GRINL1A Complex Transcription Unit Containing GCOM1, MYZAP, and POLR2M Genes Associates with Fully Penetrant Recessive Dilated Cardiomyopathy

Background: Familial dilated cardiomyopathy (DCM) is a monogenic disorder typically inherited in an autosomal dominant pattern. We have identified two Finnish families with familial cardiomyopathy that is not explained by a variant in any previously known cardiomyopathy gene. We describe the cardiac phenotype related to homozygous truncating GCOM1 variants.Methods and Results: This study included two probands and their relatives. All the participants are of Finnish ethnicity. Whole-exome sequencing was used to test the probands; bi-directional Sanger sequencing was used to identify the GCOM1 variants in probands’ family members. Clinical evaluation was performed, medical records and death certificates were obtained. Immunohistochemical analysis of myocardial samples was conducted. A homozygous GCOM1 variant was identified altogether in six individuals, all considered to be affected. None of the nine heterozygous family members fulfilled any cardiomyopathy criteria. Heart failure was the leading clinical feature, and the patients may have had a tendency for atrial arrhythmias.Conclusions: This study demonstrates the significance of GCOM1 variants as a cause of human cardiomyopathy and highlights the importance of searching for new candidate genes when targeted gene panels do not yield a positive outcome.

Downregulation of Cell Surface Major Histocompatibility Complex Class I Expression Is Mediated by the Left-End Transcription Unit of Fowl Adenovirus 9

Major histocompatibility complex class I (MHC-I) molecules play a critical role in the host’s antiviral response by presenting virus-derived antigenic peptides to cytotoxic T lymphocytes (CTLs), enabling the clearance of virus-infected cells. Human adenoviruses evade CTL-mediated cell lysis, in part, by interfering directly with the MHC-I antigen presentation pathway through the expression of E3-19K, which binds both MHC-I and the transporter associated with antigen processing protein and sequestering MHC-I within the endoplasmic reticulum. Fowl adenoviruses have no homologues of E3-19K. Here, we show that representative virus isolates of the species Fowl aviadenovirus C, Fowl aviadenovirus D, and Fowl aviadenovirus E downregulate the cell surface expression of MHC-I in chicken hepatoma cells, resulting in 71%, 11%, and 14% of the baseline expression level, respectively, at 12 h post-infection. Furthermore, this work reports that FAdV-9 downregulates cell surface MHC-I through a minimum of two separate mechanisms—a lysosomal-independent mechanism that requires the presence of the fowl adenovirus early 1 (FE1) transcription unit located within the left terminal genomic region between nts 1 and 6131 and a lysosomal-dependent mechanism that does not require the presence of FE1. These results establish a new functional role for the FE1 transcription unit in immune evasion. These studies provide important new information about the immune evasion of FAdVs and will enhance our understanding of the pathogenesis of inclusion body hepatitis and advance the progress made in next-generation FAdV-based vectors.

Enhancer-promoter competition between homologous alleles leads to reduced transcription in early Drosophila embryos

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. Here, we ask if inter-allelic interactions between two homologous alleles can affect gene regulation. Using MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcription of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the hemizygous allele, suggesting the possibility of allelic competition in homozygotes. Moreover, the MS2-yellow reporter gene showed reduced transcriptional activity when a partial transcription unit (enhancer or promoter only) was in the homologous position. We propose that the transcriptional machinery that binds to both the enhancer and promoter region, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. To support this idea, we showed that the homologous alleles did not interfere with each other in earlier nuclear cycles when Pol II is in excess, while the degree of interference gradually increased in nuclear cycle 14. Such allelic competition was observed for endogenous snail as well. Our study provides new insights into the role of 3D inter-allelic interactions in gene regulation.

Development of an ASO therapy for Angelman syndrome by targeting an evolutionarily conserved region at the start of the UBE3A-AS transcript

Angelman syndrome is a devastating neurogenetic disorder for which there is currently no effective treatment. It is caused by mutations or epimutations affecting the expression or function of the maternally inherited allele of the ubiquitin-protein ligase E3A (UBE3A) gene. The paternal UBE3A allele is imprinted in neurons of the central nervous system (CNS) by the UBE3A antisense (UBE3A-AS) transcript, which represents the distal end of the SNHG14 transcription unit. Reactivating the expression of the paternal UBE3A allele in the CNS has long been pursued as a therapeutic option for Angelman syndrome. Here, we designed and optimized antisense oligonucleotides (ASO) targeting an evolutionarily conserved region demarcating the start of the human UBE3A-AS transcript and show that ASOs targeting this region can reverse imprinting of UBE3A in cultured Angelman syndrome neurons and throughout the CNS of a non-human primate model. Findings from this study advanced the first investigational molecular therapy for Angelman syndrome into clinical development (ClinicalTrials.gov, NCT04259281).

Comparative analysis of the characteristics of Haplorchis taichui mitochondrial genome with Metagonimus yokogawai and its ribosomal transcription unit with H. pumilio (family Heterophyidae)

Minute intestinal flukes, Haplorchis taichui and H. pumilio, belong to the family Heterophyidae (Trematoda: Platyhelminthes), which have been studied very limited, especially the molecular markers of the mitochondrial genomes (mtDNA) and the ribosome transcription units (rTU or rDNA). We have obtained the complete mitochondrial genome of H. taichui and the coding part of ribosome transcription unit of H. taichui and H. pumilio of Vietnam. Nucleotide and amino acid data were compared between H. taichui and Metagonimus yokogawai for genomic/gene composition, codon usage (skew/skewness), and tandem repeat units (TRU). The complete mtDNA of H. taichui (strain Htai-QT3-VN) with the length of 15,120 bp and M. yokogawai (15,258 bp; Korea; KC330755) contains 36 genes, including 12 protein-coding genes (cox1, cox2, cox3, nad1, nad2, nad3, nad4L, nad4, nad5, nad6, atp6 and cob), 2 ribosomal RNA genes (rRNA); 22 transfer RNA (tRNA or trn) and a noncoding region (NCR) between trnE and trnG, divided into 2 sub-regions containing 5 TRUs (182–183 bp/TRU). H. taichui (Vietnam and Laos) uses A = 19.56%, T = 39.71%, G = 28.34%, C = 12.39% (A + T is 59.27% ​​and G + C is 40.73%) for mtDNA construction, whose skew/skewness value at A+T is negative (–0,340) and G+C is positive (0.392); for 12 protein-coding genes (PCGs) is similar; but for the mito-ribosomal genes (MRGs, of 16S/rrnL and 12S/rrnS) it is less for A+T (57.22%) and more for G+C (42.78%). M. yokogawai had lower A+T (mtDNA/55.68%; PCGs/55.96%; MRGs/54.15%) and higher G+C usage rate than H. taichui. H. taichui of Vietnam and Laos has 10,164 bp encoding for 3,376 amino acids to construct 12 PCGs with the mostly used codons as Phenylalanine (Phe-TTT) and Leucine (Leu-TTG), and the leastly used codonsas Glutamine (Gln-CAA), Arginine (Arg-CGC). Additional condon, Thr-ACA/ACC can be added as the least used in M. yokogawai. The rTU (from 5 '18S to 3' 28S) of H. taichui (7,268 bp) and H. pumilio (7,416 bp) were identified with 5 genomic regions including 18S rDNA, ITS1, 5.8S rDNA, ITS2 and 28S rDNA. The 18S and 5.8S genes of both species were of the same length (1,992 bp for 18S, 160 bp for 5.8S), but different for 28S genes (3,875 bp for H. taichui and 3,870 bp for H. pumilio). ITS1 in H. taichui (797 bp) and ITS2 in H. pumilio (280 bp) do not contain TRUs, whilst ITS1 in H. pumilio (1,106 bp) contains 5 TRUs(136 bp for 3 TRU and 116 bp for 2 TRUs); and ITS2 in H. taichui (444 bp) contain 3 TRUs (83–85 bp/each).

Differential Regulation of Cellular FAM111B by Human Adenovirus C Type 5 E1 Oncogenes

The adenovirus type 5 (HAdV-C5) E1 transcription unit encodes regulatory proteins that are essential for viral replication and transformation. Among these, E1A and E1B-55K act as key multifunctional HAdV-C5 proteins involved in various steps of the viral replication cycle and in virus-induced cell transformation. In this context, HAdV-C5-mediated dysregulations of cellular factors such as the tumor suppressors p53 and pRB have been intensively investigated. However, cellular components of downstream events that could affect infection and viral transformation are widely unknown. We recently observed that cellular FAM111B is highly regulated in an E1A-dependent fashion. Intriguingly, previous reports suggest that FAM111B might play roles in tumorigenesis, but its exact functions are not known to date. Here, we set out to investigate the role of FAM111B in HAdV-C5 infections. We found that (i) FAM111B levels are upregulated early and downregulated late during infection, that (ii) FAM111B expression is differentially regulated, that (iii) FAM111B expression levels depend on the presence of E1B-55K and E4orf6 and that (iv) a FAM111B knockdown increases HAdV-C5 replication. Our data indicate that FAM111B acts as an anti-adenoviral host factor that is involved in host cell defense mechanisms in productive HAdV-C5 infection. Moreover, these findings suggest that FAM111B might play an important role in the host antiviral immune response that is counteracted by HAdV-C5 E1B-55K and E4orf6 oncoproteins.

MPRAdecoder: Processing of the Raw MPRA Data With a priori Unknown Sequences of the Region of Interest and Associated Barcodes

Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of numerous DNA regulatory elements and/or their mutant variants. The assays are based on the construction of reporter plasmid libraries containing two variable parts, a region of interest (ROI) and a barcode (BC), located outside and within the transcription unit, respectively. Importantly, each plasmid molecule in a such a highly diverse library is characterized by a unique BC–ROI association. The reporter constructs are delivered to target cells and expression of BCs at the transcript level is assayed by RT-PCR followed by next-generation sequencing (NGS). The obtained values are normalized to the abundance of BCs in the plasmid DNA sample. Altogether, this allows evaluating the regulatory potential of the associated ROI sequences. However, depending on the MPRA library construction design, the BC and ROI sequences as well as their associations can be a priori unknown. In such a case, the BC and ROI sequences, their possible mutant variants, and unambiguous BC–ROI associations have to be identified, whereas all uncertain cases have to be excluded from the analysis. Besides the preparation of additional “mapping” samples for NGS, this also requires specific bioinformatics tools. Here, we present a pipeline for processing raw MPRA data obtained by NGS for reporter construct libraries with a priori unknown sequences of BCs and ROIs. The pipeline robustly identifies unambiguous (so-called genuine) BCs and ROIs associated with them, calculates the normalized expression level for each BC and the averaged values for each ROI, and provides a graphical visualization of the processed data.

Sequence and phylogenetic analysis revealed structurally conserved domains and motifs in lincRNA-p21

Long Intergenic Non-coding RNAs (lincRNAs) are the largest class of long non-coding RNAs in the eukaryotes, which originate from the intergenic regions of the genome. A ~4kb long lincRNA-p21 is derived from a transcription unit next to the p21/Cdkn1a gene locus. LincRNA-p21 plays key regulatory roles in p53 dependent transcriptional repression and translational repression through its physical association with proteins such as hnRNP-K and HuR. It is also involved in the aberrant gene expression in different cancers. However, detailed information on its structure, recognition, and trans-regulation by proteins is not well known. In this study, we have carried out a complete gene analysis and annotation of lincRNA-p21. This analysis showed that lincRNA-p21 is highly conserved in primates, and its conservation drops significantly in lower organisms. Furthermore, our analysis has revealed two structurally conserved domains in the 5’ and 3’ terminal regions of lincRNA-p21. Phylogenetic analysis has revealed discrete evolutionary dynamics in these conserved domains for orthologous sequences of lincRNA-p21, which have evolved slowly across primates compared to other mammals. Using Infernal based covariance analysis, we have computed the secondary structures of these domains. The secondary structures were further validated by energy minimization criteria for individual orthologous sequences as well as the full-length human lincRNA-p21. In summary, this analysis has led to the identification of sequence and structural motifs in the conserved fragments, indicating the functional importance for these regions.

Deconvolution of Expression for Nascent RNA Sequencing Data (DENR) Highlights Pre-RNA Isoform Diversity in Human Cells

Quantification of mature-RNA isoform abundance from RNA-seq data has been extensively studied, but much less attention has been devoted to quantifying the abundance of distinct precursor RNAs based on nascent RNA sequencing data. Here we address this problem with a new computational method called Deconvolution of Expression for Nascent RNA sequencing data (DENR). DENR models the nascent RNA read counts at each locus as a mixture of user-provided isoforms. The performance of the baseline algorithm is enhanced by the use of machine-learning predictions of transcription start sites (TSSs) and an adjustment for the typical “shape profile” of read counts along a transcription unit. We show using simulated data that DENR clearly outperforms simple read-count-based methods for estimating the abundances of both whole genes and isoforms. By applying DENR to previously published PRO-seq data from K562 and CD4+ T cells, we find that transcription of multiple isoforms per gene is widespread, and the dominant isoform frequently makes use of an internal TSS. We also identify > 200 genes whose dominant isoforms make use of different TSSs in these two cell types. Finally, we apply DENR and StringTie to newly generated PRO-seq and RNA-seq data, respectively, for human CD4+ T cells and CD14+ monocytes, and show that entropy at the pre-RNA level makes a disproportionate contribution to overall isoform diversity, especially across cell types. Altogether, DENR is the first computational tool to enable abundance quantification of pre-RNA isoforms based on nascent RNA sequencing data, and it reveals high levels of pre-RNA isoform diversity in human cells.

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.