Polyadenylation Signal Research Articles

How to make the end of a gene, the simple way

Transcription termination of nearly all protein-coding genes in mammals requires 3’ end processing by a multiprotein complex that will cleave and polyadenylate the messenger RNA precursor. Because a variety of enzyme complexes intervene, 3’ end processing was thought to be fundamentally complex and subject to a multitude of regulatory effects. The possibility to select just one out of several polyadenylation sites, in particular, has caused much questioning and speculation. What appear to be separate mechanisms however can be combined into a defined set of rules, allowing for a relatively simple interpretation of 3’ end processing. Ultimately, readiness of the terminal exon splice site determines when a transcript reaches the maturity to select a nearby polyadenylation signal. Transcriptional pausing then acts in concert, extending the timeframe during which the transcription complex is close to polyadenylation sites. Since RNA polymerase pausing is governed by the same type of sequences in bacteria and metazoans, mammalian transcription termination resembles its prokaryote counterpart more than generally thought.

High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis.

Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.

Molecular Characterization of Small Ruminant Lentiviruses in Polish Mixed Flocks Supports Evidence of Cross Species Transmission, Dual Infection, a Recombination Event, and Reveals the Existence of New Subtypes within Group A.

Small ruminant lentiviruses (SRLVs) are a group of highly divergent viruses responsible for global infection in sheep and goats. In a previous study we showed that SRLV strains found in mixed flocks in Poland belonged to subtype A13 and A18, but this study was restricted only to the few flocks from Małopolska region. The present work aimed at extending earlier findings with the analysis of SRLVs in mixed flocks including larger numbers of animals and flocks from different part of Poland. On the basis of gag and env sequences, Polish SRLVs were assigned to the subtypes B2, A5, A12, and A17. Furthermore, the existence of a new subtypes, tentatively designed as A23 and A24, were described for the first time. Subtypes A5 and A17 were only found in goats, subtype A24 has been detected only in sheep while subtypes A12, A23, and B2 have been found in both sheep and goats. Co-infection with strains belonging to different subtypes was evidenced in three sheep and two goats originating from two flocks. Furthermore, three putative recombination events were identified within gag and env SRLVs sequences derived from three sheep. Amino acid (aa) sequences of immunodominant epitopes in CA protein were well conserved while Major Homology Region (MHR) had more alteration showing unique mutations in sequences of subtypes A5 and A17. In contrast, aa sequences of surface glycoprotein exhibited higher variability confirming type-specific variation in the SU5 epitope. The number of potential N-linked glycosylation sites (PNGS) ranged from 3 to 6 in respective sequences and were located in different positions. The analysis of LTR sequences revealed that sequences corresponding to the TATA box, AP-4, AML-vis, and polyadenylation signal (poly A) were quite conserved, while considerable alteration was observed in AP-1 sites. Interestingly, our results revealed that all sequences belonging to subtype A17 had unique substitution T to A in the fifth position of TATA box and did not have a 11 nt deletion in the R region which was noted in other sequences from Poland. These data revealed a complex picture of SRLVs population with ovine and caprine strains belonging to group A and B. We present strong and multiple evidence of dually infected sheep and goats in mixed flocks and present evidence that these viruses can recombine in vivo.

Generation of duck Tembusu virus using a simple reverse genetic system in duck embryo fibroblast cells

Outbreaks of duck Tembusu virus (DTMUV) have caused serious economic losses in China since 2010. In this study, an infectious clone of the DTMUV BZ-2010strain, isolated from layer cherry duck in China, was constructed using the bacterium-free infectious subgenomic-amplicons method. The subgenomic-amplicons of the human cytomegalovirus promoter (pCMV) at the 5′ terminus of the first DNA fragment, the entire genome of DTMUV, and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) at the 3′ terminus of the last DNA fragment were synthesized and amplified by PCR in three DNA fragments. The pCMV and HDR/SV40pA were used to drive the viral RNA transcription and generate a full-length RNA transcript of the virus, and were found to be effective in reassembling DTMUV in duck embryo fibroblast cells. The RNA transcripts from the infection clone were infectious in duck embryo fibroblast cells, generating the reconstituted DTMUV. This study provided a valuable reverse genetic tool for the further study DTMUV pathogenesis.

Sequence Diversity, Locus Structure, and Evolutionary History of the SpTransformer Genes in the Sea Urchin Genome.

The generation of large immune gene families is often driven by evolutionary pressure exerted on host genomes by their pathogens, which has been described as the immunological arms race. The SpTransformer (SpTrf) gene family from the California purple sea urchin, Strongylocentrotus purpuratus, is upregulated upon immune challenge and encodes the SpTrf proteins that interact with pathogens during an immune response. Native SpTrf proteins bind both bacteria and yeast, and augment phagocytosis of a marine Vibrio, while a recombinant SpTrf protein (rSpTrf-E1) binds a subset of pathogens and a range of pathogen associated molecular patterns. In the sequenced sea urchin genome, there are four SpTrf gene clusters for a total of 17 genes. Here, we report an in-depth analysis of these genes to understand the sequence complexities of this family, its genomic structure, and to derive a putative evolutionary history for the formation of the gene clusters. We report a detailed characterization of gene structure including the intron type and UTRs with conserved transcriptional start sites, the start codon and multiple stop codons, and locations of polyadenylation signals. Phylogenetic and percent mismatch analyses of the genes and the intergenic regions allowed us to predict the last common ancestral SpTrf gene and a theoretical evolutionary history of the gene family. The appearance of the gene clusters from the theoretical ancestral gene may have been driven by multiple duplication and deletion events of regions containing SpTrf genes. Duplications and ectopic insertion events, indels, and point mutations in the exons likely resulted in the extant genes and family structure. This theoretical evolutionary history is consistent with the involvement of these genes in the arms race in responses to pathogens and suggests that the diversification of these genes and their encoded proteins have been selected for based on the survival benefits of pathogen binding and host protection.

Targeting IgE polyadenylation signal with antisense oligonucleotides decreases IgE secretion and plasma cell viability

Allergy regroups numerous complex and various diseases classified as IgE-dependent or non-IgE-dependent hypersensitivities. IgEs are expressed as membrane and secreted forms by B cells and plasma cells, respectively. In IgE-mediated hypersensitivity, IgE secretion and binding to the high-affinity IgE receptor FcεRI on effector cells are responsible for the onset of allergic symptoms; in contrast, surface IgE expression as a B-cell receptor is barely detectable. Our aim was to test an innovative antisense approach to reducing IgE secretion. We designed an antisense oligonucleotide (ASO) targeting the polyadenylation signal of human secreted IgE to redirect IgE transcript polyadenylation from the secreted form to the membrane form. ASO treatments were performed on B cells from transgenic mice expressing humanized IgE (InEps mice), as well as on human primary B cells and myeloma cells. Invivo ASO delivery was tested by using an InEps mouse model. We demonstrated that treatment with a morpholino ASO targeting the secreted IgE polyadenylation signal drastically decreased IgE secretion and inversely increased membrane IgE mRNA expression. In addition, ASO treatment induced apoptosis of IgE-expressing U266 myeloma cells, and RNA sequencing revealed attenuation of their plasma cell phenotype. Remarkably, systemic administration of an ASO coupled with Pip6a as an arginine-rich cell-penetrating peptide decreased IgE secretion invivo. Altogether, this ASO strategy could be an effective way to decrease IgE secretion and allergic symptoms in patients with IgE-dependent allergies, and it could also promote allergen tolerance through apoptosis of IgE+ antibody-secreting cells.

Analysis of differential gene expression and transcript usage in hippocampus of Apoe null mutant mice: Implications for Alzheimer’s disease

A dataset of single-nucleus RNA sequencing (snRNAseq) data was analyzed using Seurat, Sierra, and Ingenuity Pathway Analysis (IPA) programs to assess differentially expressed genes (DEGs) and differential transcript usage (DTU) in mouse hippocampal cell types. Seurat identified DEGs between the wild type (WT) and Apoe knockout (EKO) mice. IPA identified 11 statistically significant canonical pathways in >1 cell type. Sierra identified Sipa1l1 with DTU between WT and EKO samples. Analysis of the Sipa1l1 peak region identified an alternative non-canonical polyadenylation signal and a putative cytoplasmic polyadenylation element. APOE regulation of gene transcription and co-transcriptional RNA processing may underlie Alzheimer’s disease.

A standard knockout procedure alters expression of adjacent loci at the translational level

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the ‘head-to-head’ orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5′ untranslated region (5′ UTR). In the ‘tail-to-tail’ arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3′ UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

Comprehensive characterization of somatic variants associated with intronic polyadenylation in human cancers.

Somatic single nucleotide variants (SNVs) in cancer genome affect gene expression through various mechanisms depending on their genomic location. While somatic SNVs near canonical splice sites have been reported to cause abnormal splicing of cancer-related genes, whether these SNVs can affect gene expression through other mechanisms remains an open question. Here, we analyzed RNA sequencing and exome data from 4,998 cancer patients covering ten cancer types and identified 152 somatic SNVs near splice sites that were associated with abnormal intronic polyadenylation (IPA). IPA-associated somatic variants favored the localization near the donor splice sites compared to the acceptor splice sites. A proportion of SNV-associated IPA events overlapped with premature cleavage and polyadenylation events triggered by U1 small nuclear ribonucleoproteins (snRNP) inhibition. GC content, intron length and polyadenylation signal were three genomic features that differentiated between SNV-associated IPA and intron retention. Notably, IPA-associated SNVs were enriched in tumor suppressor genes (TSGs), including the well-known TSGs such as PTEN and CDH1 with recurrent SNV-associated IPA events. Minigene assay confirmed that SNVs from PTEN, CDH1, VEGFA, GRHL2, CUL3 and WWC2 could lead to IPA. This work reveals that IPA acts as a novel mechanism explaining the functional consequence of somatic SNVs in human cancer.

Flies in the ointment: AAV vector preparations and tumor risk

In an article previously published in Molecular Therapy, a high incidence of cancer formation was reported in mice transduced with adeno-associated viral (AAV) vector.1 Tumors from hepatectomized mice transduced with a self-complementary AAV (scAAV) vector containing a strong enhancer/promoter (CMV/beta-actin) but lacking a transgene or polyadenylation signal (“CBA-null,” Figure 1A) were more frequently associated with vector DNA than tumors in the control group that had been transduced with a conventional scAAV vector expressing a reporter gene.

Two cases of T cell lymphoma following Piggybac-mediated CAR T cell therapy

Recently, Micklethwaite et al. 1 Micklethwaite K.P. Gowrishankar K. Gloss B.S. Li Z. Street J.A. Moezzi L. Mach M.A. Sutrave G. Clancy L.E. Bishop D.C. et al. Investigation of product derived lymphoma following infusion of piggyBac modified CD19 chimeric antigen receptor T-cells. Blood. 2021; (Published online May 11, 2021)https://doi.org/10.1182/blood.2021010858 Crossref Scopus (28) Google Scholar and Bishop et al. 2 Bishop D.C. Clancy L.E. Simms R. Burgess J. Mathew G. Moezzi L. Street, J.A., Sutrave, G., Atkins, E., McGuire, H.M. et al. Development of CAR T-cell lymphoma in two of ten patients effectively treated with piggyBac modified CD19 CAR T-cells. Blood. 2021; (Published online May 19, 2021)https://doi.org/10.1182/blood.2021010813 Crossref Scopus (25) Google Scholar reported results of a phase I first-in-human clinical trial of CD19-directed allogeneic chimeric antigen receptor (CAR)-T cells in 10 patients with relapsed or persistent B cell malignancies after matched-related allogeneic hematopoietic stem cell transplantation (HSCT). CAR-T cells were produced via electroporation with a Piggybac (PB) transposon vector, encoding a CD19 CAR (second generation, with a 4-1BB co-stimulatory domain) driven by a human elongation factor-1 alpha (EF1a) promoter, terminated by an SV40 polyadenylation signal, and flanked by chicken hypersensitivity site 4 (cHS4) β-globin insulator sequences. Clinical responses (five patients with continuous complete remission at median follow-up of 18 months) were promising and comparable to those seen with retroviral CD19-CAR vectors. Surprisingly, two patients developed donor-derived T cell lymphoma. While the exact causes are still under investigation, clinical trials with PB-based CAR-T cells were voluntarily suspended at the investigators’ research centers. The authors discuss a number of mechanisms that may have contributed to T cell transformation and have performed work to address potential underlying causes, which we put into perspective here regarding lessons learned in gene therapy trials with integrating retroviral vectors.

Alternative polyadenylation events differ dramatically between Tongcheng and Large White pigs in response to PRRSV infection.

Alternative polyadenylation (APA) is a widespread post-transcriptional regulation mechanism that increases the biological complexity of transcriptome and proteome. However, it is unclear whether APA regulation plays a role in genetic resistance to porcine reproductive and respiratory syndrome virus (PRRSV). Here, we reported genome-wide APA regulation of porcine alveolar macrophages in PRRSV-resistant Tongcheng (TC) pigs and PRRSV-susceptible Large White (LW) pigs upon PRRSV infection. Using 3' mRNA sequencing strategy, we detected 75981 high-quality APA sites in porcine alveolar macrophages of TC and LW pigs. Furthermore, 1202 and 1089 differentially expressed APA sites, as well as 79 and 117 untranslated region-APA switching genes were identified in TC pigs and LW pigs upon PRRSV infection respectively. The APA events in TC pigs and LW pigs were involved in different biological pathways, while APA events in TC pigs are directly associated with the immune response to PRRSV infection. In addition, we identified genetic variations affecting polyadenylation signal between TC pigs and LW pigs. These findings would provide helpful information on APA regulation for further understanding of genetic resistance to PRRSV.

QuantifyPoly(A): reshaping alternative polyadenylation landscapes of eukaryotes with weighted density peak clustering.

The dynamic choice of different polyadenylation sites in a gene is referred to as alternative polyadenylation, which functions in many important biological processes. Large-scale messenger RNA 3' end sequencing has revealed that cleavage sites for polyadenylation are presented with microheterogeneity. To date, the conventional determination of polyadenylation site clusters is subjective and arbitrary, leading to inaccurate annotations. Here, we present a weighted density peak clustering method, QuantifyPoly(A), to accurately quantify genome-wide polyadenylation choices. Applying QuantifyPoly(A) on published 3' end sequencing datasets from both animals and plants, their polyadenylation profiles are reshaped into myriads of novel polyadenylation site clusters. Most of these novel polyadenylation site clusters show significantly dynamic usage across different biological samples or associate with binding sites of trans-acting factors. Upstream sequences of these clusters are enriched with polyadenylation signals UGUA, UAAA and/or AAUAAA in a species-dependent manner. Polyadenylation site clusters also exhibit species specificity, while plants ones generally show higher microheterogeneity than that of animals. QuantifyPoly(A) is broadly applicable to any types of 3' end sequencing data and species for accurate quantification and construction of the complex and dynamic polyadenylation landscape and enables us to decode alternative polyadenylation events invisible to conventional methods at a much higher resolution.

Efficient translation of Eggplant mottled dwarf nucleorhabdovirus N and X genes requires both 5\u2032 and 3\u2032 UTRs

BackgroundCircularization of RNA mediated by association of translation factors and RNA elements in 5′ and 3′ UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses.MethodsThe role of 5′ and 3′ UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing.ResultsWe have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5′ and 3′ UTRs. Luciferase reporter constructs containing precise 5′ and 3′ UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5′ or 3′ UTR. The 3′UTR of carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3′ UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5′ cap was higher than the capped construct. No RNA-RNA interactions between 5′ and 3′ UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3′ terminus negated the synergistic activity of the 3′UTR.ConclusionsThe results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3′UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.

CPSF4 regulates circRNA formation and microRNA mediated gene silencing in hepatocellular carcinoma.

CircRNAs play essential roles in various physiological processes and involves in many diseases, in particular cancer. Global downregulation of circRNA expression has been observed in hepatocellular carcinoma (HCC) in many studies. Previous studies revealed that the pre-mRNA 3' end processing complex participates in circRNA cyclization and plays an important role in HCC tumorigenesis. Therefore, we explored the role of CPSF4, for 3' end formation and cleavage, in circRNA formation. Clinical research has shown that CPSF4 expression is upregulated in HCC and that high expression of CPSF4 is associated with poor prognosis in HCC patients. Mechanistic studies have demonstrated that CPSF4 reduces the levels of circRNAs, which possess a polyadenylation signal sequence and this decrease in circRNAs reduces the accumulation of miRNA and disrupts the miRNA-mediated gene silencing in HCC. Experiments in cell culture and xenograft mouse models showed that CPSF4 promotes the proliferation of HCC cells and enhances tumorigenicity. Moreover, CPSF4 antagonizes the tumor suppressor effect of its downstream circRNA in HCC. In summary, CPSF4 acts as an oncogene in HCC through circRNA inhibition and disruption of miRNA-mediated gene silencing.

Adenine base editing of the DUX4 polyadenylation signal for targeted genetic therapy in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation of the D4Z4 repeat resulting in misexpression of the D4Z4-encoded DUX4 gene in skeletal muscle. One of the key genetic requirements for the stable production of full-length DUX4 mRNA in skeletal muscle is a functional polyadenylation signal (ATTAAA) in exon three of DUX4 that is used in somatic cells. Base editors hold great promise to treat DNA lesions underlying genetic diseases through their ability to carry out specific and rapid nucleotide mutagenesis even in postmitotic cells such as skeletal muscle. In this study, we present a simple and straightforward strategy for mutagenesis of the somatic DUX4 polyadenylation signal by adenine base editing in immortalized myoblasts derived from independent FSHD-affected individuals. We show that mutating this critical cis-regulatory element results in downregulation of DUX4 mRNA and its direct transcriptional target genes. Our findings identify the somatic DUX4 polyadenylation signal as a therapeutic target and represent the first step toward clinical application of the CRISPR-Cas9 base editing platform for FSHD gene therapy.

A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758)

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5′ untranslated region (UTR) of 112 bp and 3′ UTR of 165 bp. The 5′ UTR and 3′ UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3’ loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.