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Mounting evidence suggests a prominent role for alpha-synuclein (a-syn) in neuronal cell function. Alterations in the levels of cellular a-syn have been hypothesized to play a critical role in the development of Parkinson's disease (PD); however, mechanisms that control expression of the gene for a-syn (SNCA) in cis and trans as well as turnover of a-syn are not well understood. We analyzed whether methyl-CpG binding protein 2 (MeCP2), a protein that specifically binds methylated DNA, thus regulating transcription, binds at predicted binding sites in intron 1 of the SNCA gene and regulates a-syn protein expression. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of SNCA. Site-specific methylation and introduction of localized mutations by CRISPR/Cas9 were used to investigate the binding properties of MeCP2 in human SK-N-SH neuroblastoma cells. The significance of MeCP2 for SNCA regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in MeCP2 knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of SNCA had a significant impact on the production of a-syn. A single nucleotide substitution near to CpG1 strongly increased the binding of MeCP2 to intron 1 of SNCA and decreased a-syn protein expression by 60%. In contrast, deletion of a single nucleotide closed to CpG2 led to reduced binding of MeCP2 and significantly increased a-syn levels. In accordance, knockout of MeCP2 in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that SNCA is a genomic target for MeCP2 regulation. In addition, the expression of two mutated MeCP2 variants found in Rett syndrome (RTT) showed a loss of their ability to reduce a-syn expression. This study demonstrates that methylation of CpGs and binding of MeCP2 to intron 1 of the SNCA gene plays an important role in the control of a-syn expression. In addition, the changes in SNCA regulation found by expression of MeCP2 variants carrying mutations found in RTT patients may be of importance for the elucidation of a new molecular pathway in RTT, a rare neurological disorder caused by mutations in MECP2. [ABSTRACT FROM AUTHOR]

The species of Purpureocillium are cosmopolitan and multitrophic fungi that can infect a wide range of invertebrate hosts. This study reports the mitogenome of P. atypicola, a specialized spider pathogenic fungus. The 112,465 bp mitogenome encoded genes typically found in fungal mitogenomes, and a total of 52 introns inserted into seven genes. A comparison with three other Purpureocillium species revealed significant differences in length and intron number, primarily due to intron variation; however, there was no dynamic variation in the introns of the cox1 gene within the same species of the Purpureocillium genus. Different mitochondrial protein-coding genes showed variable degrees of genetic differentiation among these species, but they were all under purifying selection. Additionally, frequent intron loss or gain events were detected to have occurred during the evolution of the Ophiocordycipitaceae mitogenomes, yet the gene arrangement remains conserved. A phylogenetic analysis of the combined mitochondrial gene set gave identical and well-supported tree topologies. The estimated age of the crown of Ophiocordycipitaceae and Purpureocillium were around the Early Cretaceous period (127 Mya) and Late Cretaceous period (83 Mya), respectively. The results of this study advance our understanding of the genomics, evolution, and taxonomy of this important fungal group. [ABSTRACT FROM AUTHOR]

tRNAs have recently gained attention for their novel regulatory roles in translation and for their diverse functions beyond translation. One of the most remarkable aspects of tRNA biogenesis is the incorporation of various chemical modifications, ranging from simple base or ribose methylation to more complex hypermodifications such as formation of queuosine and wybutosine. Some tRNAs are transcribed as intron-containing pre-tRNAs. While the majority of these modifications occur independently of introns, some are catalyzed in an intron-inhibitory manner, and in certain cases, they occur in an intron-dependent manner. This review focuses on pre-tRNA modification, including introncontaining pre-tRNA, in both intron-inhibitory and intron-dependent fashions. Any perturbations in the modification and processing of tRNAs may lead to a range of diseases and disorders, highlighting the importance of understanding these mechanisms in molecular biology and medicine. [ABSTRACT FROM AUTHOR]

Papilionoideae is the most species-rich subfamily of the third largest angiosperm family Fabaceae. One constituent large group, the inverted-repeat-lacking clade (IRLC), is well-known for the broad loss of one IR copy. Accumulating observations of massive plastomic disparities have made IRLC a well-suited model for exploring plastome evolution. However, there is still a large amount left to explore. The present study focused on the plastid tRNA (pttRNA) evolution within Papilionoideae, employing the currently densest sampling strategies for both the IRLC (156) and non-IRLC (109) lineages. Strikingly, our results revealed abundant inter-lineage variabilities in both tRNA sequences and structures, including a 3 nt difference in the average size of trnS-UGA, the consensus sequence disparities across 29 tRNAs, the distinct 3 nt indels in trnA-UGC, and an impressive 248 nt intron loss of IRLC trnI-GAU (potential markers). Additionally, there was unequal stability of the atypical secondary structures in trnS-GGA and trnS-UGA, as well as significantly diverse compositions of substitution events in all compared tRNAs (p < 0.05). Ultimately, these findings not only demonstrate the significant differences and unique markers of IRLC pttRNAs compared to other non-IRLC Papilionoideae, but also draw an important conclusion that the large losses of one IR potentially promote highly diverse evolutionary patterns of IRLC, which could partly compensate for the potential IR-lacking impacts. [ABSTRACT FROM AUTHOR]

Abstract The widely accepted hypothesis postulates that the first spliceosomal introns originated from group II self-splicing introns. However, it is evident that not all spliceosomal introns in the nuclear genes of modern eukaryotes are inherited through vertical transfer of intronic sequences. Several phenomena contribute to the formation of new introns but their most common origin seems to be the insertion of transposable elements. Recent analyses have highlighted instances of mass gains of new introns from transposable elements. These events often coincide with an increase or change in the spliceosome's tolerance to splicing signals, including the acceptance of noncanonical borders. Widespread acquisitions of transposon-derived introns occur across diverse evolutionary lineages, indicating convergent processes. These events, though independent, likely require a similar set of conditions. These conditions include the presence of transposon elements with features enabling their removal at the RNA level as introns and/or the existence of a splicing mechanism capable of excising unusual sequences that would otherwise not be recognized as introns by standard splicing machinery. Herein we summarize those mechanisms across different eukaryotic lineages.

Abstract In the present study, three mitogenomes from the Bipolaris genus (Bipolaris maydis, B. zeicola, and B. oryzae) were assembled and compared with the other two reported Bipolaris mitogenomes (B. oryzae and B. sorokiniana). The five mitogenomes were all circular DNA molecules, with lengths ranging from 106,403 bp to 135,790 bp. The mitogenomes of the five Bipolaris species mainly comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs, and a certain number of tRNAs and unidentified open reading frames (ORFs). The PCG length, AT skew and GC skew showed large variability among the 13 PCGs in the five mitogenomes. Across the 13 core PCGs tested, nad6 had the least genetic distance among the 16 Pleosporales species we investigated, indicating that this gene was highly conserved. In addition, the Ka/Ks values for all 12 core PCGs (excluding rps3) were < 1, suggesting that these genes were subject to purifying selection. Comparative mitogenomic analyses indicate that introns were the main factor contributing to the size variation of Bipolaris mitogenomes. The introns of the cox1 gene experienced frequent gain/loss events in Pleosporales species. The gene arrangement and collinearity in the mitogenomes of the five Bipolaris species were almost highly conserved within the genus. Phylogenetic analysis based on combined mitochondrial gene datasets showed that the five Bipolaris species formed well-supported topologies. This study is the first report on the mitogenomes of B. maydis and B. zeicola, as well as the first comparison of mitogenomes among Bipolaris species. The findings of this study will further advance investigations into the population genetics, evolution, and genomics of Bipolaris species.

The widely accepted hypothesis postulates that the first spliceosomal introns originated from group II self-splicing introns. However, it is evident that not all spliceosomal introns in the nuclear genes of modern eukaryotes are inherited through vertical transfer of intronic sequences. Several phenomena contribute to the formation of new introns but their most common origin seems to be the insertion of transposable elements. Recent analyses have highlighted instances of mass gains of new introns from transposable elements. These events often coincide with an increase or change in the spliceosome's tolerance to splicing signals, including the acceptance of noncanonical borders. Widespread acquisitions of transposon-derived introns occur across diverse evolutionary lineages, indicating convergent processes. These events, though independent, likely require a similar set of conditions. These conditions include the presence of transposon elements with features enabling their removal at the RNA level as introns and/or the existence of a splicing mechanism capable of excising unusual sequences that would otherwise not be recognized as introns by standard splicing machinery. Herein we summarize those mechanisms across different eukaryotic lineages. [ABSTRACT FROM AUTHOR]

Sex determination by DNA-based molecular techniques in Sumatran tigers needs to be investigated and developed for forensic and population genetic purposes. The amelogenin gene is a marker commonly used for sex determination. In some species, the difference between the AMELX and AMELY sequences has been reported to be in the intron region. However, the difference between the AMELX and AMELY sequences in the Sumatran tiger is unknown. Therefore, it is necessary to investigate the sequence differences in introns between AMELX and AMELY Sumatran tigers to determine the specific differences between male and female samples. This study aimed to analyse the sequence of nucleotide bases in the Sumatran tiger amelogenin gene introns based on the nucleotide base sequences in the amelogenin gene introns. The method in this research is descriptive, with a molecular observation of the AMELX and AMELY Sumatran tiger sequences. The amplified samples were sequenced, and it was found that the lengths of the AMELX and AMELY Sumatran tiger sequences were 215 bp and 194 bp, respectively, with a 21 bp deletion in the AMELY sequence. [ABSTRACT FROM AUTHOR]

In the present study, three mitogenomes from the Bipolaris genus (Bipolaris maydis, B. zeicola, and B. oryzae) were assembled and compared with the other two reported Bipolaris mitogenomes (B. oryzae and B. sorokiniana). The five mitogenomes were all circular DNA molecules, with lengths ranging from 106,403 bp to 135,790 bp. The mitogenomes of the five Bipolaris species mainly comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs, and a certain number of tRNAs and unidentified open reading frames (ORFs). The PCG length, AT skew and GC skew showed large variability among the 13 PCGs in the five mitogenomes. Across the 13 core PCGs tested, nad6 had the least genetic distance among the 16 Pleosporales species we investigated, indicating that this gene was highly conserved. In addition, the Ka/Ks values for all 12 core PCGs (excluding rps3) were < 1, suggesting that these genes were subject to purifying selection. Comparative mitogenomic analyses indicate that introns were the main factor contributing to the size variation of Bipolaris mitogenomes. The introns of the cox1 gene experienced frequent gain/loss events in Pleosporales species. The gene arrangement and collinearity in the mitogenomes of the five Bipolaris species were almost highly conserved within the genus. Phylogenetic analysis based on combined mitochondrial gene datasets showed that the five Bipolaris species formed well-supported topologies. This study is the first report on the mitogenomes of B. maydis and B. zeicola, as well as the first comparison of mitogenomes among Bipolaris species. The findings of this study will further advance investigations into the population genetics, evolution, and genomics of Bipolaris species. [ABSTRACT FROM AUTHOR]

The grooved carpet-shell clam is one of the most economically relevant shellfish species living in the Mediterranean and nearby Atlantic coasts. Previous studies using different types of genetic markers showed a remarkable genetic divergence of the eastern Mediterranean, western Mediterranean, and Atlantic populations, but important details remained unclear. Here, data from six nuclear introns scored for restriction fragment size polymorphisms in eight populations that have not been studied before have been pooled for the analysis with data scattered through three previous studies, totaling 32 samples from 29 locations. The results show lower levels of heterozygosity, higher mean number of alleles, and alleles with restricted distribution in the Mediterranean populations, suggesting the existence of a large, isolated population in the eastern Mediterranean at the middle Pleistocene. The data also confirm the similarity of populations from Tunisia to Western Mediterranean populations. Finally, a genetic mosaic is apparent in the Atlantic coasts of the Iberian Peninsula, with a divergence of Rias Baixas populations from more northern populations and Central Portugal populations. The effects of oceanic fronts, seasonal upwellings, river plumes, and/or fishery management operations could explain this and other features of the Atlantic populations. [ABSTRACT FROM AUTHOR]

Abstract Glucose is a critical nutrient for energy metabolism. The SLC2A2 gene is essential for glucose sensing and homeostasis, as it encodes the facilitated glucose transporter GLUT2. During diabetes treatment, the C-allele of rs8192675 in SLC2A2 has been found to regulate the action of metformin and reduce the absolute level of HbA1c more effectively than the T-allele. In this study, stable HEK293T cell lines carrying the CC, CT, and TT genotypes of rs8192675 in SLC2A2 were generated using CRISPR/Cas9-mediated genome editing. GLUT2 mRNA and protein levels were elevated in cell clones with the TC genotype compared to those with the CC genotype but were reduced relative to the TT genotype. Additionally, high concentrations of glucose or fructose induced more GLUT2 protein production in CT-genotype cells than that induced in CC-genotype cells, yet less than that induced in TT-genotype cells. Metformin induced a greater increase in GLUT2 expression and a smaller increase in activated AMPK protein expression in CC-genotype cells than those induced in TT-genotype cells, resulting in a remarkable reduction in activated mTOR and S6 levels. This study directly supports the biological mechanism linking the C-allele of rs8192675 with improved treatment outcomes in metformin therapy for diabetes.

The grooved carpet-shell clam is one of the most economically relevant shellfish species living in the Mediterranean and nearby Atlantic coasts. Previous studies using different types of genetic markers showed a remarkable genetic divergence of the eastern Mediterranean, western Mediterranean, and Atlantic populations, but important details remained unclear. Here, data from six nuclear introns scored for restriction fragment size polymorphisms in eight populations that have not been studied before have been pooled for the analysis with data scattered through three previous studies, totaling 32 samples from 29 locations. The results show lower levels of heterozygosity, higher mean number of alleles, and alleles with restricted distribution in the Mediterranean populations, suggesting the existence of a large, isolated population in the eastern Mediterranean at the middle Pleistocene. The data also confirm the similarity of populations from Tunisia to Western Mediterranean populations. Finally, a genetic mosaic is apparent in the Atlantic coasts of the Iberian Peninsula, with a divergence of Rias Baixas populations from more northern populations and Central Portugal populations. The effects of oceanic fronts, seasonal upwellings, river plumes, and/or fishery management operations could explain this and other features of the Atlantic populations.

Abstract 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. Graphical Abstract

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. [ABSTRACT FROM AUTHOR]

Plants are constantly exposed to different kinds of biotic stress, such as herbivore attack and wounding. To deal with these stresses, plants have evolved sophisticated defence mechanisms to protect themselves.Previously, we found that EIJ1 (EDS1‐interacting J protein 1) plays a negative regulatory role in plant disease resistance in Arabidopsis thaliana. Follow‐up studies revealed that EIJ1 specifically responds to wounding and herbivore stresses. The expression of EIJ1 was specifically induced by wounding or herbivore stress, as demonstrated by similar results in EIJ1 protein assay.Interestingly, GUS staining found that the promoter of EIJ1 is not involved in the induction of expression under wounding stress. Instead, we identified the first intron of EIJ1 as a key factor in response to wounding stress. Deleting the first intron of EIJ1 resulted in a loss of response to wounding stress in plants.Our results broaden the role of EIJ1 in plant resistance to biotic stress and provide new insights into plant responses to biotic stress. [ABSTRACT FROM AUTHOR]

ABSTRACT At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

Sex determination by DNA-based molecular techniques in Sumatran tigers needs to be investigated and developed for forensic and population genetic purposes. The amelogenin gene is a marker commonly used for sex determination. In some species, the difference between the AMELX and AMELY sequences has been reported to be in the intron region. However, the difference between the AMELX and AMELY sequences in the Sumatran tiger is unknown. Therefore, it is necessary to investigate the sequence differences in introns between AMELX and AMELY Sumatran tigers to determine the specific differences between male and female samples. This study aimed to analyse the sequence of nucleotide bases in the Sumatran tiger amelogenin gene introns based on the nucleotide base sequences in the amelogenin gene introns. The method in this research is descriptive, with a molecular observation of the AMELX and AMELY Sumatran tiger sequences. The amplified samples were sequenced, and it was found that the lengths of the AMELX and AMELY Sumatran tiger sequences were 215 bp and 194 bp, respectively, with a 21 bp deletion in the AMELY sequence.

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrast with long-term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far Introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with Introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Aquatic organisms were 6.5 times more likely to contain Introners than terrestrial organisms. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from nonautonomous transposable elements. Transposable elements and aquatic taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally, our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.

From the biota beneath the sea ice in Lake Saroma, which is adjacent to Sea of Okhotsk, a diatom culture of Saroma 16 was isolated. Strutted processes and a labiate process in Saroma 16 were characteristic of those in Thalassiosira nordenskioeldii. Similarity search analysis showed that the 826-bp rbcL-3P region sequence of this strain was 100% identical to multiple sequences registered as T. nordenskioeldii in a public database. The 4305-bp PCR-amplified mitochondrial cytochrome c oxidase subunit I (COI) gene (COI)-5P region of Saroma 16 included a 1060-bp open reading frame (ORF), which was interrupted by 934-bp and 2311-bp introns that included frame-shifted ORFs encoding reverse-transcriptase (RTase)-like proteins. Previous reports showed that a strain of the same species, CNS00052, originating from the East China Sea included no introns in the COI, whereas North Atlantic Ocean strains of the same species, such as CCMP992, CCMP993, and CCMP997, included a 2.3-kb intron in the same position as Saroma 16.

The species of Purpureocillium are cosmopolitan and multitrophic fungi that can infect a wide range of invertebrate hosts. This study reports the mitogenome of P. atypicola, a specialized spider pathogenic fungus. The 112,465 bp mitogenome encoded genes typically found in fungal mitogenomes, and a total of 52 introns inserted into seven genes. A comparison with three other Purpureocillium species revealed significant differences in length and intron number, primarily due to intron variation; however, there was no dynamic variation in the introns of the cox1 gene within the same species of the Purpureocillium genus. Different mitochondrial protein-coding genes showed variable degrees of genetic differentiation among these species, but they were all under purifying selection. Additionally, frequent intron loss or gain events were detected to have occurred during the evolution of the Ophiocordycipitaceae mitogenomes, yet the gene arrangement remains conserved. A phylogenetic analysis of the combined mitochondrial gene set gave identical and well-supported tree topologies. The estimated age of the crown of Ophiocordycipitaceae and Purpureocillium were around the Early Cretaceous period (127 Mya) and Late Cretaceous period (83 Mya), respectively. The results of this study advance our understanding of the genomics, evolution, and taxonomy of this important fungal group.

Variants that affect splice sites comprise 14.3% of all pathogenic variants in the SERPING1 gene; more than half of them are located outside the canonical sites. To make a clinical decision concerning patients with such variants, it is essential to know the exact way in which the effect of the variant would be realized. The optimal approach to determine the consequences is considered to be mRNA analysis. In the current study, we present the results of functional analysis of two previously non-described variants in the SERPING1 gene (NM_000062.3) affecting intron 4: c.686-1G>A and c.685+4dup, which were detected in members of two Russian families with autosomal dominant inheritance of angioedema type 1. Analysis of the patients' mRNA (extracted from whole blood) showed that the SERPING1(NM_000062.3):c.685+4dup variant leads to the loss of the donor splice site and the activation of the cryptic site in exon 4: r.710_745del (p.Gly217_Pro228del), while the SERPING1(NM_000062.3):c.686-1G>A variant leads to the skipping of exon 5: r.746_949del (p.Asp229_Ser296del). [ABSTRACT FROM AUTHOR]

Across eukaryotic genomes, multiple α- and β-tubulin genes require regulation to ensure sufficient production of tubulin heterodimers. Features within these gene families that regulate expression remain underexplored. Here, we investigate the role of the 5′ intron in regulating α-tubulin expression in Saccharomyces cerevisiae. We find that the intron in the α-tubulin, TUB1, promotes α-tubulin expression and cell fitness during microtubule stress. The role of the TUB1 intron depends on proximity to the TUB1 promoter and sequence features that are distinct from the intron in the alternative α-tubulin isotype, TUB3. These results lead us to perform a screen to identify genes that act with the TUB1 intron. We identified several genes involved in chromatin remodeling, α/β-tubulin heterodimer assembly, and the spindle assembly checkpoint. We propose a model where the TUB1 intron promotes expression from the chromosomal locus and that this may represent a conserved mechanism for tubulin regulation under conditions that require high levels of tubulin production. [ABSTRACT FROM AUTHOR]

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Diverse splicing types in nuclear and chloroplast genes of protist Euglena gracilis have been recognized for decades. However, the splicing machinery responsible for processing nuclear precursor messenger RNA introns, including trans-splicing of the 5′ terminal outron and spliced leader (SL) RNA, remains elusive. Here, we identify 166 spliceosomal protein genes and two snRNA genes from E. gracilis by performing bioinformatics analysis from a combination of next-generation and full-length transcriptomic RNA sequencing (RNAseq) data as well as draft genomic data. With the spliceosomal proteins we identified in hand, the insensitivity of E. gracilis to some splicing modulators is revealed at the sequence level. The prevalence of SL RNA-mediated trans-splicing is estimated to be more than 70% from our full-length RNAseq data. Finally, the splicing proteomes between E. gracilis and its three evolutionary cousins within the same Excavata group are compared. In conclusion, our study characterizes the spliceosomal components in E. gracilis and provides the molecular basis for further exploration of underlying splicing mechanisms.

Papilionoideae is the most species-rich subfamily of the third largest angiosperm family Fabaceae. One constituent large group, the inverted-repeat-lacking clade (IRLC), is well-known for the broad loss of one IR copy. Accumulating observations of massive plastomic disparities have made IRLC a well-suited model for exploring plastome evolution. However, there is still a large amount left to explore. The present study focused on the plastid tRNA (pttRNA) evolution within Papilionoideae, employing the currently densest sampling strategies for both the IRLC (156) and non-IRLC (109) lineages. Strikingly, our results revealed abundant inter-lineage variabilities in both tRNA sequences and structures, including a 3 nt difference in the average size of trnS-UGA, the consensus sequence disparities across 29 tRNAs, the distinct 3 nt indels in trnA-UGC, and an impressive 248 nt intron loss of IRLC trnI-GAU (potential markers). Additionally, there was unequal stability of the atypical secondary structures in trnS-GGA and trnS-UGA, as well as significantly diverse compositions of substitution events in all compared tRNAs (p < 0.05). Ultimately, these findings not only demonstrate the significant differences and unique markers of IRLC pttRNAs compared to other non-IRLC Papilionoideae, but also draw an important conclusion that the large losses of one IR potentially promote highly diverse evolutionary patterns of IRLC, which could partly compensate for the potential IR-lacking impacts.

Abstract Right temporal variant frontotemporal dementia, also called right-predominant semantic dementia, often has an unclear position within the framework of the updated diagnostic criteria for behavioral variant frontotemporal dementia or primary progressive aphasia. Recent studies have suggested that this population may be clinically, neuropathologically, and genetically distinct from those with behavioral variant frontotemporal dementia or left-predominant typical semantic variant primary progressive aphasia. Here we describe a Japanese case of right temporal variant frontotemporal dementia with novel heterozygous MAPT mutation Adenine to Thymidine in intervening sequence (IVS) 9 at position -7 from 3ʹ splicing site of intron 9/exon 10 boundary (MAPT IVS9-7A > T). Postmortem neuropathological analysis revealed a predominant accumulation of 4 repeat tau, especially in the temporal lobe, amygdala, and substantia nigra, but lacked astrocytic plaques or tufted astrocytes. Immunoelectron microscopy of the tau filaments extracted from the brain revealed a ribbon-like structure. Moreover, a cellular MAPT splicing assay confirmed that this novel variant promoted the inclusion of exon 10, resulting in the predominant production of 4 repeat tau. These data strongly suggest that the MAPT IVS9-7 A > T variant found in our case is a novel mutation that stimulates the inclusion of exon 10 through alternative splicing of MAPT transcript and causes predominant 4 repeat tauopathy which clinically presents as right temporal variant frontotemporal dementia.

Abstract Background Intracellular symbionts often undergo genome reduction, losing both coding and non-coding DNA in a process that ultimately produces small, gene-dense genomes with few genes. Among eukaryotes, an extreme example is found in microsporidians, which are anaerobic, obligate intracellular parasites related to fungi that have the smallest nuclear genomes known (except for the relic nucleomorphs of some secondary plastids). Mikrocytids are superficially similar to microsporidians: they are also small, reduced, obligate parasites; however, as they belong to a very different branch of the tree of eukaryotes, the rhizarians, such similarities must have evolved in parallel. Since little genomic data are available from mikrocytids, we assembled a draft genome of the type species, Mikrocytos mackini, and compared the genomic architecture and content of microsporidians and mikrocytids to identify common characteristics of reduction and possible convergent evolution. Results At the coarsest level, the genome of M. mackini does not exhibit signs of extreme genome reduction; at 49.7 Mbp with 14,372 genes, the assembly is much larger and gene-rich than those of microsporidians. However, much of the genomic sequence and most (8075) of the protein-coding genes code for transposons, and may not contribute much of functional relevance to the parasite. Indeed, the energy and carbon metabolism of M. mackini share several similarities with those of microsporidians. Overall, the predicted proteome involved in cellular functions is quite reduced and gene sequences are extremely divergent. Microsporidians and mikrocytids also share highly reduced spliceosomes that have retained a strikingly similar subset of proteins despite having reduced independently. In contrast, the spliceosomal introns in mikrocytids are very different from those of microsporidians in that they are numerous, conserved in sequence, and constrained to an exceptionally narrow size range (all 16 or 17 nucleotides long) at the shortest extreme of known intron lengths. Conclusions Nuclear genome reduction has taken place many times and has proceeded along different routes in different lineages. Mikrocytids show a mix of similarities and differences with other extreme cases, including uncoupling the actual size of a genome with its functional reduction.

ABSTRACTIntrons are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to

Metagenomics provides a tool to assess the functional potential of environmental and host‐associated microbiomes based on the analysis of environmental DNA: assembly, gene prediction and annotation. While gene prediction is straightforward for most bacterial and archaeal taxa, it has limited applicability in the majority of eukaryotic organisms, including fungi that contain introns in gene coding sequences. As a consequence, eukaryotic genes are underrepresented in metagenomics datasets and our understanding of the contribution of fungi and other eukaryotes to microbiome functioning is limited. Here, we developed a machine intelligence‐based algorithm that predicts fungal introns in environmental DNA with reasonable precision and used it to improve the annotation of environmental metagenomes. Intron removal increased the number of predicted genes by up to 9.1% and improved the annotation of several others. The proportion of newly predicted genes increased with the share of eukaryotic genes in the metagenome and—within fungal taxa—increased with the number of introns per gene. Our approach provides a tool named SVMmycointron for improved metagenome annotation, especially of microbiomes with a high proportion of eukaryotes. The scripts described in the paper are made publicly available and can be readily utilized by microbiome researchers analysing metagenomics data. [ABSTRACT FROM AUTHOR]

Simple Summary: The zoonotic liver flukes Opisthorchis viverrini and Clonorchis sinensis infect small mammals, such as cats, dogs, pigs, rodents, and rabbits, as well as humans. Human infection subsequently develops into bile duct malignancy, also referred to as cholangiocarcinoma (CCA). Understanding the molecular systematics and population genetics of these liver flukes has an important role in prevention and control, and is important in comprehending their roles in zoonotic transmission. Different molecular markers have varying evolution rates and contain different genetic information. Polymorphic genetic markers are necessary and more suitable for such investigations. Therefore, we screened seven intron regions of the taurocyamine kinase gene (TK) to determine their potential as genetic markers for population genetic investigations of the liver flukes O. viverrini and C. sinensis which were collected from a range of geographical isolates and animal hosts. We identified a suitable intron region of TK, i.e., intron 5 of domain 1 (TkD1Int5) as having the most potential as a polymorphic marker. Results showed that TkD1Int5 is effective in examining the genetic variation and heterozygosity of O. viverrini and C. sinensis, but further studies are required to better understand the role of different species of animals as reservoir hosts of these zoonotic liver flukes. Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called "O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts. [ABSTRACT FROM AUTHOR]

The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot. The receptor binding domain (RBD) sequence from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein was used to replace the eGFP sequence in the expression vector and the RBD expression was detected by qRT-PCR and western blot. The results showed that protein expression can be regulated by optimizing the combination of cis-acting elements. The vector with the CMV enhancer, EF-1α core promoter, and TEF terminator was found to express approximately threefold higher eGFP than the unmodified vector in different animal cells, as well as 2.63-fold higher recombinant RBD protein than the original vector in HEK-293T cells. Moreover, we suggest that combinations of multiple regulatory elements capable of regulating gene expression do not necessarily exhibit synergistic effects to enhance expression further. Overall, our findings provide insights into biological applications that require the regulation of gene expression and will help to optimize expression vectors for biosynthesis and other fields. Additionally, we provide valuable insights into the production of RBD proteins, which may aid in developing reagents for diagnosis and treatment during the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

The aim of the study was to clarify the phylogenetic relationships among Northern European Ancistrocerus and comparison of the applicability of evolutionarily neutral and non‐neutral markers for reconstruction of phylogeny. We used a 19,400 bp long dataset that included parts of mitochondrial DNA, nuclear rDNA operon, and 10 nuclear protein‐coding genes. Application of molecular barcoding and species delimitation algorithms unveiled a presence of cryptic species, A. balticus sp. n., in the trap‐nesting wasp communities of the centre of Europe. We assessed the morphological, biological, and ecological differences of it from the sibling A. trifasciatus and updated the regional identification key. Phylogeny reconstruction using the neutral and the presumably non‐neutral markers resulted in different tree topologies. Evolutionary congruence of the rDNA operon with the other markers was relatively low. Evolutionary rate of the mitochondrial genes was 7–8 times as high as that of the exons of the nuclear genes, therefore, the mitochondrial markers overshadowed the nuclear ones in the phylogeny reconstructions. We assumed that at the speciation level, we might consider two different patterns of phylogeny: one based on evolutionary time and neutral changes, and the other based on adaptive evolutionary pathways under directional selection pressures. We assessed the effect of directional selection on the nuclear protein‐coding genes, applying the Spearman's rank correlation between pairwise phylogenetic distances among species, estimated using exons, and these distances, estimated using introns. One of these markers demonstrated a lack of positive correlation, implying a variable directional selection pressure on the coded protein. The publication has been registered on ZooBank: urn:lsid:zoobank.org:pub:13BD28D0‐736D‐4B2A‐B5CF‐4824BD4CDCFB. [ABSTRACT FROM AUTHOR]

Introduction: Conventional genetic investigation fails to identify the F8 causal variant in 2.5%‐10% of haemophilia A (HA) patients with non‐severe phenotypes. In these cases, F8 deep intronic variants could be causal. Aim: To identify pathogenic F8 deep intronic variants in genetically unresolved families with non‐severe HA analysed in the haematology laboratory of the Hospices Civils de Lyon. Methods: The whole F8 was analysed by next generation sequencing. The pathogenic impact of candidate variants identified was assessed using both in silico analysis (MaxEntScan and spliceAI) and functional analysis (RNA or minigene assay). Results: Sequencing was performed in 49/55 families included for which a DNA sample from a male propositus was available. In total, 33 candidate variants from 43 propositi were identified. These variants corresponded to 31 single nucleotide substitutions, one 173‐bp deletion, and an 869‐bp tandem triplication. No candidate variant was found in six propositi. The most frequent variants found were the association of [c.2113+1154G>C and c.5374‐304C>T], identified in five propositi, and the c.2114‐6529C>G identified in nine propositi. Four variants had been previously described as HA‐causing. Splicing functional assay found a deleterious impact for 11 substitutions (c.671‐94G>A, c.788‐312A>G, c.2113+1154G>C, c.2114‐6529C>G, c.5999‐820A>T, c.5999‐786C>A, c.5999‐669G>T, c.5999‐669G>A, c.5999‐669G>C, c.6900+4104A>C, and c.6901‐2992A>G). The HA‐causing variant was identified in 33/49 (67%) cases. In total, F8 deep intronic variants caused 8.8% of the non‐severe HA among the 1643 families analysed in our laboratory. Conclusion: The results emphasise the value of whole F8 gene sequencing combined with splicing functional analyses to improve the diagnosis yield for non‐severe HA. [ABSTRACT FROM AUTHOR]

Progress in eupulmonate gastropod taxonomy is limited by absence of single- or low-copy nuclear DNA markers that provide species-scale insights. We detail here the single-copy intron 8 of the embryonic lethality and abnormal visual system gene (ELAVI8). High sequence conservation within flanking exons 8 and 9 allowed design of non-redundant primers that enable PCR amplification across a broad phylogenetic extent. Across the Eupulmonata and Hygrophila ELAVI8 product ranged from 602 to 802 base pairs in length. A multiple sequence alignment across 62 taxa, representing 36 genera and 12 major clades, showed 1296 sites with 865 variable positions. Typically, 2–10 base pair differences were noted between closely related species within a genus, with 74–200 bases differing between major infra-order clades. ELAVI8 maximum-likelihood trees showed essentially identical topologies at high levels of support compared to those created from concatenated ITS1 + 2 (within a genus) or 28S (across Eupulmonata and Hygrophila). ELAVI8 is superior to these two phylogenetic marker regions, however, because it (1) resolves phylogenetic pattern from species- to sub-cohort scales in a single amplicon, (2) was better able to resolve family- and deeper-clades at high support, (3) allows for outgroup rooting between genera, and (4) possesses no intercopy haplotype variability. [ABSTRACT FROM AUTHOR]

Abstract Apiotrichum is a diverse anamorphic basidiomycetous yeast genus, and its mitogenome characterization has not been revealed. In this study, we assembled two Apiotrichum mitogenomes and compared them with mitogenomes from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. The mitogenomes of Apiotrichum gracile and A. gamsii comprised circular DNA molecules, with sizes of 34,648 bp and 38,096 bp, respectively. Intronic regions were found contributed the most to the size expansion of A. gamsii mitogenome. Comparative mitogenomic analysis revealed that 6.85–38.89% of nucleotides varied between tRNAs shared by the two Apiotrichum mitogenomes. The GC content of all core PCGs in A. gamsii was lower than that of A. gracile, with an average low value of 4.97%. The rps3 gene differentiated the most among Agaricomycotina, Pucciniomycotina and Ustilaginomycotina species, while nad4L gene was the most conserved in evolution. The Ka/Ks values for cob and rps3 genes were > 1, indicating the two genes may be subjected to positive selection in Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. Frequent intron loss/gain events and potential intron transfer events have been detected in evolution of Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. We further detected large-scale gene rearrangements between the 19 mitogenomes from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina, and fifteen of the 17 mitochondrial genes shared by Apiotrichum varied in gene arrangements. Phylogenetic analyses based on maximum likelihood and Bayesian inference methods using a combined mitochondrial gene dataset revealed different taxonomic assignment of two Apiotrichum species, wherein A. gamsii had a more closely relationship with Trichosporon asahii. This study served as the first report on mitogenomes from the genus Apiotrichum, which promotes the understanding of evolution, genomics, and phylogeny of Apiotrichum.

Introduction: Genetic and environmental factors play a significant role in the development of Cardiovascular Disease (CVD). With advancements in molecular techniques, it is now possible to study genes that may provide evidence of the role of genetic factors in Coronary Artery Disease (CAD). Genetic variants of the Apo A-1 gene might play an important role in regulating lipid profiles and their alteration, leading to the subsequent development of Myocardial Infarction (MI). Aim: The aim of this study was to evaluate the association between C+83T sequence variations in the first intron of the Apo A-1 gene and lipid variables in patients with MI. Materials and Methods: A case-control study was conducted at the Department of Biochemistry in association with the Department of Cardiology, Kilpauk Medical College Hospital, Chennai, Tamil Nadu, India, from October 2013 to March 2014. The study included 52 MI cases and 52 age-, gender-, and risk factor-matched controls. Fasting venous blood samples were collected from each patient, and routine investigations, lipid profile assessments, and polymorphic studies were carried out. Allele frequencies between cases and controls were compared using the Chi-square test. Analysis of Variance (ANOVA) was performed to determine the association between fasting serum lipid variables and allele distribution. Results: Both study groups were matched for age, gender, and risk factors such as alcohol consumption, smoking, Hypertension (HT), and Diabetes Mellitus (DM). The results were compared and found to be statistically insignificant. Genotype analysis between the groups disclosed that the TT homozygous genotype was more prevalent in cases, while the ‘CC’ genotype was more prevalent in controls, and the CT genotype was approximately equal in both cases and controls. The genotype difference between cases and controls was statistically significant (p-value=0.006). The ‘T’ allele frequency was higher among cases (0.36) compared to controls (0.13), while the frequency of the ‘C’ allele was higher among controls (0.86) compared to cases (0.63). In the CC genotype, there was a higher mean value of Apo A-1 and a lower ApoB/Apo A-1 ratio compared to the TT genotype. It was observed that carriers of the C allele and T allele showed no statistical difference in lipid variables except for High-density Lipoprotein (HDL). Conclusion: This study found a significantly increased T allele frequency in cases compared to controls, suggesting that the presence of the T allele in the C+83T (first intron) of the Apo A-1 gene may increase the risk of developing MI.

Right temporal variant frontotemporal dementia, also called right-predominant semantic dementia, often has an unclear position within the framework of the updated diagnostic criteria for behavioral variant frontotemporal dementia or primary progressive aphasia. Recent studies have suggested that this population may be clinically, neuropathologically, and genetically distinct from those with behavioral variant frontotemporal dementia or left-predominant typical semantic variant primary progressive aphasia. Here we describe a Japanese case of right temporal variant frontotemporal dementia with novel heterozygous MAPT mutation Adenine to Thymidine in intervening sequence (IVS) 9 at position -7 from 3ʹ splicing site of intron 9/exon 10 boundary (MAPT IVS9-7A > T). Postmortem neuropathological analysis revealed a predominant accumulation of 4 repeat tau, especially in the temporal lobe, amygdala, and substantia nigra, but lacked astrocytic plaques or tufted astrocytes. Immunoelectron microscopy of the tau filaments extracted from the brain revealed a ribbon-like structure. Moreover, a cellular MAPT splicing assay confirmed that this novel variant promoted the inclusion of exon 10, resulting in the predominant production of 4 repeat tau. These data strongly suggest that the MAPT IVS9-7 A > T variant found in our case is a novel mutation that stimulates the inclusion of exon 10 through alternative splicing of MAPT transcript and causes predominant 4 repeat tauopathy which clinically presents as right temporal variant frontotemporal dementia. [ABSTRACT FROM AUTHOR]

Background: Intracellular symbionts often undergo genome reduction, losing both coding and non-coding DNA in a process that ultimately produces small, gene-dense genomes with few genes. Among eukaryotes, an extreme example is found in microsporidians, which are anaerobic, obligate intracellular parasites related to fungi that have the smallest nuclear genomes known (except for the relic nucleomorphs of some secondary plastids). Mikrocytids are superficially similar to microsporidians: they are also small, reduced, obligate parasites; however, as they belong to a very different branch of the tree of eukaryotes, the rhizarians, such similarities must have evolved in parallel. Since little genomic data are available from mikrocytids, we assembled a draft genome of the type species, Mikrocytos mackini, and compared the genomic architecture and content of microsporidians and mikrocytids to identify common characteristics of reduction and possible convergent evolution. Results: At the coarsest level, the genome of M. mackini does not exhibit signs of extreme genome reduction; at 49.7 Mbp with 14,372 genes, the assembly is much larger and gene-rich than those of microsporidians. However, much of the genomic sequence and most (8075) of the protein-coding genes code for transposons, and may not contribute much of functional relevance to the parasite. Indeed, the energy and carbon metabolism of M. mackini share several similarities with those of microsporidians. Overall, the predicted proteome involved in cellular functions is quite reduced and gene sequences are extremely divergent. Microsporidians and mikrocytids also share highly reduced spliceosomes that have retained a strikingly similar subset of proteins despite having reduced independently. In contrast, the spliceosomal introns in mikrocytids are very different from those of microsporidians in that they are numerous, conserved in sequence, and constrained to an exceptionally narrow size range (all 16 or 17 nucleotides long) at the shortest extreme of known intron lengths. Conclusions: Nuclear genome reduction has taken place many times and has proceeded along different routes in different lineages. Mikrocytids show a mix of similarities and differences with other extreme cases, including uncoupling the actual size of a genome with its functional reduction. [ABSTRACT FROM AUTHOR]

The emergence of introns was a significant evolutionary leap that is a major distinguishing feature between prokaryotic and eukaryotic genomes. While historically introns were regarded merely as the sequences that are removed to produce spliced transcripts encoding functional products, increasingly data suggests that introns play important roles in the regulation of gene expression. Here, we use an intron-centric lens to review the role of introns in eukaryotic gene expression. First, we focus on intron architecture and how it may influence mechanisms of splicing. Second, we focus on the implications of spliceosomal snRNAs and their variants on intron splicing. Finally, we discuss how the presence of introns and the need to splice them influences transcription regulation. Despite the abundance of introns in the eukaryotic genome and their emerging role regulating gene expression, a lot remains unexplored. Therefore, here we refer to introns as the “dark matter” of the eukaryotic genome and discuss some of the outstanding questions in the field. [ABSTRACT FROM AUTHOR]

RNA splicing refers to a process by which introns of a pre-mRNA are excised and the exons at both ends are joined together. Chloroplast introns are inherently self-splicing ribozymes, but over time, they have lost self-splicing ability due to the degeneration of intronic elements. Thus, the splicing of chloroplast introns relies heavily on nuclear-encoded splicing factors, which belong to diverse protein families. Different splicing factors and their shared intron targets are supposed to form ribonucleoprotein particles (RNPs) to facilitate intron splicing. As characterized in a previous review, around 14 chloroplast intron splicing factors were identified until 2010. However, only a few genetic and biochemical evidence has shown that these splicing factors are required for the splicing of one or several introns. The roles of splicing factors are generally believed to facilitate intron folding; however, the precise role of each protein in RNA splicing remains ambiguous. This may be because the precise binding site of most of these splicing factors remains unexplored. In the last decade, several new splicing factors have been identified. Also, several splicing factors were found to bind to specific sequences within introns, which enhanced the understanding of splicing factors. Here, we summarize recent progress on the splicing factors in land plant chloroplasts and discuss their possible roles in chloroplast RNA splicing based on previous studies.

Abstract In the present study, we assembled and analyzed the mitogenomes of two Ramaria species. The assembled mitogenomes of Ramaria cfr. rubripermanens and R. rubella were circularized, with sizes of 126,497 bp and 143,271 bp, respectively. Comparative mitogenome analysis showed that intron region contributed the most (contribution rate, 43.74%) to the size variations of Ramaria mitogenomes. The genetic contents, gene length, tRNAs, and codon usages of the two Ramaria mitogenomes varied greatly. In addition, the evolutionary rates of different core protein coding genes (PCGs) in Phallomycetidae mitogenomes varied. We detected large-scale gene rearrangements between Phallomycetidae mitogenomes, including gene displacement and tRNA doubling. A total of 4499 bp and 7746 bp aligned fragments were detected between the mitochondrial and nuclear genomes of R. cfr. rubripermanens and R. rubella, respectively, indicating possible gene transferring events. We further found frequent intron loss/gain and potential intron transfer events in Phallomycetidae mitogenomes during the evolution, and the mitogenomes of R. rubella contained a novel intron P44. Phylogenetic analyses using both Bayesian inference (BI) and Maximum Likelihood (ML) methods based on a combined mitochondrial gene dataset obtained an identical and well-supported phylogenetic tree for Basidiomycota, wherein R. cfr. rubripermanens and Turbinellus floccosus are sister species. This study served as the first report on mitogenomes from the genus Ramaria, which provides a basis for understanding the evolution, genetics, and taxonomy of this important fungal group.