Re-analysis of Next-generation Sequencing Data in Patients with Hypertrophic Cardiomyopathy: Contribution of Spliceogenic MYBPC3 Variants in an Italian Cohort.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac muscle disease characterized by clinical and genetic heterogeneity. Genetic testing can reveal the presence of disease-causing variants in genes encoding sarcomere proteins. However, it yields inconclusive or negative results in 40-60% of HCM cases, owing to, among other causes, technical limitations such as the inability to detect pathogenic intronic variants. Therefore, we aimed to increase the diagnostic yield of molecular analysis for HCM by improving the in-silico detection of intronic variants in MYBPC3 that may escape detection by algorithms normally used with tagged diagnostic panels. We included 142 HCM probands with negative results in Illumina TruSight Cardio panel analysis, including exonic regions of 174 cardiomyopathy genes. Raw data were re-analyzed using existing bioinformatics tools. The spliceogenic variant c.1224-80G>A was detected in three patients (2.1%), leading us to reconsider their molecular diagnosis. These patients showed late onset and mild symptoms, although no peculiar phenotypic characteristics were shared. Collectively, rare spliceogenic MYBPC3 variants may play a role in causing HCM, and their systematic detection should be performed to provide more comprehensive solutions in genetic testing using multigenic panels.

Genomic compliance with Chargaff's second parity rule may have originated non-adaptively, but stem-loops now function adaptively.

Of Chargaff's four rules on DNA base quantity, his second parity rule (PR-2) is the most contentious. Various biometricians (e.g., Sueoka, Lobry) regarded PR-2 compliance as a non-adaptive feature of modern genomes that could be modeled through interrelations among mutation rates. However, PR-2 compliance with stem-loop potential was considered adaptively relevant by biochemists familiar with analyses of nucleic acid structure (e.g., of Crick) and of meiotic recombination (e.g., of Kleckner). Meanwhile, other biometricians had shown that PR-2 complementarity extended beyond individual bases (1-mers) to oligonucleotides (k-mers), possibly reflecting "advantageous DNA structure" (Nussinov). An "introns early" hypothesis (Reanney, Forsdyke) had suggested a primordial nucleic acid world with recombination-mediated error-correction requiring genome-wide stem-loop potential to have evolved prior to localized intrusions of protein-encoding potential (exons). Thus, a primordial genome was equivalent to one long intron. Indeed, when assessed as the base order-dependent component (correcting for local influences of GC%), modern genes, especially when evolving rapidly under positive Darwinian selection, display high intronic stem-loop potential. This suggests forced migration from neighboring exons by competing protein-encoding potential. PR-2 compliance may have first arisen non-adaptively. Primary prototypic structures were later strengthened by their adaptive contribution to recombination. Thus, contentious views may actually be in harmony.

Can stable introns and noncoding RNAs be harnessed to improve health through activation of mitohormesis?

Ever since their introduction a decade ago, stable introns, a type of noncoding (nc)RNAs, are found to be key players in different important cellular processes acting through regulation of gene expression and feedback loops to maintain cellular homeostasis. Despite being commonly regarded as useless byproducts, recent studies in yeast suggested that stable introns are essential for cell survivability under starvation. In Drosophila, we found that a stable intron, sisR-1, has a direct effect in regulating mitochondrial dynamics during short-term fasting and subsequently improved overall oocyte quality. We speculated that the beneficial effects implicated by sisR-1 is through the activation of mitohormesis, an interesting phenomenon in mitochondrial biology. Mitohormesis is suggested to improve health span and lifespan of cells and organisms, but the involvement of ncRNAs is not well-documented. Here, we discuss the potential role of sisR-1 and other ncRNAs in activating mitohormesis and the possible applications in improving cellular and organismal health.

The polyphyletic origins of glycyl-tRNA synthetase and lysyl-tRNA synthetase and their implications.

I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), making plausible the following implications. The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. Indeed, as suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages, as analyzed here. Furthermore, this late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. Indeed, this polyphyletic origin would imply that the genetic code was still evolving because at least two ARSs, i.e. proteins that make the genetic code possible today, were still evolving. This would imply that the evolutionary stages involved were characterized not by cells but by protocells, that is, by progenotes because this is precisely the definition of a progenote. This conclusion would be strengthened by the observation that both GlyRS and LysRS originating in the phyletic lineages leading to bacteria and archaea, would demonstrate that, more generally, proteins were most likely still in rapid and progressive evolution. Namely, a polyphyletic origin of proteins which would qualify at least the initial phase of the evolutionary stage of the ancestor of bacteria and that of archaea as stages belonging to the progenote.

Intra-Phenotypic and -Genotypic Variations of Beauveria bassiana (Bals.) Vuill. Strains Infecting Aedes aegypti L. Adults.

Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain's geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL50) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification.

Molecular impact of mutations in RNA splicing factors in cancer.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing.

Interconnected roles of fungal nuclear- and intron-encoded maturases: at the crossroads of mitochondrial intron splicing.

Group I and II introns are large catalytic RNAs (ribozymes) that are frequently encountered in fungal mitochondrial genomes. The discovery of respiratory mutants linked to intron splicing defects demonstrated that for the efficient removal of organellar introns there appears to be a requirement of protein splicing factors. These splicing factors can be intron-encoded proteins with maturase activities that usually promote the splicing of the introns that encode them (cis-acting) and/or nuclear-encoded factors that can promote the splicing of a range of different introns (trans-acting). Compared to plants organellar introns, fungal mitochondrial intron splicing is still poorly explored, especially in terms of the synergy of nuclear factors with intron-encoded maturases that has direct impact on splicing through their association with intron RNA. In addition, nuclear-encoded accessory factors might drive the splicing impetus through translational activation, mitoribosome assembly, and phosphorylation-mediated RNA turnover. This review explores protein-assisted splicing of introns by nuclear and mitochondrial-encoded maturases as a means of mitonuclear interplay that could respond to environmental and developmental factors promoting phenotypic adaptation and potentially speciation. It also highlights key evolutionary events that have led to changes in structure and ATP-dependence to accommodate the dual functionality of nuclear and organellar splicing factors.

Molecular taxonomic characterization and infra-specific diversity of entomopathogenic Beauveria bassiana fungi from Argentina.

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

Historical fragmentation and stepping-stone gene flow led to population genetic differentiation in a coastal seabird.

Understanding the forces that shape population genetic structure is fundamental both for understanding evolutionary trajectories and for conservation. Many factors can influence the geographic distribution of genetic variation, and the extent to which local populations differ can be especially difficult to predict in highly mobile organisms. For example, many species of seabirds are essentially panmictic, but some show strong structure. Pigeon Guillemots (Cepphus columba; Charadriiformes: Alcidae) breed in small colonies scattered along the North Pacific coastline and feed in shallow nearshore waters year-round. Given their distribution, gene flow is potentially lower and population genetic structure is stronger than in most other high-latitude Northern Hemisphere seabirds. We screened variation in the mitochondrial control region, four microsatellite loci, and two nuclear introns in 202 Pigeon Guillemots representing three of five subspecies. Mitochondrial sequences and nuclear loci both showed significant population differences, although structure was weaker for the nuclear loci. Genetic differentiation was correlated with geographic distance between sampling locations for both the mitochondrial and nuclear loci. Mitochondrial gene trees and demographic modeling both provided strong evidence for two refugial populations during the Pleistocene glaciations: one in the Aleutian Islands and one farther east and south. We conclude that historical fragmentation combined with a stepping-stone model of gene flow led to the relatively strong population differentiation in Pigeon Guillemots compared to other high-latitude Northern Hemisphere seabird species. Our study adds to growing evidence that Pleistocene glaciation events affected population genetic structure not only in terrestrial species but also in coastal marine animals.

Circular code in introns.

A massive statistical analysis based on the autocorrelation function of the circular code X observed in genes is performed on the (eukaryotic) introns. Surprisingly, a circular code periodicity 0 modulo 3 is identified in 5 groups of introns: birds, ascomycetes, basidiomycetes, green algae and land plants. This circular code periodicity, which is a property of retrieving the reading frame in (protein coding) genes, may suggest that these introns have a coding property. In a well-known way, a periodicity 1 modulo 2 is observed in 6 groups of introns: amphibians, fishes, mammals, other animals, reptiles and apicomplexans. A mixed periodicity modulo 2 and 3 is found in the introns of insects. Astonishing, a subperiodicity 3 modulo 6 is a common statistical property in these 3 classes of introns. When the particular trinucleotides N1N2N1 of the circular code X are not considered, the circular code periodicity 0 modulo 3, hidden by the periodicity 1 modulo 2, is now retrieved in 5 groups of introns: amphibians, fishes, other animals, reptiles and insects. Thus, 10 groups of introns, taxonomically different, out of 12 have a coding property related to the reading frame retrieval. The trinucleotides N1N2N1 are analysed in the 216 maximal C3 self-complementary trinucleotide circular codes. A hexanucleotide code (words of 6 letters) is proposed to explain the periodicity 3 modulo 6. It could be a trace of more general circular codes at the origin of the circular code X.

Human exons and introns classification using pre-trained Resnet-50 and GoogleNet models and 13-layers CNN model.

BACKGROUND: Examining functions and characteristics of DNA sequences is a highly challenging task. When it comes to the human genome, which is made up of exons and introns, this task is more challenging. Human exons and introns contain millions to billions of nucleotides, which contributes to the complexity observed in this sequences. Considering how complicated the subject of genomics is, it is obvious that using signal processing techniques and deep learning tools to build a strong predictive model can be very helpful for the development of the research of the human genome. RESULTS: After representing human exons and introns with color images using Frequency Chaos Game Representation, two pre-trained convolutional neural network models (Resnet-50 and GoogleNet) and a proposed CNN model having 13 hidden layers were used to classify our obtained images. We have reached a value of 92% for the accuracy rate for Resnet-50 model in about 7 h for the execution time, a value of 91.5% for the accuracy rate for the GoogleNet model in 2 h and a half for the execution time. For our proposed CNN model, we have reached 91.6% for the accuracy rate in 2 h and 37 min. CONCLUSIONS: Our proposed CNN model is faster than the Resnet-50 model in terms of execution time. It was able to slightly exceed the GoogleNet model for the accuracy rate value.

Minor intron-containing genes as an ancient backbone for viral infection?

Minor intron-containing genes (MIGs) account for <2% of all human protein-coding genes and are uniquely dependent on the minor spliceosome for proper excision. Despite their low numbers, we surprisingly found a significant enrichment of MIG-encoded proteins (MIG-Ps) in protein-protein interactomes and host factors of positive-sense RNA viruses, including SARS-CoV-1, SARS-CoV-2, MERS coronavirus, and Zika virus. Similarly, we observed a significant enrichment of MIG-Ps in the interactomes and sets of host factors of negative-sense RNA viruses such as Ebola virus, influenza A virus, and the retrovirus HIV-1. We also found an enrichment of MIG-Ps in double-stranded DNA viruses such as Epstein-Barr virus, human papillomavirus, and herpes simplex viruses. In general, MIG-Ps were highly connected and placed in central positions in a network of human-host protein interactions. Moreover, MIG-Ps that interact with viral proteins were enriched with essential genes. We also provide evidence that viral proteins interact with ancestral MIGs that date back to unicellular organisms and are mainly involved in basic cellular functions such as cell cycle, cell division, and signal transduction. Our results suggest that MIG-Ps form a stable, evolutionarily conserved backbone that viruses putatively tap to invade and propagate in human host cells.

Organellar genomic characterization of Anunuuluaehu liula representing a new genus and species of Phyllophoraceae (Gigartinales, Rhodophyta) from the mesophotic zone of Hawai'i.

Over the last 2 decades, routine collections in the Hawaiian Archipelago have expanded to mesophotic reefs, leading to the discovery of a new red algal genus and species, here described as Anunuuluaehu liula gen. et sp. nov. This study provides a detailed genus and species description and characterizes chloroplast and mitochondrial organellar genomes. The new genus, Anunuuluaehu, shares many characteristics with the family Phyllophoraceae and shows close similarities to Archestennogramma and Stenogramma, including habit morphology, nemathecia forming proliferations at the outer cortex with terminal chains of tetrasporangia, and carposporophytes with multi-layered pericarps. The single species in this genus exhibits distinctive features within the Phyllophoraceae: the presence of single-layer construction of large medullary cells and the development of long, tubular gonimoblastic filaments. Multi-gene phylogenetic analyses confirmed it as a unique, monophyletic lineage within the family. Cis-splicing genes, interrupted by intron-encoded proteins within group II introns, are present in both the chloroplast and mitochondrial genomes of A. liula. Notably, a specific region of the coxI group II intron exhibits similarity to fungal introns. Anunuuluaehu liula is presumed to be endemic to the Hawaiian Archipelago and thus far is known to live solely at mesophotic depths from Holaniku to Kaho'olawe ranging from 54 to 201 m, which is the deepest collection record of any representative in the family. Overall, this study enhances our understanding of the genomic and taxonomic complexities of red algae in mesophotic habitats, emphasizing the significance of continued research in this area to uncover further insights into evolutionary processes and biogeographic patterns.

Genomic imprints of unparalleled growth.

Chlorella ohadii was isolated from desert biological soil crusts, one of the harshest habitats on Earth, and is emerging as an exciting new green model for studying growth, photosynthesis and metabolism under a wide range of conditions. Here, we compared the genome of C. ohadii, the fastest growing alga on record, to that of other green algae, to reveal the genomic imprints empowering its unparalleled growth rate and resistance to various stressors, including extreme illumination. This included the genome of its close relative, but slower growing and photodamage sensitive, C. sorokiniana UTEX 1663. A larger number of ribosome-encoding genes, high intron abundance, increased codon bias and unique genes potentially involved in metabolic flexibility and resistance to photodamage are all consistent with the faster growth of C. ohadii. Some of these characteristics highlight general trends in Chlorophyta and Chlorella spp. evolution, and others open new broad avenues for mechanistic exploration of their relationship with growth. This work entails a unique case study for the genomic adaptations and costs of exceptionally fast growth and sheds light on the genomic signatures of fast growth in photosynthetic cells. It also provides an important resource for future studies leveraging the unique properties of C. ohadii for photosynthesis and stress response research alongside their utilization for synthetic biology and biotechnology aims.

Spliceosomal introns in the diplomonad parasite Giardia duodenalis revisited.

Complete reference genomes, including correct feature annotations, are a fundamental aspect of genomic biology. In the case of protozoan species such as Giardia duodenalis, a major human and animal parasite worldwide, accurate genome annotation can deepen our understanding of the evolution of parasitism and pathogenicity by identifying genes underlying key traits and clinically relevant cellular mechanisms, and by extension, the development of improved prevention strategies and treatments. This study used bioinformatics analyses of Giardia mRNA libraries to characterize known introns and identify new intron candidates, working towards completion of the G. duodenalis assemblage A strain 'WB' genome and further elucidating Giardia's gene expression. By using a set of experimentally validated positive control loci to calibrate our intron detection pipeline, we were able to detect evidence of previously missed candidate splice junctions directly from expressed transcript data. These intron candidates were further studied in silico using NMDS (non-metric multidimensional scaling) clustering to determine shared characteristics and their relative importance such as secondary structure, splicing efficiency and motif conservation, and thus to refine intron models. Results from this study identified 34 new intron candidates, with several potential introns showing evidence that secondary structure of the mRNA molecule might play a more significant role in splicing than previously reported eukaryotic splicing activity mediated by a reduced spliceosome present in G. duodenalis.

Exploring the Relationship between Fusion Genes and MicroRNAs in Cancer.

Fusion genes are key cancer driver genes that can be used as potential drug targets in precision therapies, and they can also serve as accurate diagnostic and prognostic biomarkers. The fusion genes can cause microRNA (miRNA/miR) aberrations in many types of cancer. Nevertheless, whether fusion genes incite miRNA aberrations as one of their many critical oncogenic functionalities for driving carcinogenesis needs further investigation. Recent discoveries of miRNA genes that are present within the regions of genomic rearrangements that initiate fusion gene-based intronic miRNA dysregulation have brought the fusion genes into the limelight and revealed their unexplored potential in the field of cancer biology. Fusion gene-based 'promoter-switch' event aberrantly activate the miRNA-related upstream regulatory signals, while fusion-based coding region alterations disrupt the original miRNA coding loci. Fusion genes can potentially regulate the miRNA aberrations regardless of the protein-coding capability of the resultant fusion transcript. Studies on out-of-frame fusion and nonrecurrent fusion genes that cause miRNA dysregulation have attracted the attention of researchers on fusion genes from an oncological perspective and therefore could have potential implications in cancer therapies. This review will provide insights into the role of fusion genes and miRNAs, and their possible interrelationships in cancer.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Root Primordium Defective 1 Encodes an Essential PORR Protein Required for the Splicing of Mitochondria Encoded Group II Introns and for Respiratory Complex I Biogenesis.

Cellular respiration involves complex organellar metabolic activities that are pivotal for plant growth and development. Mitochondria contain their own genetic system (mitogenome, mtDNA), which encodes key elements of the respiratory machinery. Plant mtDNAs are notably larger than their counterparts in Animalia, with complex genome organization and gene-expression characteristics. The maturation of the plant mitochondrial transcripts involves extensive RNA editing, trimming and splicing events. These essential processing steps rely on the activities of numerous nuclear-encoded cofactors, which may also play key regulatory roles in mitochondrial biogenesis and function, and hence in plant physiology. Proteins that harbor the Plant Organelle RNA Recognition (PORR) domain are represented in a small gene family in plants. Several PORR members, including WTF1, WTF9 and LEFKOTHEA, are known to act in the splicing of organellar group II introns in angiosperms. The AT4G33495 gene-locus encodes an essential PORR-protein in Arabidopsis, termed as ROOT PRIMORDIUM DEFECTIVE 1 (RPD1). A null mutation of At.RPD1 causes arrest in early embryogenesis, while the missense mutant lines, rpd1.1 and rpd1.2, exhibit a strong impairment in root development and retarded growth phenotypes, especially under high-temperature conditions. Here, we further show that RPD1 functions in the splicing of introns that reside in the coding regions of various complex I (CI) subunits (i.e., nad2, nad4, nad5 and nad7), as well as in the maturation of the ribosomal rps3 pre-RNA in Arabidopsis mitochondria. The altered growth and developmental phenotypes and modified respiration activities are tightly correlated with respiratory chain CI defects in rpd1 mutants.

The intragenic cis-elements mediate temperature response of RrKSN.

Gene regulation via intragenic sequences is becoming more recognized in many eukaryotes. However, the intragenic sequences mediated gene expressions in response to environmental stimuli have been largely uncharacterized. Here, we showed that the first intron of RrKSN from the Rosa rugosa cultivar 'Purple branch' had a positive effect on RrKSN expression, and the effect depends on its position and orientation. Further analyses revealed that the four adjacent cis-elements (T)CGATT/AATCG(A) within the first intron were critical for the positive regulation, and the RrKSN promotion was significantly suppressed with mutations of these elements. These cis-elements were further evidenced as binding sites for RrARR1, the homologous of Arabidopsis type-B ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) transcription factor. The first intron-mediated RrKSN expression was enhanced with over-expressing of RrARR1, but abolished with RrARR1 silencing in rose seedlings. Moreover, the expression difference of RrKSN between 16°C and 28°C was eliminated along with RrARR1-silencing. Taken together, these results suggested both RrARR1 and its binding elements are required for the first intron-mediated RrKSN expression in response to varying temperatures. Therefore, our results reveal a unique intragenic regulation mechanism of gene expression by which plants perceive the signal of ambient temperature in rose.

Promoters and introns as key drivers for enhanced gene expression in Saccharomyces cerevisiae.

The transcription of genes in the yeast Saccharomyces cerevisiae is governed by multiple layers of regulatory elements and proteins, cooperating to ensure optimum expression of the final protein product based on the cellular requirements. Promoters have always been regarded as the most important determinant of gene transcription, but introns also play a key role in the expression of intron-encoding genes. Some introns can enhance transcription when introduced either promoter-proximal or embedded in the open reading frame of genes. However, the outcome is seldom predictable, with some introns increasing or decreasing transcription depending on the promoter and reporter gene employed. This chapter provides an overview of the general structure and function of promoters and introns and how they may cooperate during transcription to allow intron-mediated enhancement of gene expression. Since S. cerevisiae is a suitable host for recombinant protein production on a commercial level, stronger and more controllable promoters are in high demand. Enhanced gene expression can be achieved via promoter engineering, which may include introns that increase the efficacy of recombinant expression cassettes. Different models for the role of introns in transcription are briefly discussed to show how these intervening sequences can actively interact with the transcription machinery. Furthermore, recent examples of improved protein production via the introduction of promoter-proximal introns are highlighted to showcase the potential value of intron-mediated enhancement of gene expression.