Developmental and Housekeeping Genes: Two Types of Genetic Organization in the Drosophila Genome.

We developed a procedure for locating genes on Drosophila melanogaster polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the Drosophila genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.

Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.

Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Intron detention tightly regulates the stemness/differentiation switch in the adult neurogenic niche.

The adult mammalian brain retains some capacity to replenish neurons and glia, holding promise for brain regeneration. Thus, understanding the mechanisms controlling adult neural stem cell (NSC) differentiation is crucial. Paradoxically, adult NSCs in the subependymal zone transcribe genes associated with both multipotency maintenance and neural differentiation, but the mechanism that prevents conflicts in fate decisions due to these opposing transcriptional programmes is unknown. Here we describe intron detention as such control mechanism. In NSCs, while multiple mRNAs from stemness genes are spliced and exported to the cytoplasm, transcripts from differentiation genes remain unspliced and detained in the nucleus, and the opposite is true under neural differentiation conditions. We also show that m6A methylation is the mechanism that releases intron detention and triggers nuclear export, enabling rapid and synchronized responses. m6A RNA methylation operates as an on/off switch for transcripts with antagonistic functions, tightly controlling the timing of NSCs commitment to differentiation.

A concerted increase in readthrough and intron retention drives transposon expression during aging and senescence.

Aging and senescence are characterized by pervasive transcriptional dysfunction, including increased expression of transposons and introns. Our aim was to elucidate mechanisms behind this increased expression. Most transposons are found within genes and introns, with a large minority being close to genes. This raises the possibility that transcriptional readthrough and intron retention are responsible for age-related changes in transposon expression rather than expression of autonomous transposons. To test this, we compiled public RNA-seq datasets from aged human fibroblasts, replicative and drug-induced senescence in human cells, and RNA-seq from aging mice and senescent mouse cells. Indeed, our reanalysis revealed a correlation between transposons expression, intron retention, and transcriptional readthrough across samples and within samples. Both intron retention and readthrough increased with aging or cellular senescence and these transcriptional defects were more pronounced in human samples as compared to those of mice. In support of a causal connection between readthrough and transposon expression, analysis of models showing induced transcriptional readthrough confirmed that they also show elevated transposon expression. Taken together, our data suggest that elevated transposon reads during aging seen in various RNA-seq dataset are concomitant with multiple transcriptional defects. Intron retention and transcriptional readthrough are the most likely explanation for the expression of transposable elements that lack a functional promoter.

Mutations and intron polymorphisms in voltage-gated sodium channel genes of different geographic populations of Culex pipiens pallens/Culex pipiens quinquefasciatus in China.

BACKGROUND: Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution. METHODS: Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network. RESULTS: There were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns. CONCLUSIONS: Cx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.

The regulatory roles of small nucleolar RNAs within their host locus.

Small nucleolar RNAs (snoRNAs) are a class of conserved noncoding RNAs forming complexes with proteins to catalyse site-specific modifications on ribosomal RNA. Besides this canonical role, several snoRNAs are now known to regulate diverse levels of gene expression. While these functions are carried out in trans by mature snoRNAs, evidence has also been emerging of regulatory roles of snoRNAs in cis, either within their genomic locus or as longer transcription intermediates during their maturation. Herein, we review recent findings that snoRNAs can interact in cis with their intron to regulate the expression of their host gene. We also explore the ever-growing diversity of longer host-derived snoRNA extensions and their functional impact across the transcriptome. Finally, we discuss the role of snoRNA duplications into forging these new layers of snoRNA-mediated regulation, as well as their involvement in the genomic imprinting of their host locus.

Characterization of the novel HLA-DQB1*02:01:01:21Q allele by sequencing-based typing.

HLA-DQB1*02:01:01:21Q differs from HLA-DQB1*02:01:01:01 by one nucleotide substitution in the splice site in the beginning of intron 3.

On a kneading theory for gene-splicing.

Two well-known facets in protein synthesis in eukaryotic cells are transcription of DNA to pre-RNA in the nucleus and the translation of messenger-RNA (mRNA) to proteins in the cytoplasm. A critical intermediate step is the removal of segments (introns) containing ∼97% of the nucleic-acid sites in pre-RNA and sequential alignment of the retained segments (exons) to form mRNA through a process referred to as splicing. Alternative forms of splicing enrich the proteome while abnormal splicing can enhance the likelihood of a cell developing cancer or other diseases. Mechanisms for splicing and origins of splicing errors are only partially deciphered. Our goal is to determine if rules on splicing can be inferred from data analytics on nucleic-acid sequences. Toward that end, we represent a nucleic-acid site as a point in a plane defined in terms of the anterior and posterior sub-sequences of the site. The "point-set" representation expands analytical approaches, including the use of statistical tools, to characterize genome sequences. It is found that point-sets for exons and introns are visually different, and that the differences can be quantified using a family of generalized moments. We design a machine-learning algorithm that can recognize individual exons or introns with 91% accuracy. Point-set distributions and generalized moments are found to differ between organisms.

Understanding and Rescuing the Splicing Defect Caused by the Frequent ABCA4 Variant c.4253+43G>A Underlying Stargardt Disease.

Pathogenic variants in ABCA4 are the underlying molecular cause of Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by a progressive loss of central vision. Among intronic ABCA4 variants, c.4253+43G>A is frequently detected in STGD1 cases and is classified as a hypomorphic allele, generally associated with late-onset cases. This variant was previously reported to alter splicing regulatory sequences, but the splicing outcome is not fully understood yet. In this study, we attempted to better understand its effect on splicing and to rescue the aberrant splicing via antisense oligonucleotides (AONs). Wild-type and c.4253+43G>A variant-harboring maxigene vectors revealed additional skipping events, which were not previously detected upon transfection in HEK293T cells. To restore exon inclusion, we designed a set of 27 AONs targeting either splicing silencer motifs or the variant region and screened these in maxigene-transfected HEK293T cells. Candidate AONs able to promote exon inclusion were selected for further testing in patient-derived photoreceptor precursor cells. Surprisingly, no robust splicing modulation was observed in this model system. Overall, this research helped to adequately characterize the splicing alteration caused by the c.4253+43G>A variant, although future development of AON-mediated exon inclusion therapy for ABCA4 is needed.

PPR596 Is Required for nad2 Intron Splicing and Complex I Biogenesis in Arabidopsis.

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.

Comparative Mitogenomics Analysis Revealed Evolutionary Divergence among Neopestalotiopsis Species Complex (Fungi: Xylariales).

The genus Neopestalotiopsis consists of obligate parasites that cause ring spot, scab, and leaf blight diseases in higher plant species. We assembled the three complete mitogenomes for the guava fruit ring spot pathogen, Neopestalotiopsis cubana. The mitogenomes are circular, with sizes of 38,666 bp, 33,846 bp, and 32,593 bp. The comparative analyses with Pestalotiopsis fici showed that N. cubana differs greatly from it in the length of the mitogenomes and the number of introns. Moreover, they showed significant differences in the gene content and tRNAs. The two genera showed little difference in gene skewness and codon preference for core protein-coding genes (PCGs). We compared gene sequencing in the mitogenomes of the order Xylariales and found large-scale gene rearrangement events, such as gene translocations and the duplication of tRNAs. N. cubana shows a unique evolutionary position in the phylum Ascomycota constructed in phylogenetic analyses. We also found a more concentrated distribution of evolutionary pressures on the PCGs of Neopestalotiopsis in the phylum Ascomycota and that they are under little selective pressure compared to other species and are subjected to purifying selection. This study explores the evolutionary dynamics of the mitogenomes of Neopestalotiopsis and provides important support for genetic and taxonomic studies.

Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research.

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

The first A-to-I RNA editome of hemipteran species Coridius chinensis reveals overrepresented recoding and prevalent intron editing in early-diverging insects.

BACKGROUND: Metazoan adenosine-to-inosine (A-to-I) RNA editing resembles A-to-G mutation and increases proteomic diversity in a temporal-spatial manner, allowing organisms adapting to changeable environment. The RNA editomes in many major animal clades remain unexplored, hampering the understanding on the evolution and adaptation of this essential post-transcriptional modification. METHODS: We assembled the chromosome-level genome of Coridius chinensis belonging to Hemiptera, the fifth largest insect order where RNA editing has not been studied yet. We generated ten head RNA-Seq libraries with DNA-Seq from the matched individuals. RESULTS: We identified thousands of high-confidence RNA editing sites in C. chinensis. Overrepresentation of nonsynonymous editing was observed, but conserved recoding across different orders was very rare. Under cold stress, the global editing efficiency was down-regulated and the general transcriptional processes were shut down. Nevertheless, we found an interesting site with "conserved editing but non-conserved recoding" in potassium channel Shab which was significantly up-regulated in cold, serving as a candidate functional site in response to temperature stress. CONCLUSIONS: RNA editing in C. chinensis largely recodes the proteome. The first RNA editome in Hemiptera indicates independent origin of beneficial recoding during insect evolution, which advances our understanding on the evolution, conservation, and adaptation of RNA editing.

Structural basis of U12-type intron engagement by the fully assembled human minor spliceosome.

The minor spliceosome, which is responsible for the splicing of U12-type introns, comprises five small nuclear RNAs (snRNAs), of which only one is shared with the major spliceosome. In this work, we report the 3.3-angstrom cryo-electron microscopy structure of the fully assembled human minor spliceosome pre-B complex. The atomic model includes U11 small nuclear ribonucleoprotein (snRNP), U12 snRNP, and U4atac/U6atac.U5 tri-snRNP. U11 snRNA is recognized by five U11-specific proteins (20K, 25K, 35K, 48K, and 59K) and the heptameric Sm ring. The 3' half of the 5'-splice site forms a duplex with U11 snRNA; the 5' half is recognized by U11-35K, U11-48K, and U11 snRNA. Two proteins, CENATAC and DIM2/TXNL4B, specifically associate with the minor tri-snRNP. A structural analysis uncovered how two conformationally similar tri-snRNPs are differentiated by the minor and major prespliceosomes for assembly.

Detection of two HLA-B*35 variants: HLA-B*35:01:01:39 and -B*35:03:01:32.

Two nucleotide substitutions in intronic regions give rise to the novel alleles: HLA-B*35:01:01:39 and -B*35:03:01:32.

Comprehensive Evolutionary Analysis of the SMXL Gene Family in Rosaceae: Further Insights into Its Origin, Expansion, Diversification, and Role in Regulating Pear Branching.

SMXL genes constitute a conserved gene family that is ubiquitous in angiosperms and involved in regulating various plant processes, including branching, leaf elongation, and anthocyanin biosynthesis, but little is known about their molecular functions in pear branching. Here, we performed genome-wide identification and investigation of the SMXL genes in 16 angiosperms and analyzed their phylogenetics, structural features, conserved motifs, and expression patterns. In total, 121 SMXLs genes were identified and were classified into four groups. The number of non-redundant SMXL genes in each species varied from 3 (Amborella trichopoda Baill.) to 18 (Glycine max Merr.) and revealed clear gene expansion events over evolutionary history. All the SMXL genes showed conserved structures, containing no more than two introns. Three-dimensional protein structure prediction revealed distinct structures between but similar structures within groups. A quantitative real-time PCR analysis revealed different expressions of 10 SMXL genes from pear branching induced by fruit-thinning treatment. Overall, our study provides a comprehensive investigation of SMXL genes in the Rosaceae family, especially pear. The results offer a reference for understanding the evolutionary history of SMXL genes and provide excellent candidates for studying fruit tree branching regulation, and in facilitating pear pruning and planting strategies.

Transcription and splicing variations of SR genes accompany with genome-wide accumulation of long-introns in pine.

Most of mRNAs in Eukaryote were matured after the removal of introns in their pre-mRNA transcripts. Serine/arginine-rich (SR) proteins are a group of splicing regulators regulating the splicing processes globally. Expressions of SR proteins themselves were extensively regulated, at both transcription and splicing levels, under different environmental conditions, specially heat stress conditions. The pine genome is characterized by super-long and easily methylated introns in a large number of genes that derived from the extensive accumulation of transposons (TEs). Here, we identified and analyzed the phylogenetic characteristics of 24 SR proteins and their encoding genes from the pine genome. Then we explored transcription and pre-mRNA splicing expression patterns of SR genes in P. massoniana seedlings under normal and heat stress temperature conditions. Our results showed that the transcription patterns of SR genes in pine exhibited significant changes compared to other plant species, and these changes were not strictly correlated with the intron length and DNA methylation intensity of the SR genes. Interestingly, none of the long introns of SR genes underwent alternative splicing (AS) in our experiment. Furthermore, the intensity of AS regulation may be related to the potential DNA methylation intensity of SR genes. Taken together, this study explores for the first time the characteristics of significant variations in the transcription and splicing patterns of SR proteins in a plant species with an over-accumulation of super-long introns.

A novel intronic TCOF1 pathogenic variant in a Chinese family with Treacher Collins syndrome.

BACKGROUND: Treacher Collins syndrome (TCS; OMIM 154500) is a craniofacial developmental disorder. METHODS: To investigate the genetic features of a four-generation Chinese family with TCS, clinical examinations, hearing tests, computed tomography, whole-exome sequencing (WES), Sanger sequencing, reverse transcription (RT)-PCR, and the Minigene assay were performed. RESULTS: The probands, an 11-year-old male and his cousin exhibited typical clinical manifestations of TCS including conductive hearing loss, downward slanting palpebral fissures, and mandibular hypoplasia. Computed tomography revealed bilateral fusion of the anterior and posterior stapedial crura and malformation of the long crura of the incus. WES of both patients revealed a novel heterozygous intronic variant, i.e., c.4342 + 5_4342 + 8delGTGA (NM_001371623.1) in TCOF1. Minigene expression analysis revealed that the c.4342 + 5_4342 + 8delGTGA variant in TCOF1 caused a partial deletion of exon 24 (c.4115_4342del: p.Gly1373_Arg1448del), which was predicted to yield a truncated protein. The deletion was further confirmed via RT-PCR and sequencing of DNA from proband blood cells. A heterozygous variant in the POLR1C gene (NM_203290; exon6; c.525delG) was found almost co-segregated with the TCOF1 pathogenic variant. CONCLUSIONS: In conclusion, we identified a heterozygous TCOF1 splicing variant c.4342 + 5_4342 + 8delGTGA (splicing) in a Chinese TSC family with ossicular chain malformations and facial anomalies. Our findings broadened the spectrum of TCS variants and will facilitate diagnostics and prognostic predictions.