Abstract Nucleotide repeat expansions support production of toxic peptides from putatively non-coding regions of the human genome without use of an AUG initiation codon. This process, known as repeat-associated non-AUG (RAN) translation, was originally described at CAG repeats with products generated in all three possible reading frames. Recently, cryptic mis-splicing of messenger RNAs in which the CAG repeat acts as a splice acceptor was proposed as an alternative mechanism for RAN protein production. To investigate this, we generated DNA- and RNA-based reporters to assess RAN translation in the context of a CAG repeat expansion in exon 1 of the Huntingtin gene (HTT), associated with Huntington disease. HTT CAG repeats support RAN translation in both the alanine (GCA) and glutamine (CAG) frames, with or without upstream AUG start codons or near-AUG cognate codons, and at levels comparable to RAN translation from CGG and GGGGCC repeats. CAG RAN translation products were readily detectable from in vitro transcribed RNAs transfected into human cells and rodent neurons—suggesting that plasmid-based aberrant splicing into CAG repeats cannot fully explain the observed phenomena. These findings indicate that RAN translation from HTT CAG repeats shares key mechanistic parameters with other disease-associated repeat expansions.
Abstract Mutations in the LMNA gene, encoding lamin A (LMNA), cause laminopathies. Cardiac involvement is common and a major cause of mortality and morbidity in laminopathies. LMNA interacts with chromatin through lamin-associated domains (LADs) and regulates gene expression. We report that LMNA deficiency increased genome-wide CTCF occupancy, predominantly outside of the constitutive LAD (cLAD) regions. CTCF occupancy was associated with increased gene expression and enrichment of double-stranded breaks (DSBs). LMNA deficiency had a modest effect on chromatin compartments but led to mergers, splitting, and shifting boundaries of one-third of the topologically associated domains (TADs) without affecting gene expression significantly. LMNA deficiency led to a gain of ∼600 and a loss of ∼1000 loops, which were associated with increased and decreased transcript levels, respectively. Reorganization of chromatin loops affected ∼25% of the differentially expressed genes in the LMNA-deficient cardiac myocytes. In multivariate analysis, CTCF-binding sites, chromatin loops, and cLADs were independent predictors of gene expression in the LMNA-deficient cardiac myocytes, whereas TADs and chromatin compartments were not. Thus, LMNA deficiency changes genome-wide CTCF occupancy and loop organization, which affect gene expression in cardiac myocytes. The findings provide new insights into the pathogenesis of cardiac involvement in laminopathies.
Abstract The induction of DNA damage by HIV-1 prior to integration suggests a role for DNA damage responses (DDRs) during early infection; however, this role remains incompletely understood. Initial experiments, using specific inhibitors for DDR pathways, demonstrate that both ATM and ATR are necessary for efficient HIV-1 infection of macrophages with ATM acting at the late reverse transcription step. To identify DDR factors associated with ATM/ATR pathways that influence HIV-1 infection, a CRISPR knockout screen using a DDR-focused sgRNA library was performed. Approximately 30 DDR genes that impacted HIV-1 infection were identified with 13 factors that facilitated HIV-1 infection and 17 DDR factors that restrict HIV-1 infection. Several hits were factors associated with the Fanconi anemia pathway, such as BTR complex proteins, including the RecQ helicase Bloom syndrome helicase (BLM). BLM was specifically demonstrated to enhance HIV-1 infection and replication with knockdown of BLM expression diminishing integration and the establishment of intact HIV-1 proviruses in macrophages by 50%. BLM is associated with HIV-1 late reverse transcription intermediates, the step that proceeds HIV-1 integration, identifying BLM as a cofactor in this key step required for efficient infection.
Abstract Epigenetic gene regulation is central to the development and function of immune cells. For example, B cell differentiation and function require high levels of EZH2, the catalytic subunit of epigenetic regulator polycomb repressive complex 2 (PRC2). Our clinical evaluation identified two sisters with adult-onset recurrent infections and memory B cell deficiency who share a heterozygous variant (Leu50Ser, L50S) in a conserved region of EZH2. Most EZH2-catalyzed histone H3 lysine 27 methylation (H3K27me) was maintained with EZH2-L50S, in patient samples, and cell lines expressing EZH2-L50S. However, gene promoters that normally have the highest H3K27me3 levels are deficient with L50S, despite high EZH2-L50S recruitment. EZH2-L50S stably forms PRC2, but the variant affects methylation of nucleosomes and disrupts allosteric stimulation that normally amplifies H3K27me3, consistent with the location of L50 in the PRC2 allosteric regulatory region. Our molecular characterization supports a role for EZH2-L50S in contributing to the observed adult B cell immunodeficiency through epigenetic mis-regulation. Given our findings, at a frequency of ~1 in 10–20 000 in the human population, EZH2-L50S may more broadly contribute to adult immunodeficiency.
Abstract Dengue virus (DENV), a mosquito-borne virus of the Flaviviridae family, is a major cause of global morbidity and mortality. In our previous study, we performed RNA sequencing analysis, which showed that DENV infection increases tRNA fragmentation, leading to the generation of tRNA-derived small RNAs (tsRNAs). From RNA sequencing analysis, we identified differentially expressed tsRNAs, predominantly comprising of tRNA-derived fragments (tRFs), a class of tsRNAs. In this study, we have performed orthogonal validation of highly differentially expressed tRFs using northern blotting and have further investigated the functional role of these tRFs during DENV infection. Antisense oligos (ASOs) were used to inhibit the selected tRFs, and the effect of tRF inhibition on DENV replication was observed. Our results show that tRFs exhibit dual regulatory roles by either promoting or inhibiting the DENV replication through modulation of the PI3K pathway. Overall, this study identifies novel regulatory roles for tsRNAs in DENV pathogenesis and provides new insights into host–virus interactions.
