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Pathogenic PDE12 variants impair mitochondrial RNA processing causing neonatal mitochondrial disease.

Pathogenic variants in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial function. Within this group, an increasing number of families have been identified, where Mendelian genetic disorders implicate defective mitochondrial RNA biology. The PDE12 gene encodes the poly(A)-specific exoribonuclease, involved in the quality control of mitochondrial non-coding RNAs. Here, we report that disease-causing PDE12 variants in three unrelated families are associated with mitochondrial respiratory chain deficiencies a

A Chemically Defined TLR3 Agonist with Anticancer Activity.

Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized dou

The emerging function and clinical significance of circRNAs in Thyroid Cancer and Autoimmune Thyroid Diseases.

Thyroid cancer (TC) is one of the most common malignant tumors, with high morbidity and mortality rates worldwide. The incidence of TC, especially that of papillary thyroid carcinoma (PTC); has increased rapidly in recent decades. Autoimmune thyroid disease (AITD) is closely related to TC and has an estimated prevalence of 5%. Thus, it is becoming increasingly important to identify potential diagnostic biomarkers and therapeutic targets for TC and AITD. Circular RNAs (circRNAs) are a class of non-coding RNAs with covalently bonded circular structures that lack 5'-3' polarity and polyadenylated

A Fresh Look at Huntingtin mRNA Processing in Huntington's Disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a mutation that expands the polyglutamine (CAG) repeat in exon 1 of the huntingtin (HTT) gene. Wild-type HTT protein interacts with other proteins to protect cells against toxic stimuli, mediate vesicle transport and endocytosis, and modulate synaptic activity. Mutant HTT protein disrupts autophagy, vesicle transport, neurotransmitter signaling, and mitochondrial function. Although many of the activities of wild-type HTT protein and the toxicities of mutant HTT protein are characterized, less is known about the acti

Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes.

OBJECTIVE: Analyses of single oocytes are essential for a fine dissection of molecular features governing developmental competence. We adapted the phenol-chloroform procedure for the purification of total RNA from single oocytes. RESULTS: Key modifications include the use of Phasemaker™ tubes, a second chloroform wash of the aqueous phase, and the precipitation of the RNA with glyclogen in a 200 μl micro-centrifuge tube. Assessment of the RNA profile from single oocytes showed distinct peaks for 18S and 28S ribosomal subunits. This approach permitted the extraction of small RNAs from single o

Epstein-Barr virus-associated gastric carcinoma in Mexico: analysis of 135 consecutive gastrectomies in two hospitals.

Epstein-Barr virus (EBV) has been implicated in the genesis of gastric carcinoma. The presence of clonal episomal viral forms in the nuclei of neoplastic gastric epithelial cells suggests that viral infection occurs before the development of gastric carcinoma. Mexico is a country at high risk for gastric cancer-it is the second cause of death among patients who die of cancer in that country. A series of 135 consecutive non-selected gastrectomies from two hospitals in Mexico City were analyzed to search for EBV in gastric carcinomas. EBV-encoded small non-polyadenylated RNA (EBER) in situ hybri

Molecular cloning of Lassa virus RNA: nucleotide sequence and expression of the nucleocapsid protein gene.

Lassa virus RNA isolated from purified virus particles was polyadenylated and reverse transcripts were cloned into the PstI site of plasmid pUC9. Clones containing sequences of the smaller (S) segment of the Lassa virus genome were identified by hybridization with purified S RNA. They were characterized by their ability to hybridize with fragments of 3'-labeled Lassa virus S RNA and with each other, and by restriction mapping. The largest insert was 1830 bp long and began with the 3'-terminal 19-base sequence characteristic of all arenavirus S RNAs so far analyzed. The virus complementary stra

Screening for clinically significant non-deletional alpha thalassaemia mutations by pyrosequencing.

Non-deletional α(+)-thalassaemia is associated with a higher degree of morbidity and mortality than deletional forms of α(+)-thalassaemia. Screening for the common deletional forms of α-thalassaemia by Gap-PCR is widely practiced; however, the detection of non-deletional α-thalassaemia mutations is technically more labour-intensive and expensive, as it requires DNA sequencing. In addition, the presence of four very closely homologous alpha globin genes and the frequent co-existence of deletional forms of α-thalassaemia present another layer of complexity in the detection of these mutations. Wi

Structure-guided design of an LC3B-targeting PNA oligomer modulating autophagy and LC3B-mediated mRNA decay.

Autophagy plays a central role in cellular degradation and recycling pathways, and the LC3B protein is essential for this process. Dysregulation of LC3B has been implicated in oncogenesis. In this study, we used a computational approach to design a novel peptide nucleic acid (PNA) targeting the RNA-binding domain of LC3B, with the aim of inhibiting its function. The RNA AAUAAA polyadenylation signal was used as a starting point to design new PNAs with high affinity for LC3B. Molecular dynamics simulations and binding free-energy calculations on the RNA-AAUAAA/LC3B complex enabled the identific…

MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic de

RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy.

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequenci

tRNA Derivatives in Multiple Myeloma: Investigation of the Potential Value of a tRNA-Derived Molecular Signature.

Multiple myeloma (MM) is a hematologic malignancy arising from the clonal proliferation of malignant plasma cells. tRNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNAs, deriving from specific enzymatic cleavage of tRNAs. To the best of our knowledge, this is one of few studies to uncover the potential clinical significance of tRFs in MM. Total RNA was extracted from CD138+ plasma cells of MM and smoldering MM patients, and in vitro polyadenylated. First-strand cDNA synthesis was performed, priming from an oligo-dT-adaptor sequence. Next, real-time quantitative PCR (qPC

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… Total RNA Extraction, In Vitro Polyadenylation, and cDNA Synthesis Total RNA was isolated from CD138+ plasma cells, using TRI Reagent ® (Molecular Research Center Inc., Cincinnati, OH, USA). … Next, 200 ng of each total extract were subjected to in vitro polyadenylation, using E. coli poly(A) polymerase (New England Biolabs Ltd., Ontario, ON, Canada) and 80 μM ATP. Each sample was incubated at 37 °C for 60 min, followed by an inactivation step at 65 °C for 10 min. …

Toxoplasma ubiquitin-like protease 1, a key enzyme in sumoylation and desumoylation pathways, is under the control of non-coding RNAs.

Sumoylation and desumoylation are reversible pathways responsible for modification of protein structures and functions by the reversible covalent attachment of a small ubiquitin-like modifier (SUMO) peptide. These pathways are important for a wide range of cellular processes and require a steady supply of SUMO, which is generated by an enzymatic reaction catalysed by the ubiquitin-like protease (Ulp) family. Here we show by functional complementation analysis that the Ulp1 of Toxoplasma gondii (TgUlp1) can rescue a growth-deficient phenotype of a yeast-Ulp1 knockout. Recombinant TgUlp1 is an a

Modulation of risk of squamous cell carcinoma head and neck in North Indian population with polymorphisms in xeroderma pigmentosum complementation Group C gene.

BACKGROUND: Genetic variations in nucleotide excision repair genes can alter the risk of squamous cell carcinoma of head and neck (SCCHN). MATERIALS AND METHODS: The present study has genotyped 334 subjects from North Indian population for xeroderma pigmentosum complementation Group C (XPC) rs2228001A>C, XPC rs77907221 polyadenylate (PAT) deletion/insertion (D/I), xeroderma pigmentosum complementation Group D - rs13181A>C, and xeroderma pigmentosum complementation Type G rs17655 G>C polymorphisms with polymerase chain reaction (PCR)-restriction-fragment length polymorphism or allele-specific

Generation of an estrogen receptor beta-iCre knock-in mouse.

A novel knock-in mouse that expresses codon-improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatiotemporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the en…

Recombineering-based dissection of flanking and paralogous Hox gene functions in mouse reproductive tracts.

Hox genes are key regulators of development. In mammals, the study of these genes is greatly confounded by their large number, overlapping functions and interspersed shared enhancers. Here, we describe the use of a novel recombineering strategy to introduce simultaneous frameshift mutations into the flanking Hoxa9, Hoxa10 and Hoxa11 genes, as well as their paralogs on the HoxD cluster. The resulting Hoxa9,10,11 mutant mice displayed dramatic synergistic homeotic transformations of the reproductive tracts, with the uterus anteriorized towards oviduct and the vas deferens anteriorized towards ep

The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication.

The influenza A virus RNA genome segments are packaged in ribonucleoprotein complexes containing RNA polymerase and nucleoprotein. The ribonucleoprotein is involved in the transcription of viral genes and replication of the viral RNA genome in the nucleus of the infected cells, and represents the minimal transcriptional and replicative machinery of an influenza virus. During transcription, the viral RNA polymerase synthesizes capped and polyadenylated mRNA using 5΄ capped RNA primers. During replication, the viral RNA polymerase generates a complementary RNA (cRNA) replication intermediate, a

Poikilodermatous mycosis fungoides with a CD8+ CD56+ immunophenotype: a case report and literature review.

Mycosis fungoides (MF) represents the most common type of cutaneous lymphoma. MF shows varieties in both its clinical presentation and immunophenotype. We herein report one case of poikilodermatous MF with a CD8+ CD56+ immunophenotype and present a literature review. A 20-year-old Japanese woman presented with a 10-year history of multiple poikilodermatous and reddish or brownish patches with mild pruritus on the chest, abdomen, back, buttock and thighs. Histopathologically, small- to medium-sized atypical lymphocytes infiltrated into the epidermis, indicating epidermotropism, along the basal

The Rift Valley Fever virus protein NSm and putative cellular protein interactions.

Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causa … wth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an …

Transcriptional patterns of the pCD41 (U27) locus of human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is a lymphotropic herpesvirus, and in vitro, it can productively infect many of the same cell types that human immunodeficiency virus (HIV) infects. Simultaneous infection of T cells by HIV and HHV-6 can lead to both activation of the HIV promoter and acceleration of the cytopathic effects. Several HHV-6 genes have been demonstrated to activate HIV promoter expression. Among them is a cDNA clone, pCD41 (U27), which codes for the HHV-6 DNA polymerase accessory protein. We have now further characterized the transcription pattern in the pCD41 locus and identified at le