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Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes that are involved in fundamental DNA repair processes, such as homologous recombination, or in the transposition of foreign genetic material by viruses and mobile genetic elements1,2. Here we report that IS110 insertion sequences, a family of minimal and autonomous mobile genetic elements, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and the donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables the insertion of DNA into genomic target sites, as well as programmable DNA excision and inversion. The IS110 bridge recombination system expands the diversity of nucleic-acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements-insertion, excision and inversion-that are required for genome design.

Populations and species are threatened by human pressure, but their fate is variable. Some depleted populations, such as that of the northern elephant seal (Mirounga angustirostris), recover rapidly even when the surviving population was small. The northern elephant seal was hunted extensively and taken by collectors between the early 1800s and 1892, suffering an extreme population bottleneck as a consequence. Recovery was rapid and now there are over 200,000 individuals. We sequenced 260 modern and 8 historical northern elephant seal nuclear genomes to assess the impact of the population bottleneck on individual northern elephant seals and to better understand their recovery. Here we show that inbreeding, an increase in the frequency of alleles compromised by lost function, and allele frequency distortion, reduced the fitness of breeding males and females, as well as the performance of adult females on foraging migrations. We provide a detailed investigation of the impact of a severe bottleneck on fitness at the genomic level and report on the role of specific gene systems.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

The genus Acacia is a large group of woody legumes containing an enormous amount of morphological diversity in leaf shape. This diversity is at least in part the result of an innovation in leaf development where many Acacia species are capable of developing leaves of both bifacial and unifacial morphologies. While not unique in the plant kingdom, unifaciality is most commonly associated with monocots, and its developmental genetic mechanisms have yet to be explored beyond this group. In this study, we identify an accession of Acacia crassicarpa with high regeneration rates and isolate a clone for genome sequencing. We generate a chromosome-level assembly of this readily transformable clone, and using comparative analyses, confirm a whole-genome duplication unique to Caesalpinoid legumes. This resource will be important for future work examining genome evolution in legumes and the unique developmental genetic mechanisms underlying unifacial morphogenesis in Acacia.

Pathogen lineage nomenclature systems are a key component of effective communication and collaboration for researchers and public health workers. Since February 2021, the Pango dynamic lineage nomenclature for SARS-CoV-2 has been sustained by crowdsourced lineage proposals as new isolates were sequenced. This approach is vulnerable to time-critical delays as well as regional and personal bias. Here we developed a simple heuristic approach for dividing phylogenetic trees into lineages, including the prioritization of key mutations or genes. Our implementation is efficient on extremely large phylogenetic trees consisting of millions of sequences and produces similar results to existing manually curated lineage designations when applied to SARS-CoV-2 and other viruses including chikungunya virus, Venezuelan equine encephalitis virus complex and Zika virus. This method offers a simple, automated and consistent approach to pathogen nomenclature that can assist researchers in developing and maintaining phylogeny-based classifications in the face of ever-increasing genomic datasets.

At the core of molecular biology lies the intricate interplay between sequence, structure, and function. Single-molecule techniques provide in-depth dynamic insights into structure and function, but laborious assays impede functional screening of large sequence libraries. We introduce high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), integrating single-molecule fluorescence with next-generation sequencing. We applied SPARXS to study the sequence-dependent kinetics of the Holliday junction, a critical intermediate in homologous recombination. By examining the dynamics of millions of Holliday junctions, covering thousands of distinct sequences, we demonstrated the ability of SPARXS to uncover sequence patterns, evaluate sequence motifs, and construct thermodynamic models. SPARXS emerges as a versatile tool for untangling the mechanisms that underlie sequence-specific processes at the molecular scale.

The ability to both sensitively and specifically assess the sequence composition of a nucleic acid strand is an ever-growing field. Designing a detection scheme that can perform this function when the sequence of the target being detected deviates significantly from the canonical sequence however is difficult in part because probe/primer design is based on established Watson-Crick base-pairing rules. We present here a robust and tunable toehold-based exchange probe that can detect a sequence with a variable number of SNPs of unknown identity by inserting a series of controlled, sequential mismatches into the protector seal of the toehold probe, in an effort to make the protector seal "sloppy". We show that the mismatch-tolerant system follows predicted behavior closely even with targets containing up to four mismatches that thermodynamically deviate from the canonical sequence by up to 15 kcal/mole. The system also performs faithfully regardless of the global mismatch position on either the protector seal or target. Lastly, we demonstrate the generalizability of the approach by testing the increasingly mismatch-tolerant protectors on HIV clinical samples to show that the system is capable of resolving multiple, iteratively mutated sequences derived from numerous HIV sub-populations with remarkable precision., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bugga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA., (© 2024. The Author(s).)

Morganella psychrotolerans is a histamine-producing bacterium that causes histamine poisoning. In this study, we isolated and characterized a novel phage, MopsHU1, that infects M. psychrotolerans. MopsHU1 is a podovirus with a limited host spectrum. Genomic analysis showed that MopsHU1 belongs to the family Autographiviridae, subfamily Studiervirinae, and genus Kayfunavirus. Comparative analysis revealed that the MopsHU1 genome is similar to those of Citrobacter phage SH3 and Cronobacter phage Dev2. Moreover, the Escherichia coli phage K1F genome is also similar, except for its tailspike gene sequence. These results expand our understanding of the Kayfunavirus phages that infect Morganella spp. Note: The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank database under the accession number LC799501., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The family Convolvulaceae comprises approximately 50-60 genera with approximately 1600-1700 species, exhibiting a rich diversity of morphological characteristics and occupying a broad range of ecological habitats. High-throughput sequencing identified a tentative new virus in the family Geminiviridae infecting Calystegia sepium var. japonica in South Korea. The 2,706 nt long genome comprises six open reading frames (ORFs). The analysis of the nucleotide sequence of the genome and the putative amino acid sequences of ORFs indicated that the virus was closely related to the members of the family Geminiviridae. The genome organization of the virus was similar to that of members of the genus Topilevirus in terms of the number of ORFs and splicing signal. However, the virus, tentatively named bindweed mottle virus (BWMV), may not be assigned to current genera into the family Geminiviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, p recise R NA-mediated in sertion of t ransgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells., (© 2024 Palm et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98 nt sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three or two stem-loop structures that result from the different base-pairing interactions within the first 55 nt. Here, we used single-molecule Förster resonance energy transfer spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes., (© 2024 Sperstad and Holmstrom; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD-DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease., (© 2024. The Author(s).)

Background: While melanomas commonly harbor losses of 9p21, on which CDKN2A resides, the presence of additional tumor suppressor elements at this locus is incompletely characterized. Here we assess the expression levels and functional role of microRNA-876-3p (miR-876), whose gene also maps to 9p21., Methods: Expression of miR-876 was assessed in human tissues and cell lines using quantitative miRNA reverse transcriptase polymerase chain reaction (qRT-PCR). MIR876 copy number was determined in The Cancer Genome Atlas (TCGA) melanoma cohort. The consequences of regulation of miR-876 expression were assessed on melanoma cell colony formation, migration, invasion, apoptosis, cell cycle progression, and drug sensitivity in culture, and on in vivo tumor growth in a xenograft model. Genome-wide transcriptomic changes induced by miR-876 overexpression were determined using RNA sequencing (RNA-Seq)., Results: miR-876 expression was significantly decreased in primary melanoma samples when compared with nevi, and in human melanoma cell lines when compared with human melanocytes. Analysis of the TCGA cohort revealed deletions in MIR876 in > 50% of melanomas. miR-876 overexpression resulted in decreased melanoma cell colony formation, migration, and invasion, which was accompanied by cell cycle arrest and increased apoptosis. Intra-tumoral injections of miR-876 significantly suppressed melanoma growth in vivo. RNA-Seq analysis of miR-876-treated tumors revealed downregulation of several growth-promoting genes, along with upregulation of tumor suppressor genes, which was confirmed by qRT-PCR analysis. Computational analyses identified MAPK1 (or ERK2) as a possible target of miR-876 action. Overexpression of miR-876 significantly suppressed luciferase expression driven by the MAPK1/ERK2 3' UTR, and resulted in decreased ERK protein expression in melanoma cells. MAPK1/ERK2 cDNA overexpression rescued the effects of miR-876 on melanoma colony formation. miR-876 overexpression sensitized melanoma cells to treatment with the BRAF inhibitor vemurafenib., Conclusions: These studies identify miR-876 as a distinct tumor suppressor on 9p21 that is inactivated in melanoma and suggest miR-876 loss as an additional mechanism to activate ERK and the mitogen activated protein kinase (MAPK) pathway in melanoma. In addition, they suggest the therapeutic potential of combining miR-876 overexpression with BRAF inhibition as a rational therapeutic strategy for melanoma., (© 2024. The Author(s).)

Population-scale genome modification can alter the composition or fate of wild populations. Synthetic gene drives provide one set of tools, but their use is complicated by scientific, regulatory, and social issues associated with transgene persistence and flow. Here we propose an alternative approach. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits, and is inherited in a Mendelian fashion. Meanwhile, the edits (the Sail) experience an arithmetic, Super-Mendelian increase in frequency. We model this system and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can bring about population suppression. In regulatory frameworks that distinguish between transgenics (GMO) and their edited non-transgenic progeny (non-GMO) Allele Sails may prove useful since the spread and persistence of the GM component can be limited., (© 2024. The Author(s).)

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action., Competing Interests: Declaration of interests D.P.B. has equity in Alnylam Pharmaceuticals, where he is a co-founder and advisor. D.P.B. is a member of Molecular Cell's advisory board., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Objective: In past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3' end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3' end of genes in promoting yFACT dissociation upon transcription termination., Results: We generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3' end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene's 3' end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3' ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture., (© 2024. The Author(s).)

With the increasing age of the population worldwide, the incidence rate of Parkinson's disease (PD) is increasing annually. Currently, the treatment strategy for PD only improves clinical symptoms. No effective treatment strategy can slow down the progression of the disease. In the present study, whole transcriptome sequencing was used to obtain the mRNA and miRNA expression profiles in a PD mouse model, which revealed the pathogenesis of PD. The transcription factor RUNX3 upregulated the miR-186-3p expression in the PD model. Furthermore, the high miR-186-3p expression in PD can be targeted to inhibit the DAT expression, resulting in a decrease in the dopamine content of dopaminergic neurons. Moreover, miR-186-3p can be targeted to inhibit the IGF1R expression and prevent the activation of the IGF1R-P-PI3K-P-AKT pathway, thus increasing the apoptosis of dopaminergic neurons by regulating the cytochrome c-Bax-cleaved caspase-3 pathway. Our research showed that the RUNX3-miR-186-3p-DAT-IGF1R axis plays a key role in the pathogenesis of PD, and miR-186-3p is a potential target for the treatment of PD., (© 2024. The Author(s).)

The non-classical HLA-G*01:55 allele differs from G*01:01:12 at one position in exon 4., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nucleotide substitution in the intron 2 of HLA-DPB1*04:02:01:01 results in a novel allele, HLA-DPB1*04:02:01:44., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Two novel HLA class II, HLA-DRB1*13:357 and HLA-DQB1*03:525, characterised in Russian individuals., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Full genomic sequence shows HLA-G*01:19 differs from HLA-G*01:04:01:01 only at position 99 in exon 2., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The new HLA-DRB3*03:69 allele differs from DRB3*03:01:01:03 by change of C → G in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-A*24:617 differs from HLA-A*24:02:01:01 by one nucleotide in exon 4., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-A*02:1146 differs from HLA-A*02:01:01:01 by one nucleotide in exon 1., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A missense nucleotide substitution at codon 95 of HLA-C*18:01:01:01 creates a novel allele HLA-C*18:20., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The coding sequence of HLA-B*40:540N differs from HLA-B*40:02:01:01 by a non-synonymous nucleotide substitution in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-A*24:630 differs from HLA-A*24:20:01:01 by one nucleotide substitution in codon 131 in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*17:78 differs from HLA-C*17:03:01:03 by one nucleotide change C>T in exon 3 (GCG>GTG)., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The novel HLA-C*07:1132 allele differs from HLA-C*07:01:01 by one nucleotide substitution in Exon 5., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

MICB*002:06 differs from MICB*002:01:01 by one nucleotide change at nucleotide 33 in exon 1 from C to T., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-A*33:33:02 differs from HLA-A*33:33:01 by one synonymous nucleotide change C>T in exon 3 (TCC>TCT)., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DQB1*02:211 allele differs from DQB1*02:02:01:02 by change of C → A in exon 2., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DQB1*06:02:61 differs from HLA-DQB1*06:02:01:01 by one nucleotide substitution in exon 4-5512 G>A., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*07:02:151 differs from HLA-C*07:02:01:01 by one nucleotide substitution in codon 166 in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Two novel HLA-DQB1 alleles, HLA-DQB1*05:01:50 and HLA-DQB1*06:486, characterised in bone marrow volunteers., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*15:689, HLA-B*35:603 and HLA-B*49:01:25, three novel HLA class I alleles detected by next-generation sequencing., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Le Scornet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The novel allele, HLA-DQB1*03:517, differs by a single nucleotide substitution in exon 3 to HLA-DQB1*03:02:01:02., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DRB1*08:126 differs from HLA-DRB1*08:04:01:01 by one nucleotide substitution in codon 152 in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The novel HLA-DPB1*14:01:15 allele differs from DPB1*14:01:01:01 by change of C > T in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The novel HLA-C*15:279 allele differs from HLA-C*15:02:01:01 by five nucleotide substitutions in exons 4 and 5., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We identified a novel C-type lectin (CTL) from Macrobrachium nipponense, designated as Mn-clip-Lec. It consists of 1315 bp with an open reading frame of 1098 bp, encoding a polypeptide of 365 amino acids. Mn-clip-Lec contains 6 exons and 5 introns. Mn-clip-Lec possessed a CLIP domain at the N-terminal and two carbohydrate recognition domains at the C-terminal. Interaction between Mn-clip-Lec and MnLec was found by Yeast two-hybrid analysis. The expressions of Mn-clip-Lec, MnLec, prophenoloxidase (proPO)-activating system-associated genes (MnPPAF, MnPPAE, and MnPO), and antimicrobial peptides (AMPs) (MnALF and MnCRU) were up-regulated after the challenge with Staphylococcus aureus. RNA interference (RNAi)-mediated suppression of the Mn-clip-Lec and MnLec genes in S. aureus-challenged prawns reduced the transcripts of MnPPAF, MnPPAE, MnPO, MnALF and MnCRU. Knockdown of Mn-clip-Lec and MnLec resulted in decrease in PO activity in M. nipponense infected with S. aureus. The recombinant Mn-clip-Lec (rMn-clip-Lec) protein bound all tested bacteria and agglutinated S. aureus. A sugar-binding assay revealed that rMn-clip-Lec could bind to LPS or PGN. rMn-clip-Lec accelerated the clearance of S. aureus in vivo. Our findings suggest that Mn-clip-Lec and its interacting MnLec play important roles in the induction of the proPO system and AMPs expression in M. nipponense during bacterial infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Heat shock factor binding protein 1 (HSBP1) is known to regulate the activity of heat shock factor 1 (HSF1) and the early development of organisms. To understand the involvement of HSBP1 in the heat shock response and embryonic and larval development of Pacific abalone (Haliotis discus hannai), the Hdh-HSBP1 gene was sequenced from the digestive gland (DG) tissue. The full-length sequence of Hdh-HSBP1 encompassed 738 nucleotides, encoding an 8.42 kDa protein consisting of 75 deduced amino acids. The protein contains an HSBP1 domain and a coiled-coil domain, which are conserved features in the HSBP1 protein family. Protein-protein molecular docking revealed that the coiled-coil region of Hdh-HSBP1 binds to the coiled-coil region of Hdh-HSF1. Tissue expression analysis demonstrated that the highest Hdh-HSBP1 expression occurred in the DG, whereas seasonal expression analysis revealed that this gene was most highly expressed in summer. In heat-stressed abalone, the highest expression of Hdh-HSBP1 occurred at 30 °C. Moreover, time-series analysis revealed that the expression of this gene began to increase significantly at 6 h post-heat stress, with higher expression observed at 12 h and 24 h post-heat stress. Furthermore, Hdh-HSBP1 mRNA expression showed a link to ROS production. Additionally, the expression of Hdh-HSBP1 showed significantly higher expression in the early stages of embryonic development in Pacific abalone. These results suggest that Hdh-HSBP1 plays a crucial role in the stress physiology of Pacific abalone by interacting with Hdh-HSF1, as well as its embryonic development., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs., (© 2024. The Society for In Vitro Biology.)