Salmon sperm DNA increases sample recovery from cotton swabs.

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001-1-10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

PABPN1 aggregation is driven by Ala expansion and poly(A)-RNA binding, leading to CFIm25 sequestration that impairs alternative polyadenylation.

Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear aggregates in oculopharyngeal muscular dystrophy. The factors that drive PABPN1 aggregation and its cellular consequences remain largely unknown. Here, we investigated the roles of Ala stretch and poly(A) RNA in the phase transition of PABPN1 using biochemical and molecular cell biology methods. We have revealed that the Ala stretch controls its mobility in nuclear speckles, and Ala expansion leads to aggregation from the dynamic speckles. Poly(A) nucleotide is essential to the early-stage condensation that thereby facilitates speckle formation and transition to solid-like aggregates. Moreover, the PABPN1 aggregates can sequester CFIm25, a component of the pre-mRNA 3'-UTR processing complex, in an mRNA-dependent manner and consequently impair the function of CFIm25 in alternative polyadenylation. In conclusion, our study elucidates a molecular mechanism underlying PABPN1 aggregation and sequestration, which will be beneficial for understanding PABPN1 proteinopathy.

An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

The enigma of ultraviolet radiation stress granules: Research challenges and new perspectives.

Stress granules (SGs) are non-membrane bound cytoplasmic condensates that form in response to a variety of different stressors. Canonical SGs are thought to have a cytoprotective role, reallocating cellular resources during stress by activation of the integrated stress response (ISR) to inhibit translation and avoid apoptosis. However, different stresses result in compositionally distinct, non-canonical SG formation that is likely pro-apoptotic, though the exact function(s) of both SGs subtypes remain unclear. A unique non-canonical SG subtype is triggered upon exposure to ultraviolet (UV) radiation. While it is generally agreed that UV SGs are bona fide SGs due to their dependence upon the core SG nucleating protein Ras GTPase-activating protein-binding protein 1 (G3BP1), the localization of other key components of UV SGs are unknown or under debate. Further, the dynamics of UV SGs are not known, though unique properties such as cell cycle dependence have been observed. This Perspective compiles the available information on SG subtypes and on UV SGs in particular in an attempt to understand the formation, dynamics, and function of these mysterious stress-specific complexes. We identify key gaps in knowledge related to UV SGs, and examine the unique aspects of their formation. We propose that more thorough knowledge of the distinct properties of UV SGs will lead to new avenues of understanding of the function of SGs, as well as their roles in disease.

Inhibition of mitochondrial complex III or dihydroorotate dehydrogenase (DHODH) triggers formation of poly(A)+ RNA foci adjacent to nuclear speckles following activation of ATM (ataxia telangiectasia mutated).

Intracellular and intercellular signalling networks play an essential role in optimizing cellular homoeostasis and are thought to be partly reflected in nuclear mRNA dynamics. However, the regulation of nuclear mRNA dynamics by intracellular and intercellular signals remains largely unexplored, and research tools are lacking. Through an original screening based on the mRNA metabolic mechanism, we discovered that eight well-known inhibitors cause significant nuclear poly(A)+ RNA accumulation. Among these inhibitors, we discovered a new mRNA metabolic response in which the addition of antimycin A, an inhibitor of mitochondrial respiratory-chain complex III (complex III), resulted in a marked accumulation of poly(A)+ RNA near the nuclear speckles. Furthermore, dihydroorotate dehydrogenase (DHODH) inhibitors, a rate-limiting enzyme in the intracellular de novo pyrimidine synthesis reaction that specifically exchanges electrons with complex III, also caused a remarkable accumulation of nuclear poly(A)+ RNA adjacent to the nuclear speckles, which was abolished by extracellular uridine supply, indicating that the depletion of intracellular pyrimidine affects poly(A)+ RNA metabolism. Further analysis revealed that ataxia telangiectasia mutated (ATM), a serine and threonine kinase and a master regulator of DNA double-strand break (DSB) and nucleolar stress, is required for this poly(A)+ RNA nuclear accumulation phenomenon. This study reports new insights into novel aspects of nuclear poly(A)+ RNA metabolism, especially the relationship between mitochondrial respiratory-chain functions, pyrimidine metabolism, and nuclear RNA metabolism.

CHERP Regulates the Alternative Splicing of pre-mRNAs in the Nucleus.

Calcium homeostasis endoplasmic reticulum protein (CHERP) is colocalized with the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or perinuclear region, and has been involved in intracellular calcium signaling. Structurally, CHERP carries the nuclear localization signal and arginine/serine-dipeptide repeats, like domain, and interacts with the spliceosome. However, the exact function of CHERP in the nucleus remains unknown. Here, we showed that poly(A)+ RNAs accumulated in the nucleus of CHERP-depleted U2OS cells. Our global analysis revealed that CHERP regulated alternative mRNA splicing events by interaction with U2 small nuclear ribonucleoproteins (U2 snRNPs) and U2 snRNP-related proteins. Among the five alternative splicing patterns analyzed, intron retention was the most frequently observed event. This was in accordance with the accumulation of poly(A)+ RNAs in the nucleus. Furthermore, intron retention and cassette exon choices were influenced by the strength of the 5' or 3' splice site, the branch point site, GC content, and intron length. In addition, CHERP depletion induced anomalies in the cell cycle progression into the M phase, and abnormal cell division. These results suggested that CHERP is involved in the regulation of alternative splicing.

TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer.

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

QPAT-seq, a rapid and deduplicatable method for quantification of poly(A) site usages.

Alternative polyadenylation (APA) is an essential regulatory mechanism for gene expression. The next generation sequencing provides ample opportunity to precisely delineate APA sites genome-wide. Various methods for profiling transcriptome-wide poly(A) sites were developed. By comparing available methods, the ways for adding sequencing adaptors to fit with the Illumina sequencing platform are different. These methods have identified more than 50% genes that undergo APA in eukaryotes. However, due to the unbalanced PCR during library preparation, accurate quantification of poly(A) sites is still a challenge. Here, we describe an updated poly(A) tag sequencing method that incorporates unique molecular identifier (UMI) into the adaptor for removing quantification bias induced by PCR duplicates. Hence, quantification of poly(A) site usages can be achieved by counting UMIs. This protocol, quantifying poly(A) tag sequencing (QPAT-seq), can be finished in 1 day with reduced cost, and is particularly useful for application with a large number of samples.

Gene expression profiling of cultured mouse testis fragments treated with ethinylestradiol.

The assessment of xenobiotic-induced testicular toxicity is important in drug development. Nonetheless, in vitro models to test drugs and chemicals that may cause testicular toxicity are lacking, requiring the continued use of animal models for those studies. We previously evaluated an in vitro mouse testis organ culture system using ethinylestradiol (EE), a well-studied testicular toxicant, and demonstrated a dose-dependent relationship between adverse effects to germ cell differentiation and increasing EE concentrations. However, we terminated that study after 20 days of culture due to oxygen deficiency during germ cell differentiation. Therefore, in the current study, we aimed to identify gene(s) with potential for supporting the histopathological evaluations of testicular toxicity using in vitro testis organ culture system. We cultured testis fragments obtained from mice at postnatal day (PND) 5 in α-Minimal Essential Medium containing 40 mg/mL AlbuMAX™ I and treated them with 0.01 or 1 nM EE on day 1 of culture. On day 20, we collected testis fragments for RNA sequencing analysis and quantitative polymerase chain reaction (qPCR). We found that phospholipase C, zeta 1 and testis-specific serine kinase 4 genes, that are involved in spermatogenesis and predominantly expressed in the testis, were significantly reduced in testis fragments treated with the highest concentration of EE. Also, cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1) and interleukin 16 (Il16) were up-regulated in the highest EE-treated groups. Further studies are needed to confirm the variations of these gene expression using other testicular toxicants.

Circular RNAs in the cardiovascular system.

Until recently considered as rare, circular RNAs (circRNAs) are emerging as important regulators of gene expression. They are ubiquitously expressed and represent a novel branch of the family of non-coding RNAs. Recent investigations showed that circRNAs are regulated in the cardiovascular system and participate in its physiological and pathological development. In this review article, we will provide an overview of the role of circRNAs in cardiovascular health and disease. After a description of the biogenesis of circRNAs, we will summarize what is known of the expression, regulation and function of circRNAs in the cardiovascular system. We will then address some technical aspects of circRNAs research, discussing how artificial intelligence may aid in circRNAs research. Finally, the potential of circRNAs as biomarkers of cardiovascular disease will be addressed and directions for future research will be proposed.

A Lucifer-Based Environment-Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs.

Fluorescence-based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment-sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8-naphthalimide) at the 5-position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells-poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.

A noncanonical poly(A) RNA polymerase gene affects morphology in Phoma medicaginis.

Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot, an important disease of alfalfa and annual medics. P. medicaginis forms uninucleate conidia in melanized pycnidia and is genetically tractable using Agrobacterium mediated transformation (ATMT), resulting in random integration of T-DNA that occasionally generates pycnidial mutants. The T-DNA tagged mutant, P265 displayed smaller pycnidia and more aerial hyphae than the wild type. A single T-DNA disrupted a putative noncanonical poly(A) RNA polymerase gene, Pmncpap1, which in yeast interacts with ribonucleotide reductase (RNR). As in yeast mutants, P265 showed sensitivity to hydroxyurea (HU), a RNR inhibitor. To characterize the role of Pmncpap1, targeted ΔPmncpap1 mutants were created using a hygromycin selectable marker flanked by 1 Kbp regions of Pmncpap1. ΔPmncpap1 mutants possessed similar morphological features to those of P265. The plasmid for rescue of PmncPAP1, pCAM-Nat1 (nourseothricin selection) was constructed and used to introduce full-length PmncPAP1 into mutants. Rescued P265 showed partial recovery of wild type and the original T-DNA was lost due to homologous integration. To our knowledge, this is the first ncPAP to be examined in a filamentous fungus.

Regulation of poly(A) RNA retention in the nucleus as a survival strategy of plants during hypoxia.

Last finding indicates that post-transcriptional processes are significant in low-oxygen conditions, but their nature is poorly understood. Here, we localized poly(A) RNA and mRNA coding proteins involved and not involved with resistance to hypoxia in Lupinus luteus and Arabidopsis thaliana during submergence and after recovery of aerobic conditions. We showed a strong nuclear accumulation of poly(A) RNA and 6 of 7 studied mRNAs with a concurrent strong reduction in RNA polymerase II transcription during hypoxia. In this study, the nucleus did not accumulate mRNA of the ADH1 (alcohol dehydrogenase 1) gene, which is a core hypoxia gene. The RNA accumulation in the nucleus is among the mechanisms of post-transcriptional gene regulation that prevents translation. However re-aeration was accompanied by a strong increase in the amount of the mRNAs in the cytoplasm and a simultaneous decrease in nuclear mRNAs. This finding indicates that the nucleus is a storage site for those of mRNAs which are not involved in the response to hypoxia for use by the plants after the hypoxic stress. In this study, the highest intensity of RNA accumulation occurred in Cajal bodies (CBs); the intensity of accumulation was inversely correlated with transcription. Under hypoxia, ncb-1 mutants of Arabidopsis thaliana with a complete absence of CBs died sooner than wild type (WT), accompanied by a strong reduction in the level of poly(A) RNA in the nucleus. These results suggest that the CBs not only participate in the storage of the nuclear RNA, but they also could take part in its stabilization under low-oxygen conditions.

Giant poly(A)-rich RNP aggregates form at terminal regions of avian lampbrush chromosomes.

The cell nucleus comprises a number of chromatin-associated domains. Certain chromatin-associated domains are nucleated by nascent RNA and accumulate non-nascent transcripts in the form of ribonucleoprotein (RNP) aggregates. In the transcriptionally active nucleus of the growing avian oocyte, RNP-rich structures, here termed giant terminal RNP aggregates (GITERA), form at the termini of lampbrush chromosomes. Using GITERA as an example, we aimed to explore mechanisms of RNP aggregate formation at certain chromosomal loci to establish whether they accumulate non-nascent RNA and to analyze protein composition in RNP aggregates. We found that GITERA on chicken and pigeon lampbrush chromosomes do not contain nascent transcripts. At the same time, RNA fluorescent in situ hybridization (FISH) and in situ reverse transcription demonstrated that GITERA accumulate poly(A)-rich RNA. Moreover, subtelomere chromosome regions adjacent to GITERA are transcriptionally active as shown by detection of incorporated BrUTP and the elongating form of RNA polymerase II. GITERA on both chicken and pigeon lampbrush chromosomes are enriched in splicing factors but not in heterogeneous nuclear RNP (hnRNP) L and K. A subtype of GITERA concentrates hnRNP I/PTB and p54nrb/NonO. Interestingly, hnRNP I/PTB and p54nrb/NonO in such subtype of GITERA were revealed in long threads. The resemblance of these threads to amyloid-like fibers is discussed. Our data suggest that transcription of subtelomeric sequences serves as a seeding event for accumulation of non-nascent RNA and associated RNP proteins. Such accumulation leads to GITERA formation in terminal chromosomal regions in avian oocyte nucleus. 3'-processed transcripts derived from other chromosomal loci may be attracted to GITERA by binding to the same RNP proteins or to their interaction partners.

PABPN1: molecular function and muscle disease.

The polyadenosine RNA binding protein polyadenylate-binding nuclear protein 1 (PABPN1) plays key roles in post-transcriptional processing of RNA. Although PABPN1 is ubiquitously expressed and presumably contributes to control of gene expression in all tissues, mutation of the PABPN1 gene causes the disease oculopharyngeal muscular dystrophy (OPMD), in which a limited set of skeletal muscles are affected. A major goal in the field of OPMD research is to understand why mutation of a ubiquitously expressed gene leads to a muscle-specific disease. PABPN1 plays a well-documented role in controlling the poly(A) tail length of RNA transcripts but new functions are emerging through studies that exploit a variety of unbiased screens as well as model organisms. This review addresses (a) the molecular function of PABPN1 incorporating recent findings that reveal novel cellular functions for PABPN1 and (b) the approaches that are being used to understand the molecular defects that stem from expression of mutant PABPN1. The long-term goal in this field of research is to understand the key molecular functions of PABPN1 in muscle as well as the mechanisms that underlie the pathological consequences of mutant PABPN1. Armed with this information, researchers can seek to develop therapeutic approaches to enhance the quality of life for patients afflicted with OPMD.

Poly A RNA status and expression of milli (m) and micro (µ) calpains in skeletal muscle of duchenne muscular dystrophy patients.

Poly A RNA status and itsin vitro translation in a rabbit reticulolysate cell free system, were assessed in the skeletal muscle of young boys with Duchenne muscular dystrophy. Compared to normals there is a significant 48% increase in poly A RNA content of dystrophic muscle and its translatability was increased by 56% based on(35)S methionine incorporation into total protein systhesised. Immunoprecipitation of the translated products with monospecific antibodies showed that there is a 2.6 fold and a 2 fold increase in m and µ calpains respectively. This underlines the importance of both synthetic and degenerative activities in the early pathology in DMD muscle.

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos.

spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)+ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)+ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)+ RNA. Mosaic females containing spz germ cells surrounded by spz + tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line.

Storage of embryonically transcribed poly(A) RNA and its utilization during metamorphosis of the hydroidHydractinia echinata.

The formation of tentacles and stolons during metamorphosis is severely disturbed if inhibitors of mRNA metabolism are applied during certain phases of development. The periods of sensitivity to α-amanitin are late gastrulation and the disk stage of metamorphosis. A cordycepin sensitive phase exists during the first hour of metamorphosis. In all drug sensitive phases an enhanced poly(A) synthesis is found indicating increased mRNA metabolism in these stages. Pulse-chase experiments show that planula larvae store a poly(A)-rich RNA population sedimenting between 28-18s. These long living molecules are of embryonic origin, are located in RNP particles and are degraded during metamorphosis. The particles in question appear to be stored mainly in interstitial cells. In early metamorphosis no uridine is incorporated but labelled poly(A) is added to preexisting molecules.

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.).

Ovaries ofC. erythrocephala synthesize large amounts of poly(A)+ and poly(A)- RNA during early and middle stages of oogenesis as shown by labelling with3H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)+ RNA and the poly(A)+ RNA present in sufficient amounts for optical density measurements (steady state poly(A)+ RNA) was observed. During early and mid-oogenesis, in the poly(A)- RNA fraction, ≧4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)+ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)+ RNA were not obvious. However, different sizes of labelled poly(A)+ RNA species were detected in 0-2h old preblastoderm embryos, after injection of3H-uridine into females either 3-4 days (stage 3-4 of oogenesis) or 24 h before oviposition (stage 5-6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)+ RNA fraction represents about 2-3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)+ RNA varied from about 30 to 200 nucleotides.

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis.

Nuclear poly(A)+ and polysomal poly(A)+ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)+ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)+ RNA hybridization reactions suggest the existence of polysomal poly(A)+ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)- RNA it could be shown that most of the poly(A)+ containing RNA sequences transcribed into cDNA were also present within the poly(A)- RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)- polysomal RNA and 3-6 more abundant within the poly(A)- nuclear RNA as compared to the poly(A)+ RNAs.