milestones, ataxia, and motor deficits. Loss-of-function pathogenic variants in the SCN1A gene are found in the majority of patients with Dravet syndrome; however, a significant number of patients remain undiagnosed even after comprehensive genetic testing. Previously, it was shown that intronic elements in the SCN1A gene called poison exons can incorporate into SCN1A mRNA, leading to haploinsufficiency and potentially causing Dravet syndrome. Here, we developed a splicing reporter assay for all described poison exons of the SCN1A gene and validated it using previously reported and artificially introduced variants. Overall, we tested 18 deep-intronic single nucleotide variants and one complex allele in the SCN1A gene. Our approach is capable of evaluating the effect of both variants affecting cis-regulatory sequences and splice-site variants, with the potential to functionally annotate every possible variant within these elements. Moreover, using antisense-modified uridine-rich U7 small nuclear RNAs, we were able to block poison exon incorporation in mutant constructs, an approach that could be used as a promising therapeutic intervention in Dravet syndrome patients with deep-intronic variants.

ExRNA-eCLIP intersection analysis reveals a map of extracellular RNA binding proteins and associated RNAs across major human biofluids and carriers

Although the role of RNA binding proteins (RBPs) in extracellular RNA (exRNA) biology is well established, their exRNA cargo and distribution across biofluids are largely unknown. To address this gap, we extend the exRNA Atlas resource by mapping exRNAs carried by extracellular RBPs (exRBPs). This map was developed through an integrative analysis of ENCODE enhanced crosslinking and immunoprecipitation (eCLIP) data (150 RBPs) and human exRNA profiles (6,930 samples). Computational analysis and experimental validation identified exRBPs in plasma, serum, saliva, urine, cerebrospinal fluid, and cell-culture-conditioned medium. exRBPs carry exRNA transcripts from small non-coding RNA biotypes, including microRNA (miRNA), piRNA, tRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Y RNA, and lncRNA, as well as protein-coding mRNA fragments. Computational deconvolution of exRBP RNA cargo reveals associations of exRBPs with extracellular vesicles, lipoproteins, and ribonucleoproteins across human biofluids. Overall, we mapped the distribution of exRBPs across human biofluids, presenting a resource for the community.

MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Bi-allelic variants in INTS11 are associated with a complex neurological disorder

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Development and validation of an m6A RNA methylation regulator-based signature for the prediction of prognosis and immunotherapy in cutaneous melanoma.

Development and validation of an m6A RNA methylation regulator-based signature for the prediction of prognosis and immunotherapy in cutaneous melanoma.

RIP-PEN-seq identifies a class of kink-turn RNAs as splicing regulators.

A kink-turn (K-turn) is a three-dimensional RNA structure that exists in all three primary phylogenetic domains. In this study, we developed the RIP-PEN-seq method to identify the full-length sequences of RNAs bound by the K-turn binding protein 15.5K and discovered a previously uncharacterized class of RNAs with backward K-turn motifs (bktRNAs) in humans and mice. All bktRNAs share two consensus sequence motifs at their fixed terminal position and have complex folding properties, expression and evolution patterns. We found that a highly conserved bktRNA1 guides the methyltransferase fibrillarin to install RNA methylation of U12 small nuclear RNA in humans. Depletion of bktRNA1 causes global splicing dysregulation of U12-type introns by impairing the recruitment of ZCRB1 to the minor spliceosome. Most bktRNAs regulate the splicing of local introns by interacting with the 15.5K protein. Taken together, our findings characterize a class of small RNAs and uncover another layer of gene expression regulation that involves crosstalk among bktRNAs, RNA splicing and RNA methylation.

Abstract 3814: In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways

Abstract 5-10% of patients with the IGHV wild type form of chronic lymphocytic leukemia (CLL) carry a g.3A&amp;gt;C driver mutation in the U1 small nuclear RNA (snRNA). We investigatedthe patterns of mis-splicing and their pathway consequences in three U1-mutant CLL cellline models using long read sequencing in order to better understand the mechanisms ofoncogenicity in tumors carrying this mutation. CLL cell lines (HG3, JVM3 and MEC1) expressing the U1 mutation and their wild-type counterparts were submitted to Oxford Nanopore sequencing to generate full-length RNA transcripts. This transcriptomic datawas evaluated, together with previously obtained short-read data. The g.3A&amp;gt;C mutation occurs at a specific location of the U1 snRNA, a core component of the eukaryotic spliceosome, and acts by altering the 5' splice site (5'SS) recognition sequence to cause consistent patterns of mis-splicing. Long read analysis identified multiple instances of intron retention and suppression of exon skipping, and in silico translation of these mis-splicing events predicted stop-gain and other loss of function mutations in several expressed genes, as well as widespread changes in gene expression levels. We performed a pathway overrepresentation analysis of the altered gene expression patterns in the mutant cells using the Reactome knowledgebase and identified an enrichment in the processes of translation, non-sense mediated decay (NMD) and immune signaling, including interferon signaling. In particular, there was a marked down-regulation of genes related to ribosomal assembly and the translational machinery. A more comprehensiveanalysis of the U1 mutation phenotype in CLL may accelerate the development ofbetter therapeutic and diagnostic approaches in patients. Citation Format: Andrea Senff-Ribeiro, Fatemeh Almodaresi, Quang Trinh, Shimin Shuai, David Spaner, Xose S. Puente, Elias Campo, Lincoln D. Stein. In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3814.

109 A Small RNA Signature from Extracellular Vesicles in Patient Plasma Correlates With Recurrence or Progression of High-Grade Gliomas

INTRODUCTION: Glial cell-derived tumors of the central nervous system make up the largest group of brain tumors. Most are high-grade gliomas (HGG) and are universally fatal despite multimodal therapy. In managing patients with HGG, predicting recurrence, or differentiating between pseudoprogression (radiation necrosis) and true tumor progression would be invaluable in improving overall prognosis. Characterizing small RNA (sRNA) expression profiles from plasma-derived extracellular vesicles (EVs) over the course of a patient’s treatments, may allow for patient-specific treatment modifications and improve outcomes. METHODS: Plasma EVs were isolated using Vn96 capture from HGG patients perioperatively and with routine, follow-up surveillance imaging. Small RNA sequencing was conducted and unsupervised hierarchal clustering of sRNA signatures were completed. Expression profiles were grouped longitudinally with the clinical status of patients. RESULTS: Cluster analysis of nine HGG patients, has revealed a sRNA signature that is able to distinguish between tumors showing evidence of progression and those remaining stable over time. Those samples obtained from patients where a clinical diagnosis of tumor progression or pseudoprogression were uncertain, were found to cluster into progression vs. stable signatures. Annotation of differentially expressed small RNAs identified from stable vs. clinically progressed patient include small nuclear RNA, ribosomal RNA, and micro RNA among others. MiRNA species we have identified correspond to previously identified miRNA, which have been ascribed functional significance to HGG aggressiveness and progression. CONCLUSIONS: These preliminary findings demonstrate the potential utility of sRNA profiling of plasma-derived EVs obtained from patients with high-grade gliomas as non-invasive biomarkers for recurrent/progressive disease or stability/pseudoprogression.

Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase.

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.

Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

The small nuclear RNAs 4.5SH and 4.5SI were characterized only in mouse-like rodents; their genes originate from 7SL RNA and tRNA, respectively. Similar to many genes transcribed by RNA polymerase III (pol III), the genes of 4.5SH and 4.5SI RNAs include boxes A and B, forming an intergenic pol III-directed promoter. In addition, their 5'-flanking sequences have TATA-like boxes at position -31/-24, also required for efficient transcription. The patterns of the three boxes notably differ in the 4.5SH and 4.5SI RNA genes. The A, B, and TATA-like boxes were replaced in the 4.5SH RNA gene with the corresponding boxes in the 4.5SI RNA gene to evaluate their effect on the transcription of transfected constructs in HeLa cells. Simultaneous replacement of all three boxes decreased the transcription level by 40%, which indicates decreased promoter activity in a foreign gene. We developed a new approach to compare the promoter strength based on the competition of two co-transfected gene constructs when the proportion between the constructs modulates their relative activity. This method demonstrated that the promoter activity of 4.5SI is 12 times that of 4.5SH. Unexpectedly, the replacement of all three boxes of the weak 4.5SH promoter with those of the strong 4.5SI gene significantly reduced, rather than enhanced, the promoter activity. Thus, the strength of a pol III-directed promoter can depend on the nucleotide environment of the gene.

Bi-allelic SNAPC4 variants dysregulate global alternative splicing and lead to neuroregression and progressive spastic paraparesis

The vast majority of human genes encode multiple isoforms through alternative splicing, and the temporal and spatial regulation of those isoforms is critical for organismal development and function. The spliceosome, which regulates and executes splicing reactions, is primarily composed of small nuclear ribonucleoproteins (snRNPs) that consist of small nuclear RNAs (snRNAs) and protein subunits. snRNA gene transcription is initiated by the snRNA-activating protein complex (SNAPc). Here, we report ten individuals, from eight families, with bi-allelic, deleterious SNAPC4 variants. SNAPC4 encoded one of the five SNAPc subunits that is critical for DNA binding. Most affected individuals presented with delayed motor development and developmental regression after the first year of life, followed by progressive spasticity that led to gait alterations, paraparesis, and oromotor dysfunction. Most individuals had cerebral, cerebellar, or basal ganglia volume loss by brain MRI. In the available cells from affected individuals, SNAPC4 abundance was decreased compared to unaffected controls, suggesting that the bi-allelic variants affect SNAPC4 accumulation. The depletion of SNAPC4 levels in HeLa cell lines via genomic editing led to decreased snRNA expression and global dysregulation of alternative splicing. Analysis of available fibroblasts from affected individuals showed decreased snRNA expression and global dysregulation of alternative splicing compared to unaffected cells. Altogether, these data suggest that these bi-allelic SNAPC4 variants result in loss of function and underlie the neuroregression and progressive spasticity in these affected individuals.