U1 snRNA Research Articles

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Evaluation of reference genes for miRNA expression analysis inGaleruca daurica(Coleoptera: Chrysomelidae) using qRT‐PCR

AbstractThe selection of suitable reference genes (RGs) for data normalization is a crucial step for obtaining reliable results from gene expression analysis using the quantitative real‐time polymerase chain reaction (qRT‐PCR). Expression stability of eleven putative RGs (miR‐100‐5p, miR‐92a‐3p, miR‐998‐3p, miR‐279d‐3p, miR‐305‐5p, miR‐276a‐3p, miR‐275‐3p, miR‐9a‐5p, miR‐2a‐3p, U6 snRNA and 5S rRNA) was assessed by using a web‐based tool: RefFinder, which integrates four algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), and the best combination number of RGs was calculated by geNorm. Validation of selected RGs was performed by an expression analysis of let‐7‐5p under different experimental conditions. The best combinations of RGs were shown as follows: miR‐100‐5p + miR‐275‐3p for temperature treatment, miR‐100‐5p + miR‐305‐5p for adult tissues, miR‐276a‐3p + miR‐9a‐5p for genders, miR‐2a‐3p + miR‐276a‐3p for days after eclosion, U6 snRNA + miR‐9a‐5p + miR‐2a‐3p for developmental stages, and U6 snRNA + miR‐279d‐3p for all above conditions, and there was no one RG suitable for all experimental conditions. Incorrect RG selection affected the evaluation of let‐7‐5p abundance. Our findings demonstrate that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for miRNA evaluation, and provide a basis for further studies on expression and function analysis of miRNAs and their target genes inGaleruca daurica, a new pest with great outbreaks in Inner Mongolia since 2009.

Cytogenetic markers using single-sequence probes reveal chromosomal locations of tandemly repetitive genes in scleractinian coral Acropora pruinosa

The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes from tandemly repetitive 5S ribosomal RNA (rRNA) and core histone coding sequences to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the 5S rDNA probe was on chromosome 5, whereas that of core histone probe was on chromosome 8. The sequence of the 5S rDNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rDNA. This is the first report of a tandemly repetitive linkage of snRNA and 5S rDNA sequences in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

A single m6A modification in U6 snRNA diversifies exon sequence at the 5\u2019 splice site

N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5’ splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5’ exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.

The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

Lack of Toxicity in Nonhuman Primates Receiving Clinically Relevant Doses of an AAV9.U7snRNA Vector Designed to Induce DMD Exon 2 Skipping.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an adeno-associated virus (AAV)-encapsidated viral vector containing four copies of the noncoding U7 small nuclear RNA (U7snRNA), each targeted to either the splice donor or the splice acceptor sites of DMD exon 2. We have previously shown that delivery of this vector (scAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length dystrophin from wild-type DMD mRNA, as well as an internal ribosome entry site (IRES)-driven isoform translated only in the absence of exon 2 (deletion exon 2 [Del2] mRNA). Here we present the data from a rigorous dose escalation toxicity study in nonhuman primates, encompassing two doses (3 × 1013 and 8 × 1013 vg/kg) and two time points (3 and 6 months postinjection). No evidence for significant toxicity was seen by biochemical, histopathologic, or clinical measures, providing evidence for safety that led to initiation of a first-in-human clinical trial.

The splicing factor U2AF84 elicits intron retention impacting the virulence of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscesses. The parasite infects approximately 1% of the human population, resulting in circa 100,000 deaths annually. The determining factors resulting in amebiasis are not well understood. The proteome of in vivo assembled Early splicing complexes identified a huge set of E. histolytica proteins involved in RNA processing, among them U2AF84, which is the ortholog of the human U2AF65. The Carboxyl-terminus domain of this factor is 240 amino acids longer than U2AF65, of which the last 136 residues correspond to the KH-QUA2 acidic domain, involved in the regulation of gene expression through Intron Retention. Such domains are able to dimerize members of the STAR family of proteins, such as Splicing Factor 1 (SF1), a factor that binds to the intronic Branch point Sequence (BS) in the Early splicing complex though its own KH-QUA2. Recent transcriptomic data showed upregulated expression of U2AF84 in amoebas recovered from liver abscesses. As a hypothesis, we propose that the interaction between the U2AF84- and SF1-KH-QUA2 domains reduces splicing via intronic retention and the virulence of E. histolytica. Our objective was analyze the impact of the interaction between the U2AF84- and SF1-KH-QUA2 domains on splicing and on pathogen virulence. KH-QUA2 deleted U2AF84 amoeba transformants displayed increased splicing of target transcripts in vivo. EMSA, UV and formaldehyde crosslinking, and overlay assays showed that recombinant SF1- and U2AF84-KH-QUA2 domains interact with each other, even in the absence of a BS radioactive probe. Also, the U2AF84-KH-QUA2 domain blocks partial replacement of the SF1-KH-QUA2 by the U2 snRNA from the BS, suggesting that splicing inhibition occurs by blockage of splicing complex E to A transition. All amoeba transformants were tested for their virulent phenotypes. Changes in the U2AF84-KH-QUA2 domain resulted in more aggressive amoebas, suggesting that an increase in splicing (therefore decreased intron retention) of target transcripts is required for appropriate amebiasis outcome. To test this, we constructed a chimeric U2AF84 protein combining the amino terminus of E. histolytica U2AF84 with the longer KH-QUA2 of the non-virulent human commensal Entamoeba dispar. The transfected chimeras were less aggressive than the U2AF84-KH-QUA2 deleted or mutated transformants, confirming, at least in part, our hypothesis. We conclude that a more extensive transcriptome resulting from intron retention, and an additional layer of gene expression regulation are necessary for appropriate amebiasis establishment. Our work links for the first-time splicing downregulation and pathogen virulence.

Kindler epidermolysis bullosa-like skin phenotype and downregulated basement membrane zone gene expression in poikiloderma with neutropenia and a homozygous USB1 mutation

Epidermolysis bullosa (EB) is a genotypically heterogeneous group of disorders characterized by cutaneous blistering and erosions with a tremendous spectrum of severity. One of the distinct forms of EB, Kindler EB (KEB), manifests with blistering and poikiloderma; this subtype of EB is caused by mutations in the FERMT1 gene encoding kindlin-1. In this study, we investigated a patient clinically diagnosed as KEB with reduced FERMT1 gene expression and intensity of immunostaining for kindlin-1. Transmission electron microscopy showed lamina densa reduplication, frequently observed in KEB. However, no mutations were identified in FERMT1 in this patient with consanguineous parents, and this gene resided outside of genomic regions of homozygosity (ROH). Instead, whole-exome sequencing and homozygosity mapping identified a homozygous sequence variant at the +4 position of intron 2 in the USB1 gene, encoding an exoribonuclease required for processing of U6 snRNA, a critical component of spliceosomes. Examination of the patient's RNA by RNA-Seq confirmed the pathogenicity of this variant, causing aberrant splicing predicted to result in loss of function of USB1. Mutations in this gene have been reported in patients with poikiloderma and neutropenia, with a few reported cases in association with skin fragility, a condition distinct from the KEB phenotype. Transcriptome analysis revealed that several genes, expressed in the cutaneous basement membrane zone and previously associated with different subtypes of EB, were differentially downregulated at the mRNA level. EB-associated mRNA downregulation was confirmed at protein levels by skin immunofluorescence. These observations provide a novel mechanism for blistering and erosions in the skin as a result reduced presence of adhesion complexes critical for stable association of epidermis and dermis at the level of cutaneous basement membrane zone.

Cellular Sticking Can Strongly Reduce Complex Binding by Speeding Dissociation.

While extensive studies have been carried out to determine protein-RNA binding affinities, mechanisms, and dynamics in vitro, such studies do not take into consideration the effect of the many weak nonspecific interactions in a cell filled with potential binding partners. Here we experimentally tested the role of the cellular environment on affinity and binding dynamics between a protein and RNA in living U-2 OS cells. Our model system is the spliceosomal protein U1A and its binding partner SL2 of the U1 snRNA. The binding equilibrium was perturbed by a laser-induced temperature jump and monitored by Förster resonance energy transfer. The apparent binding affinity in live cells was reduced by up to 2 orders of magnitude compared to in vitro. The measured in-cell dissociation rate coefficients were up to 2 orders of magnitude larger, whereas no change in the measured association rate coefficient was observed. The latter is not what would be anticipated due to macromolecular crowding or nonspecific sticking of the uncomplexed U1A and SL2 in the cell. A quantitative model fits our experimental results, with the major cellular effect being that U1A and SL2 sticking to cellular components are capable of binding, just not as strongly as the free complex. This observation suggests that high binding affinities measured or designed in vitro are necessary for proper binding in vivo, where competition with many nonspecific interactions exists, especially for strongly interacting species with high charge or large hydrophobic surface areas.

Functional analyses of human LUC7-like proteins involved in splicing regulation and myeloid neoplasms.

SUMMARYVertebrates have evolved three paralogs, termed LUC7L, LUC7L2, and LUC7L3, of the essential yeast U1 small nuclear RNA (snRNA)-associated splicing factor Luc7p. We investigated the mechanistic and regulatory functions of these putative splicing factors, of which one (LUC7L2) is mutated or deleted in myeloid neoplasms. Protein interaction data show that all three proteins bind similar core but distinct regulatory splicing factors, probably mediated through their divergent arginine-serine-rich domains, which are not present in Luc7p. Knockdown of each factor reveals mostly unique sets of significantly dysregulated alternative splicing events dependent on their binding locations, which are largely non-overlapping. Notably, knockdown of LUC7L2 alone significantly upregulates the expression of multiple spliceosomal factors and downregulates glycolysis genes, possibly contributing to disease pathogenesis. RNA binding studies reveal that LUC7L2 and LUC7L3 crosslink to weak 5′ splice sites and to the 5′ end of U1 snRNA, establishing an evolutionarily conserved role in 5′ splice site selection.

Development of CRISPR-Cas9 genome editing system in Talaromyces marneffei

Talaromyces marneffei is an important pathogenic thermally dimorphic fungus causing systemic talaromycosis mainly prevalent in Southeast Asia. The dimorphic transition between mycelium and yeast is considered crucial for the pathogenicity of T. marneffei. However, the lack of genetic toolbox has been a major impediment for understanding its pathogenicity. Here a CRISPR-Cas9 system was developed to facilitate genetic manipulations in this organism. In this study, the CRISPR-Cas9 gene editing system uses a native U6 snRNA promoter from T. marneffei to drive the expression of sgRNA. Employing this system and PEG-mediated protoplast transformation, the sakA gene was mutated. Sanger sequencing confirmed nearly 40% site-directed mutation rate. The phenotype analysis confirmed the sakA gene function in T. marneffei dimorphic transition. Our study provided a powerful genome-manipulating tool, which could accelerate studies on T. marneffei for further revealing the mechanisms of its pathogenicity.

Characterization of Pre-mRNA Splicing and Spliceosomal Machinery in Porphyridium purpureum and Evolutionary Implications for Red Algae.

Pre-mRNA splicing is a highly conserved eukaryotic process, but our understanding of it is limited by a historical focus on well-studied organisms such as humans and yeast. There is considerable diversity in mechanisms and components of pre-mRNA splicing, especially in lineages that have evolved under the pressures of genome reduction. The ancestor of red algae is thought to have undergone genome reduction prior to the lineage's radiation, resulting in overall gene and intron loss in extant groups. Previous studies on the extremophilic red alga Cyanidioschyzon merolae revealed an intron-sparse genome with a highly reduced spliceosome. To determine whether these features applied to other red algae, we investigated multiple aspects of pre-mRNA splicing in the mesophilic red alga Porphyridium purpureum. Through strand-specific RNA-Seq, we observed high levels of intron retention across a large number of its introns, and nearly half of the transcripts for these genes are not spliced at all. We also discovered a relationship between variability of 5' splice site sequences and levels of splicing. To further investigate the connections between intron retention and splicing machinery, we bioinformatically assembled the P.purpureum spliceosome, and biochemically verified the presence of snRNAs. While most other core spliceosomal components are present, our results suggest highly divergent or missing U1 snRNP proteins, despite the presence of an uncharacteristically long U1 snRNA. These unusual aspects highlight the diverse nature of pre-mRNA splicing that can be seen in lesser-studied eukaryotes, raising the importance of investigating fundamental eukaryotic processes outside of model organisms.

Structure of human RNA polymerase III elongation complex.

RNA polymerase III (Pol III) transcribes essential structured small RNAs, such as tRNAs, 5S rRNA and U6 snRNA. The transcriptional activity of Pol III is tightly controlled and its dysregulation is associated with human diseases, such as cancer. Human Pol III has two isoforms with difference only in one of its subunits RPC7 (α and β). Despite structural studies of yeast Pol III, structure of human Pol III remains unsolved. Here, we determined the structures of 17-subunit human Pol IIIα complex in the backtracked and post-translocation states, respectively. Human Pol III contains a generally conserved catalytic core, similar to that of yeast counterpart, and structurally unique RPC3-RPC6-RPC7 heterotrimer and RPC10. The N-ribbon of TFIIS-like RPC10 docks on the RPC4-RPC5 heterodimer and the C-ribbon inserts into the funnel of Pol III in the backtracked state but is more flexible in the post-translocation state. RPC7 threads through the heterotrimer and bridges the stalk and Pol III core module. The winged helix 1 domain of RPC6 and the N-terminal region of RPC7α stabilize each other and may prevent Maf1-mediated repression of Pol III activity. The C-terminal FeS cluster of RPC6 coordinates a network of interactions that mediate core-heterotrimer contacts and stabilize Pol III. Our structural analysis sheds new light on the molecular mechanism of human Pol IIIα-specific transcriptional regulation and provides explanations for upregulated Pol III activity in RPC7α-dominant cancer cells.

An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands.

U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

U7 snRNA: A tool for gene therapy.

Most U‐rich small nuclear ribonucleoproteins (snRNPs) are complexes that mediate the splicing of pre‐mRNAs. U7 snRNP is an exception in that it is not involved in splicing but is a key factor in the unique 3′ end processing of replication‐dependent histone mRNAs. However, by introducing controlled changes in the U7 snRNA histone binding sequence and in the Sm motif, it can be used as an effective tool for gene therapy. The modified U7 snRNP (U7 Sm OPT) is thus not involved in the processing of replication‐dependent histone pre‐mRNA but targets splicing by inducing efficient skipping or inclusion of selected exons. U7 Sm OPT is of therapeutic importance in diseases that are an outcome of splicing defects, such as myotonic dystrophy, Duchenne muscular dystrophy, amyotrophic lateral sclerosis, β‐thalassemia, HIV‐1 infection and spinal muscular atrophy. The benefits of using U7 Sm OPT for gene therapy are its compact size, ability to accumulate in the nucleus without causing any toxic effects in the cells, and no immunoreactivity. The risk of transgene misregulation by using U7 Sm OPT is also low because it is involved in correcting the expression of an endogenous gene controlled by its own regulatory elements. Altogether, using U7 Sm OPT as a tool in gene therapy can ensure lifelong treatment, whereas an oligonucleotide or other drug/compound would require repeated administration. It would thus be strategic to harness these unique properties of U7 snRNP and deploy it as a tool in gene therapy.

METTL16, Methyltransferase-Like Protein 16: Current Insights into Structure and Function.

Methyltransferase-like protein 16 (METTL16) is a human RNA methyltransferase that installs m6A marks on U6 small nuclear RNA (U6 snRNA) and S-adenosylmethionine (SAM) synthetase pre-mRNA. METTL16 also controls a significant portion of m6A epitranscriptome by regulating SAM homeostasis. Multiple molecular structures of the N-terminal methyltransferase domain of METTL16, including apo forms and complexes with S-adenosylhomocysteine (SAH) or RNA, provided the structural basis of METTL16 interaction with the coenzyme and substrates, as well as indicated autoinhibitory mechanism of the enzyme activity regulation. Very recent structural and functional studies of vertebrate-conserved regions (VCRs) indicated their crucial role in the interaction with U6 snRNA. METTL16 remains an object of intense studies, as it has been associated with numerous RNA classes, including mRNA, non-coding RNA, long non-coding RNA (lncRNA), and rRNA. Moreover, the interaction between METTL16 and oncogenic lncRNA MALAT1 indicates the existence of METTL16 features specifically recognizing RNA triple helices. Overall, the number of known human m6A methyltransferases has grown from one to five during the last five years. METTL16, CAPAM, and two rRNA methyltransferases, METTL5/TRMT112 and ZCCHC4, have joined the well-known METTL3/METTL14. This work summarizes current knowledge about METTL16 in the landscape of human m6A RNA methyltransferases.

The Potential Role of miRNA-19b-3p and miRNA-320c in Patients with Moderate Bronchial Asthma.

Bronchial Asthma (BA) is a complex heterogeneous disease with a number of molecular immunopathological mechanisms underlying airway inflammation, hyperreactivity, and bronchial remodeling. MicroRNAs are important regulators in the pathogenesis and progression of chronic respiratory diseases, including BA. The aim of the study is to investigate the level of expression of cell-free circulating miR-19b-3p and miR-320c in the blood plasma by comparing their plasma levels with IL-4 in the moderate BA patients and control group. The level of expression miR-19b-3p and miR-320c were evaluated by qRT-PCR using the comparative threshold cycle (Ct) method. U6 small nuclear RNA was taken as an endogenous control. The content of IL - 4 in blood plasma was determined by using ELISA. miR-19b-3p and miR-320c were significantly dysregulated in moderate asthmatic patients in comparison with control group. The area under the ROC curve of miR-19b-3p and miR-320c showed 0.8088 (95% CI 0.6925 to 0.9251, P value =0.0001) and 0,9048 (95% CI 0,7792 to 1,000, P value <0,0001), respectively. BA patients showed a considerably positive correlation between the expression level of microRNA-320c and IL-4 levels. These cell-free circulating microRNAs are probably deregulated in other inflammatory/ pathological diseases, so they could be useful to understand the molecular pathogenesis of BA or to investigate the "inflammatory reaction" in this disease.

MicroRNA-552 expression in colorectal cancer and its clinicopathological significance

BackgroundMicroRNA-552 (miR-552) has been reported to correlate with the development and progression of various cancers, including colorectal cancer (CRC). This study aimed to investigate miR-552 expression in cancer tissue samples compared to normal mucosal tissue and its role as a diagnostic or prognostic marker in CRC patients.MethodsNormal mucosal tissues and primary cancer tissues from 80 surgically resected CRC specimens were used. Quantitative real-time polymerase chain reaction was performed for miR-552 and U6 small nuclear RNA to analyze miR-552 expression and its clinicopathological significance. Immunohistochemistry for p53 and phosphatase and tension homolog (PTEN) was performed to evaluate their association with miR-552 expression.ResultsmiR-552 expression was significantly higher in primary cancer tissues compared to normal mucosal tissues (p<.001). The expression level of miR552 was inversely correlated with that of PTEN (p=.068) and p53 (p=.004). Survival analysis showed that high miR-552 expression was associated with worse prognosis but this was not statistically significant (p=.255). However, patients with CRC having high miR-552 expression and loss of PTEN expression had significantly worse prognosis than others (p=.029).ConclusionsOur results suggest that high miR-552 expression might be a potential diagnostic biomarker for CRC, and its combined analysis with PTEN expression can possibly be used as a prognostic marker.

The integrity of the U12 snRNA 3' stem-loop is necessary for its overall stability.

Disruption of minor spliceosome functions underlies several genetic diseases with mutations in the minor spliceosome-specific small nuclear RNAs (snRNAs) and proteins. Here, we define the molecular outcome of the U12 snRNA mutation (84C>U) resulting in an early-onset form of cerebellar ataxia. To understand the molecular consequences of the U12 snRNA mutation, we created cell lines harboring the 84C>T mutation in the U12 snRNA gene (RNU12). We show that the 84C>U mutation leads to accelerated decay of the snRNA, resulting in significantly reduced steady-state U12 snRNA levels. Additionally, the mutation leads to accumulation of 3′-truncated forms of U12 snRNA, which have undergone the cytoplasmic steps of snRNP biogenesis. Our data suggests that the 84C>U-mutant snRNA is targeted for decay following reimport into the nucleus, and that the U12 snRNA fragments are decay intermediates that result from the stalling of a 3′-to-5′ exonuclease. Finally, we show that several other single-nucleotide variants in the 3′ stem-loop of U12 snRNA that are segregating in the human population are also highly destabilizing. This suggests that the 3′ stem-loop is important for the overall stability of the U12 snRNA and that additional disease-causing mutations are likely to exist in this region.

RETRACTED: circTulp4 functions in Alzheimer’s disease pathogenesis by regulating its parental gene, Tulp4

Alzheimer's disease (AD)-one of the most common neurodegenerative diseases worldwide-impairs cognition, memory, and language ability and causes dementia. However, AD pathogenesis remains poorly elucidated. Recently, a potential link between AD and circular RNAs (circRNAs) has been uncovered, but only a few circRNAs that might be involved in AD have been identified. Here, we systematically investigated circRNAs in the APP/PS1 model mouse brain through deep RNA sequencing. We report that circRNAs are markedly enriched in the brain and that several circRNAs exhibit differential expression between wild-type and APP/PS1 mice. We characterized one abundant circRNA, circTulp4, derived from Intron1 of the gene Tulp4. Our results indicate that circTulp4 predominantly localizes in the nucleus and interacts with U1 small nuclear ribonucleoprotein particle (snRNP) and RNA polymerase II to modulate the transcription of its parental gene, Tulp4, and thereby regulate the function of the nervous system, and might participate in the development of AD.