snRNA Research Articles

HMGA1 Is Upregulated in Myelodysplastic Syndromes with Mutation in Pre-mRNA Splicing Genes and Inhibits Leukemia Cell Differentiation

In myelodysplastic syndrome (MDS), genes associated with pre-mRNA splicing such as SRSF2 (Serine and Arginine Rich Splicing Factor 2) and U2AF1 (U2 small nuclear RNA auxiliary factor 1) are frequently mutated. While several reports demonstrated that MDS patients with these mutations had worse prognosis, few treatments specific to these mutations are available, partly due to the dearth of material on functions of these mutated genes. To assess the role of pre-mRNA splicing gene mutations in the pathogenesis of MDS, we performed RNA-seq analysis using CD34-postitive fractions of bone marrow samples of MDS patients. Patients with mutations in either SRSF2 or U2AF1 tended to have shorter 1-year leukemia free survival compared to patients without these mutations (66.7% vs 73.7%, respectively; p=0.067). Gene set enrichment analysis revealed that MYC target genes were enriched in patients with mutations in either SRSF2 or U2AF1 compared to patients (n=4) without these mutations (n=3). Among them, we found that HMGA1 (High Mobility Group A1) was distinctly upregulated in these patients. Expression levels of HMGA1 are known to be higher in hematopoietic stem cells. HMGA1 is involved in various cellular processes such as transcriptional regulation, DNA repair, cell differentiation and regulated cell death. We confirmed that expression levels of HMGA1 in murine bone marrow cells were higher in Lineage marker-negative, Sca1-positive, and c-Kit-positive (LSK) fractions than other more differentiated fractions. To determine whether upregulation of HMGA1 contributed to pathogenesis of MDS, we retrovirally transduced HMGA1 to murine hematopoietic progenitor cell line 32D-cl3. We found that proportions of Gr-1 positive cells after treatment with G-CSF were lower in HMGA1-transduced 32D-cl3 cells. Further, expression levels of myeloid-associated genes were also suppressed in these 32D-cl3 cells. These results suggested that 32D-cl3 with extrinsic expression of HMGA1 were more resistant to G-CSF induced myeloid differentiation. Similarly, primary murine hematopoietic progenitor cells retrovirally transduced with HMGA1 showed increased colony-forming capacities. Considering that differentiation block plays a major role in the onset of MDS, these results supported our hypothesis that upregulation of HMGA1 would contribute to pathogenesis of MDS through blocking normal myeloid differentiation. To investigate whether inhibition of HMGA1 could affect the pathogenesis of myeloid malignancies, we silenced expression of HMGA1 via short-hairpin RNA (shRNA) in various cell lines of acute myeloid leukemia (AML). Human acute monocytic leukemia cell line THP-1 became more easily induced to differentiate upon phorbol12-myristate13-acetate treatment when HMGA1 was silenced. Similarly, we silenced expression of HMGA1 in HL60, which is a human acute promyelocytic leukemia (APL) cell line. We treated HL60 cells with all-trance retinoic acid (ATRA) to induce myeloid differentiation. As expected, we found that expression levels of CEBP-β and Gr-1 in ATRA-treated HL60 cells were significantly higher when HMGA1 was silenced, which meant that HL60 cells became more sensitive to ATRA-induced myeloid differentiation by inhibition of HMGA1. Considering that differentiation induction therapy has improved clinical outcome not only in APL but also in AML, these results suggested that HMGA1 would be a potential therapeutic target for myeloid malignancies. In summary, our experiments demonstrated that expression levels of HMGA1 were higher in patients with splicing mutations. Considering its inhibitory effects on differentiation demonstrated by our experiments, upregulation of HMGA1 could be involved in the pathogenesis of MDS. Further, therapeutic intervention for HMGA1 had a potential to improve clinical outcome of myeloid malignancies. Disclosures Kurokawa: Nippon Shinyaku: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Teijin: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bioverativ Japan: Consultancy; Shire Plc: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; Chugai: Consultancy, Research Funding, Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau.

Engineered U7 Small Nuclear RNA Restores Correct β-Globin Pre-mRNA Splicing in Mouse βIVS2-654-Thalassemic Erythroid Progenitor Cells

Restoration of correct splicing of βIVS2-654-globin pre-mRNA was previously accomplished in erythroid cells from β-thalassemia/HbE patients by an engineered U7 small nuclear RNA (snRNA) that carried a sequence targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP+623 snRNA. In this study, this approach was tested in thalassemic mice carrying the βIVS2-654 mutation. While correction of βIVS2-654 pre-mRNA splicing was achieved in erythroid progenitors transduced with a lentiviral vector carrying the U7.BP+623 snRNA, a high level of truncated U7.BP+623 snRNA was also observed. The discrepancy of processing of the modified U7 snRNA in human and mouse constructs hamper the evaluation of pathologic improvement in mouse model.

Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation.

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3ʹ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

Expression of miRNA-21 level in hypertrophic cardiomyopathy patients with chronic heart failure and preserved left ventricular ejection fraction: results of a 6-year follow-up

Abstract Introduction Recently it is found that circulating miRNA-21 is associated with chronic heart failure (CHF) and ischemic heart disease. Its clinical significance in hypertrophic cardiomyopathy (HCM) and CHF with preserved left ventricular ejection fraction (LVEF) (≥50%) has to be investigated. Purpose The aim of this study was to analyze the expression of miRNA-21 in the peripheral blood of HCM patients and CHF with preserved LVEF (≥50%). Materials and methods From 2014 to 2019 years we examined 180 HCM patients. The study population consisted of 60 patients ≥19 years old (51.5 [36.2; 65.7]) with symptomatic HCM and CHF with preserved LVEF (≥50%). The diagnosis of HCM was established according to the guideline of the European society of cardiology on the diagnosis and treatment of HCM, 2014. The control group included 45 healthy donors without cardiovascular diseases and other severe pathologies, matched by age and sex with the studied group. Total RNA was extracted from plasma of patients. MiRNA-21 and reference RNA U6 cDNA was prepared based on StemLoop-technology. Expression was examined using semiquantitative RT-PCR protocol. Calculation of the relative gene expression level of miRNA-21 was done according to the standard procedure 2-ΔCt. IBM SPSS software package and Microsoft Excel 2010 were used for the statistical analysis of the collected data. Results The serum expression level of miRNA-21 in HCM patients (n=60) varied from 0.13 to 477.7 (4.92 [1.77; 13]) and was significantly higher than those in the control group (0.01 - 9.85 (0.84 [0.55; 1.23]), with statistically significant difference (p=0.001). The HCM group was divided according to CHF severity: I-II functional class (NYHA) (n=42) and III-IV functional class (NYHA) (n=18) subgroups. It was found a significant increase of expression microRNA-21 level in both subgroups HCM patients, compared with control group (p=0.001). The expression level of miRNA-21 also differed between HCM patients and CHF III-IV functional class (NYHA) vs those, who had CHF I-II functional class (NYHA) – 1.1–477.7 (13 [3.88; 41]) vs 0.1–119.4 (3.25 [1.41; 6.06]), respectively (p=0.003). In HCM patients and CHF III-IV functional class (NYHA) (n=18) the expression level of miRNA-21 positively correlated with LVEF (r=0.609; p<0.05). Conclusion HCM patients with CHF III-IV functional class (NYHA) and preserved LVEF (≥50%) demonstrated high expression level of miRNA-21. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Almazov Federal Medical Research Centre, Saint-Petersburg, Russian Federation, Pavlov University, L'va Tolstogo str. 6-8, Saint Petersburg, Russian Federation

Identification of Circulating miR-22-3p and miR-93-5p as Stable Endogenous Control in Tuberculosis Study.

The diagnosis and prognosis of tuberculosis remains challenging and necessitates the development of a new test that can accurately diagnose and monitor treatment responses. In this regard, miRNA is becoming a potential diagnostic and prognostic biomarker which differentiates treatment respondents from non-respondents for various non-infectious and infectious diseases, including tuberculosis. The concentration of miRNAs varies based on cell type, disease, and site of infection, implicating that selection of an optimal reference gene is crucial, and determines the quantification of transcript level and biological interpretation of the data. Thus, the study evaluated the stability and expression level of five candidate miRNAs (let-7i-5p, let-7a-5p, miRNA-16-5p, miRNA-22-3p and miRNA-93-5p), including U6 Small Nuclear RNA (RNU6B) to normalize circulating miRNAs in the plasma of 68 participants (26 healthy controls, 23 latent, and 19 pulmonary tuberculosis infected) recruited from four health centers and three hospitals in Addis Ababa, Ethiopia. The expression levels of miRNAs isolated from plasma of culture confirmed newly diagnosed pulmonary tuberculosis patients were compared with latently infected and non-infected healthy controls. The qPCR data were analyzed using four independent statistical tools: Best Keeper, Genorm, Normfinder and comparative delta-Ct methods, and the data showed that miRNA-22-3p and miRNA-93-5p were suitable plasma reference miRNAs in a tuberculosis study.

Rescue of common exon-skipping mutations in cystic fibrosis with modified U1 snRNAs.

In cystic fibrosis (CF), the correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on 10 common mutations/variants 711+3A>G/C, 711+5G>A, TG13T3, TG13T5, TG12T5, 1863C>T, 1898+3A>G, 2789+5G>A, and 3120G>A that induce skipping of the corresponding CFTR exons 5, 10, 13, 16, and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 small nuclear RNAs (snRNAs), named Exon Specific U1s (ExSpeU1s), that was engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all 10 splicing mutations analyzed are efficiently corrected by specific ExSpeU1s. Using complementary DNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

Integrator-Dependent and Allosteric/Intrinsic Mechanisms Ensure Efficient Termination of snRNA Transcription

SummaryMany RNA polymerases terminate transcription using allosteric/intrinsic mechanisms, whereby protein alterations or nucleotide sequences promote their release from DNA. RNA polymerase II (Pol II) is somewhat different based on its behavior at protein-coding genes where termination additionally requires endoribonucleolytic cleavage and subsequent 5′→3′ exoribonuclease activity. The Pol-II-transcribed small nuclear RNAs (snRNAs) also undergo endoribonucleolytic cleavage by the Integrator complex, which promotes their transcriptional termination. Here, we confirm the involvement of Integrator but show that Integrator-independent processes can terminate snRNA transcription both in its absence and naturally. This is often associated with exosome degradation of snRNA precursors that long-read sequencing analysis reveals as frequently terminating at T-runs located downstream of some snRNAs. This finding suggests a unifying vulnerability of RNA polymerases to such sequences given their well-known roles in terminating Pol III and bacterial RNA polymerase.

The Expression of MicroRNA-21 in Bone Marrow Fluid is an Indicator of Hematological Disorders and Mortality.

Objective:Bone marrow fluid (BMF) consists of various components that establishes a microenvironment for cell differentiation and remodeling. MicroRNA-21 (miR-21) levels have recently emerged as novel biomarkers for different diseases. However, the conventional RNU6B (U6), used as the reference for intracellular miRNA, may not be appropriate for the normalization of circulating miRNAs. Methods:We measured the levels of U6, spiked-in RNA, and miR-21 in the BMF of 13 healthy controls and 37 patients with hematological disorders to investigate the reliability of either U6 or spike-in RNA as an endogenous reference and also to study the correlation between miR-21, hematological disorders and mortality. Results:Notably, the levels of U6 demonstrated a high variability in BMF of healthy controls and patients. In contrast, the levels of spiked-in RNA displayed a significantly higher stability in both cohorts. Compared with controls, the levels of miR-21 were significantly upregulated in BMF of patients with leukemia but not lymphoma. Also, using 21 as the cut-off value of miR-21, it differentiated the mortality of patients with hematologic disorders. Conclusions:Collectively, using spiked-in RNA as a reference the upregulated miR-21 levels in BMF could be an indicator of the diagnosis of leukemia and a predictor of mortality.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.207 AAV.U7 technology for two mutational hotspots of the DMD gene (∼6%) results in efficient exon skipping, protein restoration and force improvement

Mutations in the DMD gene cause two disorders, the severe Duchenne muscular dystrophy (DMD), associated with no protein expression and the milder Becker muscular dystrophy (BMD) associated with the production of an internally truncated but semi-functional protein. Genotype-phenotype correlation of DMD and BMD prompted a promising therapeutic strategy named exon skipping that intends to convert a DMD to a BMD phenotype. This strategy makes use of antisense oligonucleotides (AONs) to hide exon definition. This should transform an out-of-frame DMD reading frame into an in-frame one, leading into expression of an internally truncated but functional protein. AONs are promising but have two major drawbacks: 1. stability which required repeated injection; 2. limited penetration in heart. Alternatively, an antisense sequence can be carried by a small nuclear RNA (U7snRNA) and delivered using a viral vector such as adeno-associated virus (AAV). This approach (AAV.U7) has been demonstrated by several group including ours, to overcome AON's drawbacks. In this study, we focused on skipping of exon 2 and exon 44, two mutational hotspots of the DMD gene as their skipping could be beneficial to ∼12% of patients. Our objective was to evaluate the efficiency of an AAV.U7 exon skipping strategy in these hotspot mutations both in vitro and in vivo. For in vitro studies, we used skin punches from several DMD patients and transdifferentiated them into myotubes. Using those cells, we showed that our constructs efficiently mediated exons 2 or 44 skipping (at least ≥50%) and induced expression of a slightly truncated dystrophin. For in vivo studies, we used either a new mouse model carrying an exon 2 duplication (referred to as Dup 2 mice) or more recently a new humanized DMD mouse model that is missing human exon 45 disrupting reading frame of hDMD. This new model (referred to as hDMD/mdx del45 mice) does not express mouse and human dystrophins and presents a dystrophic muscle pathology in multiple muscles. In vivo, following intramuscular or intravenous injection, we observed an efficient exon 2 or 44 skipping (at least ≥50%) and expression of a truncated dystrophin (at least ≥40%). Muscle strength using different tests was improved (by ∼50% compared to an untreated mouse). These AAV.U7- could help ∼12% of DMD patients. One of those vectors is being evaluated in a clinical trial.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.213 Absence of toxicity with intravenous dosing of the Exon 2-skipping AAV9.U7-ACCA vector in non-human primates

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an AAV9 vector containing four copies of the non-coding 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 (rAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length dystrophin from wild-type DMD mRNA, as well as an IRES-driven isoform translated only in the absence of exon 2 (Del2 mRNA).

HEREDITARY NEUROPATHIES & ALS: P.115 Analysis of off-target effects of antisense sequence inducing exon skipping in SOD1-linked amyotrophic lateral sclerosis

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis (ALS). Given the toxic gain-of-function role of SOD1, suppressing SOD1 mRNA and protein in the affected tissues is predicted as a promising therapeutic approach. We have previously shown the high efficiency of an adeno-associated virus serotype rh10 vector (AAV10) to mediate exon skipping of the hSOD1 pre-mRNA by expression of exon-2-targeted antisense sequences (AS) embedded in a modified U7 small-nuclear RNA (AAV10-U7-hSOD1) (Biferi et al., 2017).

A mouse tissue atlas of small noncoding RNA

Small noncoding RNAs (ncRNAs) play a vital role in a broad range of biological processes both in health and disease. A comprehensive quantitative reference of small ncRNA expression would significantly advance our understanding of ncRNA roles in shaping tissue functions. Here, we systematically profiled the levels of five ncRNA classes (microRNA [miRNA], small nucleolar RNA [snoRNA], small nuclear RNA [snRNA], small Cajal body-specific RNA [scaRNA], and transfer RNA [tRNA] fragments) across 11 mouse tissues by deep sequencing. Using 14 biological replicates spanning both sexes, we identified that ∼30% of small ncRNAs are distributed across the body in a tissue-specific manner with some also being sexually dimorphic. We found that some miRNAs are subject to "arm switching" between healthy tissues and that tRNA fragments are retained within tissues in both a gene- and a tissue-specific manner. Out of 11 profiled tissues, we confirmed that brain contains the largest number of unique small ncRNA transcripts, some of which were previously annotated while others are identified in this study. Furthermore, by combining these findings with single-cell chromatin accessibility (scATAC-seq) data, we were able to connect identified brain-specific ncRNAs with their cell types of origin. These results yield the most comprehensive characterization of specific and ubiquitous small RNAs in individual murine tissues to date, and we expect that these data will be a resource for the further identification of ncRNAs involved in tissue function in health and dysfunction in disease.

Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs.

Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9∗-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade.

Non-Coding RNAs in Psychiatric Disorders and Suicidal Behavior.

It is well known that only a small proportion of the human genome code for proteins; the rest belong to the family of RNAs that do not code for protein and are known as non-coding RNAs (ncRNAs). ncRNAs are further divided into two subclasses based on size: 1) long non-coding RNAs (lncRNAs; >200 nucleotides) and 2) small RNAs (<200 nucleotides). Small RNAs contain various family members that include microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and small nuclear RNAs (snRNAs). The roles of ncRNAs, especially lncRNAs and miRNAs, are well documented in brain development, homeostasis, stress responses, and neural plasticity. It has also been reported that ncRNAs can influence the development of psychiatric disorders including schizophrenia, major depressive disorder, and bipolar disorder. More recently, their roles are being investigated in suicidal behavior. In this article, we have comprehensively reviewed the findings of lncRNA and miRNA expression changes and their functions in various psychiatric disorders including suicidal behavior. We primarily focused on studies that have been done in postmortem human brain. In addition, we have briefly reviewed the role of other small RNAs (e.g. piwiRNA, siRNA, snRNA, and snoRNAs) and their expression changes in psychiatric illnesses.

Topology of the U12-U6atac snRNA Complex of the Minor Spliceosome and Binding by NTC-Related Protein RBM22.

Splicing of precursor messenger RNA is catalyzed by the spliceosome, a dynamic ribonucleoprotein assembly including five small nuclear (sn)RNAs and >100 proteins. RNA components catalyze the two transesterification reactions, but proteins perform critical roles in assembly and rearrangement. The catalytic core comprises a paired complex of U2 and U6 snRNAs for the major form of the spliceosome and U12 and U6atac snRNAs for the minor variant (∼0.3% of all spliceosomes in higher eukaryotes); the latter shares key catalytic sequence elements and performs identical chemistry. Here we use solution NMR techniques to show that the U12–U6atac snRNA complex of both human and Arabidopsis maintain base-pairing patterns similar to those in the three-helix model of the U2–U6 snRNA complex that position key elements to form the spliceosome’s active site. However, in place of the stacked base pairs at the base of the U6 snRNA intramolecular stem loop and the central junction of the U2–U6 snRNA complex, we see altered geometry in the single-stranded hinge region opposing termini of the snRNAs to enable interaction between the key elements. We then use electrophoretic mobility shift assays and fluorescence assays to show that the protein RBM22, implicated in remodeling the human U2–U6 snRNA complex prior to catalysis, also binds the U12–U6atac snRNA complexes specifically and with similar affinity as to U2–U6 snRNA (a mean Kd for the two methods = 3.4 and 8.0 μM for U2–U6 and U12–U6atac snRNA complexes, respectively), suggesting that RBM22 performs the same role in both spliceosomes.

Isolation of Total RNA Using Monophasic Lysis Reagents

A typical mammalian cell contains ∼10-5 µg of RNA, 80%-85% of which is ribosomal RNA (rRNA; chiefly the 28S, 18S, 5.8S, and 5S species). Most of the remaining 15%-20% consists of a variety of low-molecular-weight species (e.g., transfer RNAs [tRNAs] and small nuclear RNAs). These abundant RNAs are of defined size and sequence. In contrast, messenger RNA (mRNA), which makes up between 1% and 5% of the total cellular RNA, is heterogeneous in both size-from a few hundred bases to many kilobases in length-and sequence. In this introduction, the development and use of monophasic lysis reagents for the isolation of total RNA from eukaryotic cells is discussed.

The 5S rRNA genes in Alexandrium: their use as a FISH chromosomal marker in studies of the diversity, cell cycle and sexuality of dinoflagellates

Chromosomal markers of the diversity and evolution of dinoflagellates are scarce because the genomes of these organisms are unique among eukaryotes in terms of their base composition and chromosomal structure. Similarly, a lack of appropriate tools has hindered studies of the chromosomal localization of 5S ribosomal DNA (rDNA) in the nucleosome-less chromosomes of dinoflagellates. In this study, we isolated and cloned 5S rDNA sequences from various toxin-producing species of the genus Alexandrium and developed a fluorescence in situ hybridization (FISH) probe that allows their chromosomal localization. Our results can be summarized as follows: 1) The 5S rDNA unit is composed of a highly conserved 122-bp coding region and an intergenic spacer (IGS), the length and sequence of which are variable even within strains. 2) Three different IGS types, one containing the U6 small nuclear RNA (snRNA) gene, were found among four of the studied species (A. minutum, A. tamarense, A. catenella and A. pacificum). 3) In all strains investigated by FISH (A. minutum, A. tamarense, A. pacificum, A. catenella, A. andersonii and A. ostenfeldii), 5S rDNA gene arrays were separate from the nucleolar organizer region, which contains the genes for the large 45S pre-ribosomal RNA. 4) One to three 5S rDNA sites per haploid genome were detected, depending on the strains/species. Intraspecific variability in the number of 5S rDNA sites was determined among strains of A. minutum and A. pacificum. 5) 5S rDNA is a useful chromosomal marker of mitosis progression and can be employed to differentiate vegetative (haploid) vs. planozygotes (diploid) cells. Thus, the FISH probe (oligo-Dino5Smix5) developed in this study facilitates analyses of the diversity, cell cycle and life stages of the genus Alexandrium.

Cajal body dynamics in soybean root meristem cells under chilling stress and recovery

Cajal bodies (CBs) are nuclear domains widely present in eukaryotic cells. Their main functions include processing and modifications of small nuclear RNAs (snRNAs), including those participating in mRNA splicing. CBs are dynamic structures in terms of their mobility, fusion and split, which results in their variable number and size during cell cycle. CBs are supposed to play a role in response to various stresses or at least the cell reactions to these stressors is related with changing CB number and size. That is why the number and size of CBs were analyzed in primary root meristematic cells of soybean, a subtropical plant, whose seedlings were grown at 23 °C (the control), subjected to chilling stress (10 °C) and recovered at 23 °C after chilling. These parameters were examined in three classes of cell size which corresponded to the cells in G1, S and G2 phases. The general number of CBs in nuclei of the chilled plants was halved in comparison to the control cells, however this parameter returned to the control value during recovery. Such a result suggests that chilling may hinder formation of CBs or it may provoke their fusion. Moreover, the number of small CBs decreased and the number of large CBs increased during interphase progress, while the general number of CBs was constant in each treatment group. The constant number of CBs from G1 to G2 phase proves that in soybean cells they enlarge as a result of component accumulation rather than their fusion. The reasons of such a state of soybean CBs are discussed.

Abstract 269: Small non-coding RNA profiling in stool and plasma samples to explore potential biomarkers for colorectal cancer diagnosis

Abstract Colorectal cancer (CRC) is one of the most frequent cancers worldwide. The available screening tests have reduced the incidence and mortality for this cancer; however, the research of new potential molecular biomarkers to use concomitantly could improve advanced adenomas/CRC early detection. Small non-coding RNAs (sncRNAs) are stable, easy detectable molecules that have been found dysregulated in several diseases including CRC. Besides the most studied microRNAs (miRNAs), sncRNAs include also P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transfer RNAs (tRNAs) and other miscellaneous RNAs, all with key roles in post-transcriptional regulation. The aim of the present study was to identify by next-generation sequencing (NGS) signatures of sncRNAs other than miRNAs, in fecal and plasma extracellular vesicles (EVs) samples potentially able to detect patients with adenomas or CRC. We initially performed a small RNA-sequencing in samples from 221 subjects collected at colonoscopy. The group included: i) 80 healthy subjects with negative colonoscopy results; (ii) 43 adenoma patients; (iii) 41 individuals with inflammatory bowel disease and (iv) 57 newly diagnosed CRC patients. A computational pipeline for the identification of sncRNAs after miRNA mapping reads was previously implemented by our group. In stool samples, comparing the categories of interest mentioned above, we identified 395 differentially expressed sncRNAs (DEsncRNAs), mainly piRNAs (46.3%) and tRNAs (43.6%) (likelihood ratio test adjusting for sex and age, FDR adjusted p ≤ 0.05). Similarly, a group of 32 DEsncRNAs was detected in plasma EVs with piRNAs (41%) and tRNAs (44%) resulting among the most altered. Small RNA-sequencing has also been performed in a subset of 24 paired colorectal tumor and adjacent non-malignant tissues from the CRC patients for whom stool and plasma samples were analysed. Fifty-seven DEsncRNAs were found in tumor vs. non-malignant adjacent tissue with a similar distribution of dysregulated sncRNA biotypes. Some DEsncRNAs were shared between stool and tissues (n=35), plasma EVs and tissues (n=11) and, interestingly, among the profiles of the three different biospecimens (n=10). To validate our findings, we also sequenced stool samples from an independent cohort from the Czech Republic (at present 22 healthy subjects, 19 with inflammations, 21 with adenomas and 34 CRC patients). A total of 100 DEsncRNAs were detected in this validation cohort, among which 43 overlapped with those detected in the Italian cohort. In conclusions, we show that besides the most widely performed miRNA profiling, also the detection of sncRNAs in surrogate tissues by NGS may help to identify reliable and comparable results with those of primary tissues. To further investigate these molecules may provide new accurate markers for CRC diagnostic purposes. Citation Format: Antonio Francavilla, Sonia Tarallo, Giulio Ferrero, Francesca Cordero, Gaetano Gallo, Veronika Vymetalkova, Ludmila Vodickova, Pavel Vodicka, Barbara Pardini, Alessio Naccarati. Small non-coding RNA profiling in stool and plasma samples to explore potential biomarkers for colorectal cancer diagnosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 269.