Nucleic Acids Research最新文献

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is a prevalent and conserved RNA modification. While A-to-I RNA editing is essential in mammals, in Caenorhabditis elegans, it is not, making them invaluable for RNA editing research. In C. elegans, ADR-2 is the sole catalytic A-to-I editing enzyme, and ADR-1 is an RNA editing regulator. ADAR localization is well-studied in humans but not well-established in C. elegans. In this study, we examine the cellular and tissue-specific localization of ADR-2. We show that while ADR-2 is present in most cells in the embryo, at later developmental stages, its expression is both tissue- and cell-type-specific. Additionally, both ADARs are mainly in the nucleus. ADR-2 is adjacent to the chromosomes during the cell cycle. We show that the nuclear localization of endogenous ADR-2 depends on ADBP-1, not ADR-1. In adbp-1 mutant worms, ADR-2 is mislocalized, while ADR-1 is not, leading to decreased editing levels and de-novo editing, mostly in exons, suggesting that ADR-2 is also functional in the cytoplasm. Besides, mutated ADBP-1 affects gene expression. Furthermore, we show that ADR-2 targets adenosines with different surrounding nucleotides in exons and introns. Our findings indicate that ADR-2 cellular localization is highly regulated and affects its function.

Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3'-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich RNAs. We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence features through the ROQ-ZnF tandem.

Coordination of gene regulatory networks is necessary for proper execution of cellular programs throughout development. RNA interference (RNAi) is an essential regulatory mechanism in all metazoans. Proper RNAi-mediated gene regulation requires coordination of several RNAi branches to ensure homeostasis. For example, in Caenorhabditis elegans, the Argonautes, ALG-3 and ALG-4, are expressed specifically during spermatogenesis (L4 stage) and bind small interfering RNAs (siRNAs) complementary to sperm-enriched genes. We find that alg-3 and alg-4 are regulated by siRNAs. Our work shows that gene switches are operated via these siRNAs to regulate the Argonautes' expression in a temporal manner. This RNAi-to-RNAi regulatory cascade is essential for coordinating ALG-3/4 pathway function, particularly during heat stress, to provide thermotolerant sperm-based fertility. This work provides insight into one regulatory motif used to maintain RNAi homeostasis, across developmental stages, despite environmental stressors. As RNAi pathways are evolutionarily conserved, other species likely use similar regulatory architectures to maintain RNAi homeostasis.

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.

The recent COVID-19 pandemics have demonstrated the great therapeutic potential of in vitro transcribed (IVT) mRNAs, but improvements in their biochemical properties, such as cellular stability, reactogenicity and translational activity, are critical for further practical applications in gene replacement therapy and anticancer immunotherapy. One of the strategies to overcome these limitations is the chemical modification of a unique mRNA 5'-end structure, the 5'-cap, which is responsible for regulating translation at multiple levels. This could be achieved by priming the in vitro transcription reaction with synthetic cap analogs. In this study, we combined a highly efficient trinucleotide IVT capping technology with several modifications of the 5' cap triphosphate bridge to synthesize a series of 16 new cap analogs. We also combined these modifications with epigenetic marks (2'-O-methylation and m6Am) characteristic of mRNA 5'-ends in higher eukaryotes, which was not possible with dinucleotide caps. All analogs were compared for their effect on the interactions with eIF4E protein, IVT priming, susceptibility to decapping, and mRNA translation efficiency in model cell lines. The most promising α-phosphorothiolate modification was also evaluated in an in vivo mouse model. Unexpected differences between some of the analogs were analyzed using a protein cell extract pull-down assay.

The H3K4 methyltransferase SETD1A plays an essential role in both development and cancer. However, essential components involved in SETD1A chromatin binding remain unclear. Here, we discovered that BOD1L exhibits the highest correlated SETD1A co-dependency in human cancer cell lines. BOD1L knockout reduces leukemia cells in vitro and in vivo, and mimics the transcriptional profiles observed in SETD1A knockout cells. The loss of BOD1L immediately reduced SETD1A distribution at transcriptional start sites (TSS), induced transcriptional elongation defect, and increased the RNA polymerase II content at TSS; however, it did not reduce H3K4me3. The Shg1 domain of BOD1L has a DNA binding ability, and a tryptophan residue (W104) in the domain recruits SETD1A to chromatin through the association with SETD1A FLOS domain. In addition, the BOD1L-SETD1A complex associates with transcriptional regulators, including E2Fs. These results reveal that BOD1L mediates chromatin and SETD1A, and regulates the non-canonical function of SETD1A in transcription.

Precisely modulating the kinetics of toehold-mediated DNA strand displacements (TMSD) is essential for its application in DNA nanotechnology. The sequence in the toehold region significantly influences the kinetics of TMSD. However, due to the large sample space resulting from various arrangements of base sequences and the resulted complex secondary structures, such a correlation is not intuitive. Herein, machine learning was employed to reveal the relationship between the kinetics of TMSD and the toehold sequence as well as the correlated secondary structure of invader strands. Key factors that influence the rate constant of TMSD were identified, such as the number of free hydrogen bonding sites in the invader, the number of free bases in the toehold, and the number of hydrogen bonds in intermediates. Moreover, a predictive model was constructed, which successfully achieved semi-quantitative prediction of rate constants of TMSD even with subtle distinctions in toehold sequence.

Structured RNAs often contain long-range tertiary contacts that are critical to their function. Despite the importance of tertiary contacts, methods to measure their thermodynamics are low throughput or require specialized instruments. Here, we introduce a new quantitative chemical mapping method (qMaPseq) to measure Mg2+-induced formation of tertiary contact thermodynamics in a high-throughput manner using standard biochemistry equipment. With qMaPseq, we measured the ΔG of 98 unique tetraloop/tetraloop receptor (TL/TLR) variants in a one-pot reaction. These results agree well with measurements from specialized instruments (R2= 0.64). Furthermore, the DMS reactivity of the TL directly correlates to the stability of the contact (R2= 0.68), the first direct evidence that a single DMS reactivity measurement reports on thermodynamics. Combined with structure prediction, DMS reactivity allowed the development of experimentally accurate 3D models of TLR mutants. These results demonstrate that qMaPseq is broadly accessible, high-throughput and directly links DMS reactivity to thermodynamics.

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression.