Polyadenylation Signal Research Articles

Comprehensive RNP profiling in cells identifies U1 snRNP complexes with cleavage and polyadenylation factors active in telescripting.

Full-length transcription in the majority of protein-coding and other genes transcribed by RNA polymerase II in complex eukaryotes requires U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3'-end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs). This U1 activity, termed telescripting, requires U1 to base-pair with the nascent RNA and inhibit usage of a downstream PAS. Here we describe experimental methods to determine the mechanism of U1 telescripting, involving mapping of U1 and CPA factors (CPAFs) binding locations in relation to PCPA sites, and identify U1 and CPAFs interactomes. The methods which utilizes rapid reversible protein-RNA and protein-protein chemical crosslinking, immunoprecipitations (XLIPs) of components of interest, and RNA-seq and quantitative proteomic mass spectrometry, captured U1-CPAFs complexes in cells, providing important insights into telescripting mechanism. XLIP profiling can be used for comprehensive molecular definition of diverse RNPs.

Gene Editing Targeting the DUX4 Polyadenylation Signal: A Therapy for FSHD?

Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.

Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes

Facioscapulohumeral muscular dystrophy (FSHD) arises from epigenetic changes that de-repress the DUX4 gene in muscle. The full-length DUX4 protein causes cell death and muscle toxicity, and therefore we hypothesize that FSHD therapies should center on inhibiting full-length DUX4 expression. In this study, we developed a strategy to accomplish DUX4 inhibition using U7-small nuclear RNA (snRNA) antisense expression cassettes (called U7-asDUX4). These non-coding RNAs were designed to inhibit production or maturation of the full-length DUX4 pre-mRNA by masking the DUX4 start codon, splice sites, or polyadenylation signal. In so doing, U7-asDUX4 snRNAs operate similarly to antisense oligonucleotides. However, in contrast to oligonucleotides, which are limited by poor uptake in muscle and a requirement for lifelong repeated dosing, U7-asDUX4 snRNAs can be packaged within myotropic gene therapy vectors and may require only a single administration when delivered to post-mitotic cells in vivo. We tested several U7-asDUX4s that reduced DUX4 expression in vitro and improved DUX4-associated outcomes. Inhibition of DUX4 expression via U7-snRNAs could be a new prospective gene therapy approach for FSHD or be used in combination with other strategies, like RNAi therapy, to maximize DUX4 silencing in individuals with FSHD.

EXTH-26. SECONDARY STRUCTURE ANALYSIS OF THE EGFR PRE-mRNA TRANSCRIPT TO IDENTIFY TARGETABLE REGIONS AND OPTIMIZE RNA THERAPY

Abstract Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12–15 months. Current strategies are often limited by the blood-brain barrier. This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, targeting cancer-driving genes. Epidermal growth factor receptor (EGFR) is dysregulated in 57% of all GBM. Our approach uses an adeno-associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre-mRNA transcript. The ‘pre-mRNA structurome’ can be used to uncover and determine the accessibility of targetable regions. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of EGFR and activate the expression of a stable therapeutic isoform of EGFR. To advance our therapeutic strategy, we have analyzed the EGFR secondary structure using selective 2’ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE-MaP). SHAPE-MaP reactivity profiles were generated revealing the structure of splicing and cryptic polyadenylation signal (PAS) elements within the targeted region. We identified enhancer binding motifs surrounding the 5’ splice site and hidden elements of the cryptic PAS. Based on these structural profiles, we generated RNA therapies to unravel the hidden PAS to activate expression of the short therapeutic isoform.

Aptamer-Mediated Control of Polyadenylation for Gene Expression Regulation in Mammalian Cells.

Small aptamer-based regulatory devices can be designed to control a range of RNA-dependent cellular processes and emerged as promising tools for fine-tuning gene expression in synthetic biology. Here, we design a conceptually new riboswitch device that allows for the conditional regulation of polyadenylation. By making use of ligand-induced sequence occlusion, the system efficiently controls the accessibility of the eukaryotic polyadenylation signal. Undesirable 3'-extended read-through products are counteracted by the downstream insertion of a microRNA target site. We demonstrate the modularity of the system with regard to sensor aptamers and polyadenylation signals used and combine the newly designed riboswitch with well-known aptazymes to yield superior composite systems. In addition, we show that the switches can be used to control alternative polyadenylation. The presented genetic switches require very little coding space and can be easily optimized by rational adjustments of the thermodynamic stability. The polyadenylation riboswitch extends the repertoire of RNA-based regulators and opens new possibilities for the generation of complex synthetic circuits.

Transcription Terminator-Mediated Enhancement in Transgene Expression in Maize: Preponderance of the AUGAAU Motif Overlapping With Poly(A) Signals.

The selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of Brachypodium distachyon, Setaria italica, and Zea mays. Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of Arabidopsis. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3′-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and Arabidopsis. The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and Arabidopsis gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3′ end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene expression. Research on TTs is not limited to transgene expression but could be extended to the introduction of appropriate mutations into TTs via genome editing, paving the way for expression modulation of endogenous genes.

First Report of Coinfection of Satellite and Defective RNA of Bamboo mosaic virus in the Bamboo Host in California, U.S.A.

Bamboo mosaic potexvirus (BaMV) was first reported to infect bamboo in Brazil in 1977, and the virus was later identified in Taiwan, the United States, mainland China, India, Australia, and Indonesia (Abe et al. 2019; Elliott and Zettler 1997; M. T. Lin et al. 1977; N. S. Lin et al. 1993, 1995; W. W. Lin et al. 2015). Three leaf samples of 20 plants of Beechey bamboo (Bambusa beecheyana) showing mosaic and yellowish streak symptoms were collected in Palomar College Arboretum (San Marcos, CA) in 2019. A virus was isolated by cesium chloride density gradient (Lin and Chen 1991), and sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified virions showed one major 30-kDa protein. Viral RNAs were extracted from virions, and cDNAs were synthesized using oligo(dT)15 and random primers with AMV and M-MLV RNase H− reverse transcriptase (Promega), respectively. The fragments of genomic and satellite RNA of BaMV were amplified by polymerase chain reaction (PCR) using Q5 high-fidelity DNA polymerase (NEB). The PCR primers and virus characterization are further described in supplementary materials, and amplicons were Sanger sequenced. The viral sequences of three BaMV-Yoshi clones (MT111578, MT111579, and MT111580) were aligned and compared using MAFFT and Phylo.io. The full genomic nucleotide sequence of BaMV-Yoshi shares 79.0 to 80.3% identity with other BaMV isolates in GenBank, whereas amino acids of BaMV-Yoshi ORF4 are only 71.2 to 78.9% identical to others. Phylogenetic analysis revealed that BaMV-Yoshi diverged from a common ancestor earlier than all Asian isolates and became a distinct group by itself (W. W. Lin et al. 2017; Wang et al. 2017). Six clones of BaMV satellite (satBaMV-Y; MT111572 to MT111577) RNAs show 93.2 to 95.1% identity to other satBaMV isolates in GenBank, and they contain noncanonical polyadenylation signal AAUAAU or AAGAAU instead of AAUAAA conserved in satBaMV isolates from Taiwan, China, or San Diego (CA). Northern blotting using ³²P-labeled BaMV 5′ untranslated region-specific riboprobe also detected 1.2- and 0.9-kb RNA species in virions, indicating that they are encapsidated defective RNAs (D RNAs). Two BaMV D RNAs (Y1 and Y2; MT113945 and MT113946) consist of 1,162 and 845 nucleotides in length with a single internal deletion of the helper virus genome, but their RNA recombination sites (Y1, 525/5,730; Y2, 185/5,709) are different from D RNAs generated by serial passages of BaMV-S in tobacco (Yeh et al. 1999). To our knowledge, this is the first report of full-length BaMV genome information outside Asia, and new recombination hotspots of BaMV D RNAs occurring in the bamboo host. This is also the first mixed infection of two BaMV subviral RNAs identified in nature. Contribution of mixed infection of satBaMV and D RNAs to replication competence and evolution of the helper virus warrants further investigation.

Animal-APAdb: a comprehensive animal alternative polyadenylation database.

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism that recognizes different polyadenylation signals on transcripts, resulting in transcripts with different lengths of 3′ untranslated regions and thereby influencing a series of biological processes. Recent studies have highlighted the important roles of APA in human. However, APA profiles in other animals have not been fully recognized, and there is no database that provides comprehensive APA information for other animals except human. Here, by using the RNA sequencing data collected from public databases, we systematically characterized the APA profiles in 9244 samples of 18 species. In total, we identified 342 952 APA events with a median of 17 020 per species using the DaPars2 algorithm, and 315 691 APA events with a median of 17 953 per species using the QAPA algorithm in these 18 species, respectively. In addition, we predicted the polyadenylation sites (PAS) and motifs near PAS of these species. We further developed Animal-APAdb, a user-friendly database (http://gong_lab.hzau.edu.cn/Animal-APAdb/) for data searching, browsing and downloading. With comprehensive information of APA events in different tissues of different species, Animal-APAdb may greatly facilitate the exploration of animal APA patterns and novel mechanisms, gene expression regulation and APA evolution across tissues and species.

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3′ and 5′ ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.

Systematic identification of functional SNPs interrupting 3'UTR polyadenylation signals.

Alternative polyadenylation (APA) is emerging as a widespread regulatory layer since the majority of human protein-coding genes contain several polyadenylation (p(A)) sites in their 3'UTRs. By generating isoforms with different 3'UTR length, APA potentially affects mRNA stability, translation efficiency, nuclear export, and cellular localization. Polyadenylation sites are regulated by adjacent RNA cis-regulatory elements, the principals among them are the polyadenylation signal (PAS) AAUAAA and its main variant AUUAAA, typically located ~20-nt upstream of the p(A) site. Mutations in PAS and other auxiliary poly(A) cis-elements in the 3'UTR of several genes have been shown to cause human Mendelian diseases, and to date, only a few common SNPs that regulate APA were associated with complex diseases. Here, we systematically searched for SNPs that affect gene expression and human traits by modulation of 3'UTR APA. First, focusing on the variants most likely to exert the strongest effect, we identified 2,305 SNPs that interrupt the canonical PAS or its main variant. Implementing pA-QTL tests using GTEx RNA-seq data, we identified 330 PAS SNPs (called PAS pA-QTLs) that were significantly associated with the usage of their p(A) site. As expected, PAS-interrupting alleles were mostly linked with decreased cleavage at their p(A) site and the consequential 3'UTR lengthening. However, interestingly, in ~10% of the cases, the PAS-interrupting allele was associated with increased usage of an upstream p(A) site and 3'UTR shortening. As an indication of the functional effects of these PAS pA-QTLs on gene expression and complex human traits, we observed for few dozens of them marked colocalization with eQTL and/or GWAS signals. The PAS-interrupting alleles linked with 3'UTR lengthening were also strongly associated with decreased gene expression, indicating that shorter isoforms generated by APA are generally more stable than longer ones. Last, we carried out an extended, genome-wide analysis of 3'UTR variants and detected thousands of additional pA-QTLs having weaker effects compared to the PAS pA-QTLs.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Cloning and tissue distribution of the ATP-binding cassette subfamily G member 2 gene in the marine pufferfish Takifugu rubripes

In this study, we cloned the ATP-binding cassette (ABC) subfamily G member 2 gene (Abcg2 gene; breast cancer resistance protein) from the liver of the marine pufferfish Takifugu rubripes, and examined its tissue distribution and gene structure. The full-length complementary DNA (cDNA) was 1977 base pairs (bp), comprising 15 exons in chromosome 17, and was assigned to the teleost Abcg2b paralogue. The open reading frame was 1839 bp in length, and encodes 612 amino acids constituting Walker A, Q-loop, C motif, Walker B, D-loop, and H-loop sequences in the first half and six transmembrane domains in the second half. Gene expression of Abcg2 was predominantly found in the intestines, followed by the liver and kidneys. The Kozak motif and the polyadenylation signal sequences were at respective ends of the transcript. The putative core promoter sequences including the transcription factor II B recognition elements, the TATA box, the initiator element, and the downstream core promoter element were found in the transcript and its upstream genomic DNA sequence. The hypoxia response element and the estrogen response element were located in the upstream genomic DNA sequence, suggesting that the pufferfish Abcg2 gene possesses the same regulation mechanism of transcription as the human ABCG2 gene.

A Generative Neural Network for Maximizing Fitness and Diversity of Synthetic DNA and Protein Sequences.

SUMMARYEngineering gene and protein sequences with defined functional properties is a major goal of synthetic biology. Deep neural network models, together with gradient ascent-style optimization, show promise for sequence design. The generated sequences can however get stuck in local minima and often have low diversity. Here, we develop deep exploration networks (DENs), a class of activation-maximizing generative models, which minimize the cost of a neural network fitness predictor by gradient descent. By penalizing any two generated patterns on the basis of a similarity metric, DENs explicitly maximize sequence diversity. To avoid drifting into low-confidence regions of the predictor, we incorporate variational autoencoders to maintain the likelihood ratio of generated sequences. Using DENs, we engineered polyadenylation signals with more than 10-fold higher selection odds than the best gradient ascent-generated patterns, identified splice regulatory sequences predicted to result in highly differential splicing between cell lines, and improved on state-of-the-art results for protein design tasks.

Identification and characterization of cystatin B from black rockfish, Sebastes schlegelii, indicating its potent immunological importance

Cystatins represent a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant-derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. The cystatin superfamily can be further classified into three groups: stefins, cystatins, and kininogens. Among these, cystatin B is categorized under stefins. Cystatin B lacks a signal sequence, disulfide bonds, and carbohydrate groups. However, it contains the conserved cystatin family signature, including a single cystatin-like domain, cysteine protease inhibitory signature concealing pentapeptide (QXVXG) consensus sequence, and two conserved neighboring glycine (8GG9) residues at the N-terminal. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full-length cDNA of RfCytB was 573 bp long, with a coding region of 294 bp. The 5′-untranslated region (UTR) comprised 55 bp, and the 263-bp-long 3′-UTR included a polyadenylation signal sequence and a poly-A tail. The coding sequence encodes a polypeptide comprising 97 amino acids, with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. RfCytB shared homology features with similar molecules from other teleost and vertebrate species, and was clustered with Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals, with the highest levels of expression observed in gill and spleen. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCytB showed a concentration-dependent inhibitory activity towards papain, with a high thermal stability. Transient expression of RfCytB in LPS activated murine macrophages, thereby inducing the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α. These results provide evidence for its protease inhibitory and immunity relevant roles in hosts.

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription.

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.

SAT-360 Out of Sight, out of Mind: PHEX 3’-UTR C.*231A>G X-Linked Hypophosphatemia in Adults: A Case Study of One Family Pedigree with a Widely Variable Phenotype

Title: Out of Sight, Out of Mind: PHEX 3’-UTR c.*231A>G X-Linked Hypophosphatemia in Adults: A Case Study of One Family Pedigree with a Widely Variable PhenotypeX-linked hypophosphatemia (XLH) is an inherited form of hypophosphatemia that is part of a group of disorders that leads to impaired bone mineralization, which can manifest as rickets in children and osteomalacia in adults. Mutations in PHEX, DMP1, ENPP1, and activating mutations in FGF23 have each been shown as genetic causes for XLH. PHEX is a zinc metallopeptidase which reduces expression of FGF23 through mechanisms that are not fully understood. Inactivating mutations in the PHEX gene cause elevated levels of FGF23, resulting in renal phosphate-wasting, impaired conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D and impaired bone mineralization with osteomalacia. Though the clinical significance of FGF23 levels is not clearly understood, it has also been shown that FG23 increases with age.XLH has a reported prevalence of 3.9–5 per 100,000. We speculate that this may be under recognized and that diagnosis may be delayed for many years in those with mild forms of XLH due to non-specific symptoms. Diagnosis may be complicated by normal or low-normal serum phosphorus levels. Adults may be mistakenly diagnosed with ankylosing spondylitis, polyarthritis, osteoarthritis, diffuse idiopathic skeletal hyperostosis and other disorders that cause stiffness. Some clinical manifestations of XLH in adults include dental disease, skeletal and spinal disease, sensorineural hearing loss, kidney stones, renal impairment, and hyperparathyroidism. The PHEX 3’-UTR c.231A>G near the polyadenylation signal has been previously described to cause milder severity in men and seemingly unaffecting women, but data for comparison is limited.We present a large, multigenerational family with a mutation in the PHEX gene (3’-UTR c.*231A>G) demonstrating variable penetrance in the pedigree. Of the members positive for the mutation, the most common symptoms were sensorineural hearing loss, enthesopathy, and dental disease. Most had normal or low-normal phosphorus levels in both genders. Two patients had prior diagnoses of ankylosing spondylitis/ seronegative spondyloarthropathy. In general, males with the mutation had more severe symptoms than females. However, females with a severe phenotype and low phosphorus was also observed. The disease burden was cumulative over time and correlated with serum phosphorus levels. Musculoskeletal symptoms were increased in the members over the age of 60.Sources:Chesher, Douglas et al. Outcome of Adult Patients with X-Linked Hypophosphatemia Caused by PHEX Gene Mutations. Vol. 41. Dordrecht: Springer Netherlands, 2018. Web.Zhang, Cong et al. Clinical and Genetic Analysis in a Large Chinese Cohort of Patients with X-Linked Hypophosphatemia. Vol. 121. Elsevier Inc, 2019. Web.

The Spectrum of α-Thalassemia Mutations in Kurdistan Province, West Iran

In order to identify the α-thalassemia (α-thal) mutation spectrum in Kurdistan Province, West Iran, a total of 217 individuals, including 154 α-thal carriers and 63 normal subjects were investigated in this study. Molecular analysis of α1- and α2-globin genes using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR or direct DNA sequencing, showed 11 different α-globin variants. The –α3.7 (rightward) deletion (NG_000006.1: g.34164_37967del3804) (70.32%), polyadenylation signal (polyA2) site (AATAAA>AATGAA) (αpolyA2α) (HBA2: c.*92A>G) (7.74%), –α4.2 (leftward) deletion (6.45%) and codon 59 (or Hb Adana) (G>A) (ααcodon 59) (HBA1: c.179G>A) (4.52%) were the most frequent mutations in the present study. In conclusion, the spectrum of α-thal mutations in Kurdistan Province is closest to that in western provinces of Iran (Kurdish and Laki populations). In addition, it was revealed that the codon 59 mutation is common in the Kurdish population. On the other hand, despite the same ethnic background of Kurds in Iran and Iraq, the – –MED I double gene deletion and polyA2 point mutation have different distributions in these two populations. Therefore, further studies are needed to identify the cause of these differences.

TRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly.

RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein-RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein-RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein-RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.

Rapid Generation of Attenuated Infectious Bursal Disease Virus from Dual-Promoter Plasmids by Reduction of Viral Ribonucleoprotein Activity.

Infectious bursal disease virus (IBDV) of the Birnaviridae family leads to immunosuppression of young chickens by destroying B cells in the bursa of Fabricius (BFs). Given the increasing number of variant IBDV strains, we urgently require a method to produce attenuated virus for vaccine development. To accomplish this goal, the dual-promoter plasmids in which the RNA polymerase II and RNA polymerase I (Pol I) promoters were placed upstream of the IBDV genomic sequence, which was followed by mouse Pol I terminator and a synthetic polyadenylation signal, were developed for rapid generation of IBDV. This approach did not require trans-supplementation of plasmids for the expression of VP1 and VP3, the main components of IBDV ribonucleoprotein (RNP). Based on the finding in this study that the IBDV RNP activity was partially retained by VP1-FLAG, we successfully rescued the replication-competent IBDV/1FLAG expressing VP1-FLAG. Compared with its parental counterpart, IBDV/1FLAG formed smaller size plaques in cultured cells and induced the same 100% immune protection in vivo However, neither retarded development nor severe BFs lesion was observed in the IBDV/1FLAG-inoculated chickens. Collectively, this is the first report that viral RNP activity was affected by the addition of an epitope tag on the componential viral proteins. Furthermore, this work demonstrates the rapid generation of attenuated IBDV from dual-promoter plasmids via reducing viral RNP activity by a fused FLAG tag on the C terminus of VP1. This would be a convenient strategy to attenuate epidemic variant IBDV strains for rapid and efficient vaccine development.IMPORTANCE Immunosuppression in chickens as a result of infectious bursal disease virus (IBDV) infection leads to significant economic losses in the poultry industry worldwide every year. Currently, vaccination is still the best way to prevent the prevalence of IBDV. However, with the occurrence of increasing numbers of variant IBDV strains, it is challenging to develop antigen-matched live attenuated vaccine. Here, we first developed a dual-promoter reverse-genetic system for the rapid generation of IBDV. Using this system, the attenuated IBDV/1FLAG expressing VP1-FLAG, which displays the decreased viral RNP activity, was rescued. Moreover, IBDV/1FLAG inoculation induced a similar level of neutralizing antibodies to that of its parental counterpart, protecting chickens against lethal challenge. Our study, for the first time, describes a dual-promoter reverse-genetic approach for the rapid generation of attenuated IBDV while maintaining entire parental antigenicity, suggesting a potential new method to attenuate epidemic variant IBDV strains for vaccine development.

U1 snRNP regulates cancer cell migration and invasion in vitro

Stimulated cells and cancer cells have widespread shortening of mRNA 3’-untranslated regions (3’UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates’ most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells’ migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3’UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.