Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.

Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors

Exon-intron structure and sequence variation of the calreticulin gene among Rhipicephalus sanguineus group ticks

Background: Calreticulin proteins (CRTs) are important components of tick saliva, which is involved in the blood meal success, pathogen transmission and host allergic responses. The characterization of the genes encoding for salivary proteins, such as CRTs, is pivotal to understand the mechanisms of tick-host interaction during blood meal and to develop tick control strategies based on their inhibition. In hard ticks, crt genes were shown to have only one intron with conserved position among species. In this study we investigated the exon-intron structure and variation of the crt gene in Rhipicephalus spp. ticks in order to assess the crt exon-intron structure and the potential utility of crt gene as a molecular marker. Methods: We sequenced the exon-intron region of crt gene in ticks belonging to so-called tropical and temperate lineages of Rhipicephalus sanguineus (sensu lato), Rhipicephalus sp. I, Rhipicephalus sp. III, Rhipicephalus sp. IV, R. guilhoni, R. muhsamae and R. turanicus. Genetic divergence and phylogenetic relationships between the sequences obtained were estimated. Results: All individuals belonging to the tropical lineage of R. sanguineus (s. l.), R. guilhoni, R. muhsamae, R. turanicus, Rhipicephalus sp. III and Rhipicephalus sp. IV analysed showed crt intron-present alleles. However, both crt intron-present and intron-absent alleles were found in Rhipicephalus sp. I and the temperate lineage of R. sanguineus (s. l.), showing the occurrence of an intraspecific intron presence-absence polymorphism. Phylogenetic relationships among the crt intron-present sequences showed distinct lineages for all taxa, with the tropical and temperate lineages of R. sanguineus (s. l.) being more closely related to each other. Conclusions: We expanded previous studies about the characterization of crt gene in hard ticks. Our results highlighted a previously overlooked variation in the crt structure among Rhipicephalus spp., and among hard ticks in general. Notably, the intron presence/absence polymorphism observed herein can be a candidate study-system to investigate the early stages of intron gain/loss before fixation at species level and some debated questions about intron evolution. Finally, the sequence variation observed supports the suitability of the crt gene for molecular recognition of Rhipicephalus spp. and for phylogenetic studies in association with other markers

The largest reservoir of mitochondrial introns is a relic of an ancestral split gene

In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms (ref. 1-4). Large introns (0.1 to 5 kbp) are frequent in mitochondrial genomes of plant and fungi (ref. 1,5) but scarce in Metazoa, despite these organisms are grouped with fungi among Opisthokonts. Introns are classified in two main groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism (ref. 5,6). Most of the group I introns carry a "Homing Endonuclease Gene" (ref. 7-9) encoding a DNA endonuclease acting in the transfer and site specific integration "homing") and allowing the intron spreading and gain after lateral transfer even between species from different kingdoms (ref. 10,11). Opposite to this "late intron" paradigm, the "early intron" theory indicates that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss (ref. 12,13).

Here we report the sequence of the cox1 gene of the button mushroom _Agaricus bisporus_, the most worldwide cultivated mushroom. This gene is both the longest mitochondrial gene (29,902 nt) and the largest Group I intron reservoir reported to date. An analysis of the group I introns available in _cox1_ genes shows that they are ancestral mobile genetic elements, whose frequent events of loss (according to the "late theory") and gain by lateral transfer ("early theory") must be combined to explain their wide and patchy distribution extending on several kingdoms. This allows the conciliation of the "early" and "late intron" paradigms, which are still matters of much debate (ref. 14,15). The overview of the intron distribution indicates that they evolve towards elimination. In such a landscape of eroded and lost intron sequences, the _A. bisporus_ largest intron reservoir, by its singular dynamics of intron keeping and catching, constitutes the most fitted relic of an early split gene

Peroxisome Proliferator-activated receptor alpha gene variation influences age of onset and progression of type 2 diabetes

Dysregulation of fatty acid metabolism is important in the pathogenesis of type 2 diabetes. Peroxisome proliferator-activated receptor (PPAR) is a master regulator of fatty acid catabolism, and PPAR activators delay the onset of type 2 diabetes. We examined association between three PPAR gene polymorphisms (an AC variant in intron 1, the L162V variant, and the intron 7 GC variant) and age at diagnosis of type 2 diabetes in 912 Caucasian type 2 diabetic subjects. Individually, PPAR gene variants did not influence age at diagnosis, but in combination, the rare alleles of both the intron 1 AC (P < 0.001) and intron 7 GC (P = 0.025) variants synergistically lowered age at diagnosis (interaction P < 0.001). Overall, the PPAR haplotype signficantly influenced age at diagnosis (P = 0.027), with the C-L-C and C-V-C haplotypes (intron 1-L162V-intron 7) accelerating onset of diabetes by 5.9 (P = 0.02) and 10 (P = 0.03) years, respectively, as compared with the common A-L-G haplotype, and was associated with an odds ratio for early-onset diabetes (age at diagnosis 45 years) of 3.75 (95% CI 1.65–8.56, P = 0.002). Intron 1 C-allele carriers also progressed more rapidly to insulin monotherapy (AA 9.4 ± 1.5 and AC + CC 5.3 ± 1.1 years, P = 0.002). These data indicate that PPAR gene variation influences the onset and progression of type 2 diabetes

Upregulation of Functional Kv11.1a Isoform Expression by Modified U1 Small Nuclear RNA

The KCNH2 or human ether-a go-go-related gene (hERG) encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier potassium current in the heart. The expression of Kv11.1 C-terminal isoforms is directed by the alternative splicing and polyadenylation of intron 9. Splicing of intron 9 leads to the formation of a functional, full-length Kv11.1a isoform and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO plays an important role in regulating Kv11.1 channel function. In the heart, only one-third of KCNH2 pre-mRNA is processed to Kv11.1a due to the weak 5′ splice site of intron 9. We previously showed that the weak 5′ splice site is caused by sequence deviation from the consensus, and that mutations toward the consensus sequence increased the efficiency of intron 9 splicing. It is well established that 5′ splice sites are recognized by complementary base-paring with U1 small nuclear RNA (U1 snRNA). In this study, we modified the sequence of U1 snRNA to increase its complementarity to the 5′ splice site of KCNH2 intron 9 and observed a significant increase in the efficiency of intron 9 splicing. RNase protection assay and western blot analysis showed that modified U1 snRNA increased the expression of the functional Kv11.1a isoform and concomitantly decreased the expression of the non-functional Kv11.1a-USO isoform. In patch-clamp experiments, modified U1 snRNA significantly increased Kv11.1 current. Our findings suggest that relative expression of Kv11.1 C-terminal isoforms can be regulated by modified U1 snRNA

Structural dynamics and divergence of the polygalacturonase gene family in land plants

A distinct feature of eukaryotic genomes is the presence of gene families. The polygalacturonase (PG) (EC3.2.1.15) gene family is one of the largest gene families in plants. PG is a pectin-digesting enzyme with a glycoside hydrolase 28 domain. It is involved in numerous plant developmental processes. The evolutionary processes accounting for the functional divergence and the specialized functions of PGs in land plants are unclear. Here, phylogenetic and gene structure analysis of PG genes in algae and land plants revealed that land plant PG genes resulted from differential intron gain and loss, with the latter event predominating. PG genes in land plants contained 15 homologous intron blocks and 13 novel intron blocks. Intron position and phase were not conserved between PGs of algae and land plants but conserved among PG genes of land plants from moss to vascular plants, indicating that the current introns in the PGs in land plants appeared after the split between unicellular algae and multicelluar land plants. These findings demonstrate that the functional divergence and differentiation of PGs in land plants is attributable to intronic loss. Moreover, they underscore the importance of intron gain and loss in genomic adaptation to selective pressure

In search of lost introns

Many fundamental questions concerning the emergence and subsequent evolution of eukaryotic exon-intron organization are still unsettled. Genome-scale comparative studies, which can shed light on crucial aspects of eukaryotic evolution, require adequate computational tools. We describe novel computational methods for studying spliceosomal intron evolution. Our goal is to give a reliable characterization of the dynamics of intron evolution. Our algorithmic innovations address the identification of orthologous introns, and the likelihood-based analysis of intron data. We discuss a compression method for the evaluation of the likelihood function, which is noteworthy for phylogenetic likelihood problems in general. We prove that after $O(nL)$ preprocessing time, subsequent evaluations take $O(nL/\log L)$ time almost surely in the Yule-Harding random model of $n$-taxon phylogenies, where $L$ is the input sequence length. We illustrate the practicality of our methods by compiling and analyzing a data set involving 18 eukaryotes, more than in any other study to date. The study yields the surprising result that ancestral eukaryotes were fairly intron-rich. For example, the bilaterian ancestor is estimated to have had more than 90% as many introns as vertebrates do now

Group II Intron Protein Localization and Insertion Sites Are Affected by Polyphosphate

Mobile group II introns consist of a catalytic intron RNA and an intron-encoded protein with reverse transcriptase activity, which act together in a ribonucleoprotein particle to promote DNA integration during intron mobility. Previously, we found that the Lactococcus lactis Ll.LtrB intron-encoded protein (LtrA) expressed alone or with the intron RNA to form ribonucleoprotein particles localizes to bacterial cellular poles, potentially accounting for the intron's preferential insertion in the oriC and ter regions of the Escherichia coli chromosome. Here, by using cell microarrays and automated fluorescence microscopy to screen a transposon-insertion library, we identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) whose disruption results in both an increased proportion of cells with more diffuse LtrA localization and a more uniform genomic distribution of Ll.LtrB-insertion sites. Surprisingly, we find that a common factor affecting LtrA localization in these and other disruptants is the accumulation of intracellular polyphosphate, which appears to bind LtrA and other basic proteins and delocalize them away from the poles. Our findings show that the intracellular localization of a group II intron-encoded protein is a major determinant of insertion-site preference. More generally, our results suggest that polyphosphate accumulation may provide a means of localizing proteins to different sites of action during cellular stress or entry into stationary phase, with potentially wide physiological consequences.This work was supported by National Institutes of Health R01 grants GM037949 to AML and GM076536 to EMM, Welch Foundation grants F-1607 to AML and F-1515 to EMM, and a Packard Foundation fellowship to EMM.Cellular and Molecular Biolog

Upregulation of Functional Kv11.1 Isoform Expression by Inhibition of Intronic Polyadenylation with Antisense Morpholino Oligonucleotides

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing; intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, while polyadenylationwithin intron 9 generates a non-functional, Cterminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1 isoforms plays an important role in the regulation of Kv11.1 channel function and the pathogenesis of long QT syndrome. In this study,we identified cis-acting elements that are required for KCNH2 intron 9 poly(A) signal activity. Mutation of these elements decreased Kv11.1a-USO expression and increased the expression of Kv11.1a mRNA, protein and channel current. More importantly, blocking these elements by antisense morpholino oligonucleotides shifted the alternative processing of KCNH2 intron 9 from the polyadenylation to the splicing pathway, leading to the predominant production of Kv11.1a and a significant increase in Kv11.1 current. Our findings indicate that the expression of the Kv11.1a isoform can be upregulated by an antisense approach. Antisense inhibition of KCNH2 intronic polyadenylation represents a novel approach to increase Kv11.1 channel function