A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

Background miRNAs are regulatory transcripts established as repressors of mRNA stability and translation that have been functionally implicated in carcinogenesis. miR-10b is one of the key onco-miRs associated with multiple forms of cancer. Malignant gliomas exhibit particularly striking dependence on miR-10b. However, despite the therapeutic potential of miR-10b targeting, this miRNA’s poorly investigated and largely unconventional properties hamper the clinical translation. Methods We utilized Covalent Ligation of Endogenous Argonaute-bound RNAs and their high-throughput RNA sequencing to identify miR-10b interactome and a combination of biochemical and imaging approaches for target validation. They included Crosslinking and RNA immunoprecipitation with spliceosomal proteins, a combination of miRNA FISH with protein immunofluorescence in glioma cells and patient-derived tumors, native Northern blotting, and the transcriptome-wide analysis of alternative splicing. Results We demonstrate that miR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of the direct binding between miR-10b and U6, in situ imaging of miR-10b and U6 co-localization in glioma cells and tumors, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in global splicing alterations, exemplified by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. Conclusions We identified U6 snRNA, the key RNA component of the spliceosome, as the top miR-10b target in glioblastoma. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

MicroRNA 146a is associated with diabetic complications in type 1 diabetic patients from the EURODIAB PCS

Background MicroRNA-146a-5p (miR-146a-5p) is a key regulator of inflammatory processes. Expression of miR-146a-5p is altered in target organs of diabetic complications and deficiency of miR-146a-5p has been implicated in their pathogenesis. We investigated if serum miR-146a-5p levels were independently associated with micro/macrovascular complications of type 1 diabetes (DM1). Methods A nested case–control study from the EURODIAB PCS of 447 DM1 patients was performed. Cases (n = 294) had one or more complications of diabetes, whereas controls (n = 153) did not have any complication. Total RNA was isolated from all subjects and miR-146a-5p levels measured by qPCR. Both the endogenous controls U6 snRNA and the spike (Cel-miR-39) were used to normalize the results. Logistic regression analysis was carried out to investigate the association of miR-146a-5p with diabetes complications. Results MiR-146a-5p levels were significantly lower in cases [1.15 (0.32–3.34)] compared to controls [1.74 (0.44–6.74) P = 0.039]. Logistic regression analysis showed that levels of miR-146a-5p in the upper quartile were inversely associated with reduced odds ratio (OR) of all complications (OR 0.34 [95% CI 0.14–0.76]) and particularly with cardiovascular diseases (CVD) (OR 0.31 [95% CI 0.11–0.84]) and diabetic retinopathy (OR 0.40 [95% CI 0.16–0.99]), independently of age, sex, diabetes duration, A1c, hypertension, AER, eGFR, NT-proBNP, and TNF-α. Conclusions In this large cohort of DM1 patients, we reported an inverse and independent association of miR-146a-5p with diabetes chronic complications and in particular with CVD and retinopathy, suggesting that miR-146a-5p may be a novel candidate biomarker of DM1 complications.

DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2’-O-methylation being most common. However, how U6 2’-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2’-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2′-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.

USB1 Is a miRNA Deadenylase That Regulates Hematopoietic Development

Poikiloderma with neutropenia (PN)is an autosomal-recessive bone marrow failure (BMF) syndrome in which patients harbor homozygous or compound heterozygous mutations in the human gene C16orf57, which encodes the evolutionarily conserved RNA 3' to 5' exonuclease U6 biogenesis 1 (USB1). USB1 is required for the proper maturation of U6 and U6atac snRNAs, core components of the spliceosome, and consequently, splicing defects have been observed in yeast and zebrafish models with USB1 deficiency. However, lymphoblastoid cells from PN patients do not exhibit reduced U6 snRNA levels and have normal pre-mRNA splicing, establishing PN as a singular BMF syndrome, where the underlying genetic cause has been identified but the molecular mechanisms leading to tissue failure remain obscure. To investigate the role of USB1 in a physiological context, we utilized CRISPR/Cas9 to create human embryonic stem cells (hESCs) containing a frequently occurring c.531_del_A loss-of-function mutation in the USB1 gene (USB1_c.531_del_A hESCs). USB1_c.531_del_A hESCs have normal karyotype, normal growth rate, and retain pluripotency, indicating that clinically-relevant mutations in USB1 are not deleterious in undifferentiated hESCs. To elucidate the role of USB1 during hematopoiesis, we performed serum-free hematopoietic differentiations to derive hematopoietic progenitor cells from WT and USB1_c.531_del_A hESCs. The formation of definitive hematopoietic progenitors (CD45+) was decreased in USB1 mutant cells compared to WT cells, and definitive colony potential analysis showed compromised colony formation in USB1 mutants. These observations indicate that loss-of-function mutations in USB1 negatively influence hematopoiesis. Additionally, as PN is associated with severe non-cyclic neutropenia, we analyzed the potential of neutrophil formation in WT and USB1 mutant cells. USB1 mutants have reduced formation of CD15+/CD66b+ lineages, indicating abnormal neutrophil development. Conditional expression of WT USB1 in USB1_c.531_del_A mutant cells rescued these phenotypes, leading to normal hematopoietic development. Interestingly, USB1 mutants showed no reduction in the overall levels of U6 and U6atac snRNAs, similar to what was observed in patient cells. To identify other possible targets of USB1, we sequenced the transcriptome and miRome of WT and USB1 mutant cells in different stages of hematopoietic development. Through these analyses, we demonstrate that hematopoietic failure in USB1 mutants is caused by dysregulated miRNA levels during blood development, due to a failure to remove destabilizing 3' end oligo(A) tails added by PAPD5/7. Moreover, we demonstrate that modulation of oligoadenylation through genetic or chemical inhibition of PAPD5/7 rescues the defective hematopoiesis observed in USB1 mutants. This work indicates USB1 acts as a miRNA deadenylase and suggests PAPD5/7 inhibition as a potential therapy for PN. Disclosures Parker: Faze Therapeutics: Other: Co-founder.

Molecular Determinants for RNA Release into Extracellular Vesicles

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

miRNAs are regulatory transcripts established as repressors of mRNA stability and translation. Here we demonstrate that a key onco-miRNA, miR-10b, binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of direct binding between miR-10b and U6, in situ visualizations of miR-10b and U6 co-localization in glioma cells and patient-derived tumor tissues, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in splicing alterations, illustrated by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

Identification Candidate Diagnostic Biomarkers Between Minimal Change Disease and Focal Segmental Glomerulosclerosis by Bioinformatics Analysis

Background: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are common causes of nephrotic syndrome which have similar clinical as well as histologic magnification and hard to differentiate. This study aimed to identify novel biomarkers to distinguish FSGS and MCD through bioinformatics analysis and elucidate the possible molecular mechanism. Material and Methods: Based on the microarray datasets GSE104948 and GSE108113 downloaded from the Gene Expression Omnibus database, the differentially expressed genes (DEGs) between FSGS vs healthy control, MCD vs healthy control were identified, and further defined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Hub genes were checked by protein-protein interaction networks. Results: A total of and 358 and 368 genes were identified in FSGS and MCD compared with healthy controls, among them, there were156 overlapping DEGs. GO analysis showed the DEGs in these two diseases were simultaneously enriched in mRNA splicing, RNA polymerase II transcription, mRNA export, insulin stimulus, integrin-mediated signaling pathway, viral process and phagocytosis. Module analysis showed that genes in the top 1 significant module of the PPI network were mainly associated with Spliceosome among FSGS and MCD. The top 10 hub genes analysis discovered that most of hub genes were same between two disease, while among these genes, CD2 cytoplasmic tail binding protein 2 (CD2BP2), U6 snRNA-associated Sm-like protein (LSM8) and Small nuclear ribonucleoprotein polypeptides B (SNRPB) only differential expression in FSGS and Splicing factor 3A, subunit 3 (SF3A3) only differential expression in MCD, which may be used for differential diagnosis of these two diseases in the future. Conclusions: We identified key genes and mainly pathway associated with FSGS and MCD. Our results provide a set of potential genes used for differential diagnosis of these two diseases.

A single m6A modification in U6 snRNA diversifies exon sequence at the 5’ 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.