Short Introns Research Articles

The Nix locus on the male-specific homologue of chromosome 1 in Aedes albopictus is a strong candidate for a male-determining factor

BackgroundGlobal concern over the rapid expansion of the Asian tiger mosquito, Aedes albopictus, and its vector competence has highlighted an urgent need to improve currently available population control methods, like the Sterile Insect Technique. Knowledge of the sex determination cascade is a prerequisite for the development of early-stage sexing systems. To this end, we have characterised the putative sex determination gene, Nix, in this species. In Aedes species the chromosome complement consists of three pairs of chromosomes. The sex determination alleles are linked to the smallest homomorphic chromosome.ResultsWe identified the male-specific chromosome 1 of Ae. albopictus that carries the putative male-determining gene Nix. We have also characterised the complete genomic sequence of the Nix gene which is composed of two exons and a short intron. The gene displays different levels of intron retention during development. Comparison of DNA sequences covering most of the Nix gene from individuals across the species range revealed no polymorphism.ConclusionsOur characterisation of the Nix gene in Ae. albopictus represents an initial step in the analysis of the sex determination cascade in this species. We found evidence of intron retention (IR) in Nix. IR might play a role in regulating the expression of Nix during development. Our results provide the basis for the development of new genetic control strategies.

Mathematical modeling identifies potential gene structure determinants of co-transcriptional control of alternative pre-mRNA splicing.

The spliceosome catalyzes the removal of introns from pre-messenger RNA (mRNA) and subsequent pairing of exons with remarkable fidelity. Some exons are known to be skipped or included in the mature mRNA in a cell type- or context-dependent manner (cassette exons), thereby contributing to the diversification of the human proteome. Interestingly, splicing is initiated (and sometimes completed) co-transcriptionally. Here, we develop a kinetic mathematical modeling framework to investigate alternative co-transcriptional splicing (CTS) and, specifically, the control of cassette exons’ inclusion. We show that when splicing is co-transcriptional, default splice patterns of exon inclusion are more likely than when splicing is post-transcriptional, and that certain exons are more likely to be regulatable (i.e. cassette exons) than others, based on the exon–intron structure context. For such regulatable exons, transcriptional elongation rates may affect splicing outcomes. Within the CTS paradigm, we examine previously described hypotheses of co-operativity between splice sites of short introns (i.e. ‘intron definition’) or across short exons (i.e. ‘exon definition’), and find that models encoding these faithfully recapitulate observations in the fly and human genomes, respectively.

Construction of a binary vector for the expression of the Aspergillus niger McoD laccase gene in the industrial filamentous fungus Aspergillus oryzae

The filamentous fungus Aspergillus oryzae, also named as the koji mold, is a preferred host for enzyme production due to its prominent secretion ability into the culture medium. Fungal laccases are widely used in different industrial processes, especially in removing environmental pollutants. Up till now, little was known about the roles of some laccase genes from the black mold Aspergillus niger. The McoD laccase gene from A. niger includes three exons interrupted by two short introns. The respective coding sequence of the gene is computationally predicted to encode a secreted laccase of 563 amino acids. In this study, a binary vector carrying the A. nigerMcoD gene was successfully constructed for heterologous expression in the edible fungus A. oryzae. This vector was transformed into A. oryzae using the Agrobacterium tumefaciens-mediated transformation method. The transformation efficiency was relatively high in two different auxotrophic A. oryzae strains, including RIB40∆pyrG and VS1∆pyrG. All the tested transformants possess in their genomes the construct for expression of the McoD laccase gene under control of the strong A. oryzae amyB promoter. The selected transformants were examined for the laccase activity using the ABTS substrate. The results showed that in comparison to the wild-type fungal strains, the transgenic strains could oxidise ABTS to form the sea green colour, which can be seen directly on the agar plate.

Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA.

The full length of Culex quiquefasciatus early trypsin has been cloned and sequenced and a three-dimensional (3D) model of the enzyme was built showing that the enzyme has the canonical trypsin's active pocket containing H78, D123, S129, and D128. The biosynthesis of juvenile hormone (JH) III by the corpora allata (CA) in female Cx. quiquefasciatus is sugar-dependent. Females that were maintained on water after emergence synthesize very little JH III, JH III bisepoxide, and methyl farnesoate (MF) (3.8, 1.1, and 0.8 fmol/4 hr/CA, respectively). One hour after sugar feeding, the synthesis of JH III and JH III bisepoxide reached a maximum (11.3 and 5.9 fmol/4 hr/CA, respectively) whereas MF biosynthesis reached a maximum at 24 hr (5.2 fmol/4 hr/CA). The early trypsin is transcribed with a short intron (51 nt) is spliced when JH III biosynthesis is high in sugar fed and at 1 hr after the blood meal (22 and 15 fmol/4 hr/CA, respectively). We investigated the transcriptional and posttranscriptional regulation of the early trypsin gene showing that JH III concentrations influence splicing. In the absence JH III the unspliced transcript is linked by a phosphoamide bond at the 5'-end to RNA ribonuleoprotein (RNP). The biosynthesis of the early trypsin was followed in ligated abdomens (without CA) of newly emerged females that fed blood by enema. Our results show that the early trypsin biosynthesis depends on sugar and blood feeding, whereas the late trypsin biosynthesis does not depend on sugar feeding, or JH III biosynthesis. Downregulating the early trypsin transcript does not affect the late trypsin.

Evolution and Diversity of Pre-mRNA Splicing in Highly Reduced Nucleomorph Genomes.

Eukaryotic genes are interrupted by introns that are removed in a conserved process known as pre-mRNA splicing. Though well-studied in select model organisms, we are only beginning to understand the variation and diversity of this process across the tree of eukaryotes. We explored pre-mRNA splicing and other features of transcription in nucleomorphs, the highly reduced remnant nuclei of secondary endosymbionts. Strand-specific transcriptomes were sequenced from the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans, whose plastids are derived from red and green algae, respectively. Both organisms exhibited elevated nucleomorph antisense transcription and gene expression relative to their respective nuclei, suggesting unique properties of gene regulation and transcriptional control in nucleomorphs. Marked differences in splicing were observed between the two nucleomorphs: the few introns of the G. theta nucleomorph were largely retained in mature transcripts, whereas the many short introns of the B. natans nucleomorph are spliced at typical eukaryotic levels (>90%). These differences in splicing levels could be reflecting the ancestries of the respective plastids, the different intron densities due to independent genome reduction events, or a combination of both. In addition to extending our understanding of the diversity of pre-mRNA splicing across eukaryotes, our study also indicates potential links between splicing, antisense transcription, and gene regulation in reduced genomes.

The splicing of tiny introns of Paramecium is controlled by MAGO

The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size.

Requirement for intron structures in activating the Cd8a locus

During differentiation of CD4+CD8+ double-positive (DP) thymocytes into the CD4-CD8+ single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, Cd8a transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the Cd8a gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the Cd8a gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the Cd8a locus, which was generated by insertion of hCD2 cDNA into the first exon of the Cd8a gene, requires the incorporation of intron sequences into the hCD2 transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8+ T cells, whereas hCD2 expression in DP thymocytes recapitulated the Cd8a expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the Cd4 locus restored hCD2 expression in mature CD8+ T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in Cd8a reporter alleles that failed to express hCD2 in CD8+ T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the Cd8a locus harboring hCD2 cDNA. However, the Cd8a gene was transcribed in mature CD8+ T cells, albeit at a lower level, from a mutant Cd8a locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.

The Mirtron MiR-1010 Functions in Concert with Its Host Gene SKIP to Maintain Synaptic Homeostasis

Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. Here, we found that larvae lacking the mirtron miR-1010 are unable to grow properly and pupariate. We show that miR-1010 downregulates nAcRβ2. Increase of cortical nAcRb2 mediated by neural activity elevates the level of intracellular Ca2 , which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. We reveal that Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcRβ2 mRNA levels (negative feedback loop). Our results demonstrate how a mirtron, in coordination with its host gene, contributes to maintaining homeostasis.

Detecting Agotrons in Ago CLIPseq Data.

Timely and accurate regulation of gene expression is essential to all organisms. MicroRNAs (miRNAs) are a well-characterized and important class of gene expression regulators. We recently identified a novel class of gene regulators, the agotrons. Agotrons derive from short introns and associate with Argonaute (Ago) proteins much similar to miRNAs. However, agotrons completely bypass the conventional miRNA biogenesis pathway and thus exist as full-length introns, which disobey the classical rules on Ago-substrate requirements. As a class, agotrons are conserved in mammals, and despite the non-canonical biogenesis pathway, agotrons maintain the ability to deregulate mRNAs with seed-matches in the 3'UTR. While several pipelines exist for the detection of miRNAs, no bioinformatics toolkit has yet been developed to specifically identify agotrons. Here, we describe a simple approach, termed agotron_detector ( https://github.com/ncrnalab/agotron_detector ), to identify and quantify agotrons in Ago CLIPseq datasets. Hopefully, this allows researchers worldwide to characterize agotrons in more detail and to reveal the biological relevance of these fascinating RNA species.

Functional divergence and intron variability during evolution of angiosperm TERMINAL FLOWER1 (TFL1) genes

The protein encoded by the TERMINAL FLOWER1 (TFL1) gene maintains indeterminacy in inflorescence meristem to repress flowering, and has undergone multiple duplications. However, basal angiosperms have one copy of a TFL1-like gene, which clusters with eudicot TFL1/CEN paralogs. Functional conservation has been reported in the paralogs CENTRORADIALIS (CEN) in eudicots, and ROOTS CURL IN NPA (RCNs) genes in monocots. In this study, long-term functional conservation and selective constraints were found between angiosperms, while the relaxation of selective constraints led to subfunctionalisation between paralogs. Long intron lengths of magnoliid TFL1-like gene contain more conserved motifs that potentially regulate TFL1/CEN/RCNs expression. These might be relevant to the functional flexibility of the non-duplicate TFL1-like gene in the basal angiosperms in comparison with the short, lower frequency intron lengths in eudicot and monocot TFL1/CEN/RCNs paralogs. The functionally conserved duplicates of eudicots and monocots evolved according to the duplication-degeneration-complementation model, avoiding redundancy by relaxation of selective constraints on exon 1 and exon 4. These data suggest that strong purifying selection has maintained the relevant functions of TFL1/CEN/RCNs paralogs on flowering regulation throughout the evolution of angiosperms, and the shorter introns with radical amino acid changes are important for the retention of paralogous duplicates.

Transcription-Associated Compositional Skews in Drosophila Genes.

In many organisms, local deviations from Chargaff’s second parity rule are observed around replication and transcription start sites and within intron sequences. Here, we use expression data as well as a whole-genome data set of nearly 200 haplotypes to investigate such compositional skews in Drosophila melanogaster genes. We find a positive correlation between compositional skew and gene expression, comparable in strength to similar correlations between expression levels and genome-wide sequence features. This correlation is relatively stronger for germline, compared with somatic expression, consistent with the process of transcription-associated mutation bias. We also inferred mutation rates from alleles segregating at low frequencies in short introns, and show that, whereas the overall GC content of short introns does not conform to the equilibrium expectation, the level of the observed deviation from the second parity rule is generally consistent with the inferred rates.

P.498 - Dystrophin Dp427 is lost due to multiple DMD intron retentions in rhabdomyosarcoma CRL-2061 cells

Dystrophin inactivation, responsible for Duchenne muscular dystrophy (DMD), is recently implicated as an anti-tumor suppressor factor in cancers with myogenic characteristics. Rhabdomyosarcoma (RMS), a mesodermal cancer, shows myogenic features and occurs at a higher frequency in mdx mouse, a DMD model mouse. In humans, as the clinical management of DMD advances to increase the life expectancy of patients, a subsequent issue is anticipated to be that of addressing other complications such as RMS. RMS, although displays myogenic properties, has not yet been fully evaluated for dystrophin expression. Here, we used reverse transcription PCR and western blotting to analyze dystrophin in RMS CRL-2061 cells. The 14 kb DMD transcript was fully amplified as 20 fragments as in skeletal muscle. Alternatively, spliced transcripts including 3 intron retentions and 2 exons skipping events were disclosed. In one fragment extending from exon 70 to 79, no normally spliced product was obtained. Instead, 6 alternatively spliced products including a new product deleting exon 73 were identified, and exon 78 deleted products was most abundant. However, dystrophin Dp427 was not detected by western blotting. Remarkably, analysis of 9 short DMD introns indicated retention of introns 40, 58 and 70. These intron retentions occupied 75, 15 and 20 % of the total PCR amplified products, respectively, indicating the possibility of every DMD transcript to carry an intron retention event. Multiple intron retentions in the DMD transcript are suggested to hamper translation of the Dp427 isoform and promote tumorigenesis in RMS. This study exemplifies the important significance of DMD intron retention in tumorigenesis in RMS.

Widespread intra-dependencies in the removal of introns from human transcripts.

Research into the problem of splice site selection has followed a reductionist approach focused on how individual splice sites are recognized. Early applications of information theory uncovered an inconsistency. Human splice signals do not contain enough information to explain the observed fidelity of splicing. Here, we conclude that introns do not necessarily contain ‘missing’ information but rather may require definition from neighboring processing events. For example, there are known cases where an intronic mutation disrupts the splicing of not only the local intron but also adjacent introns. We present a genome-wide measurement of the order of splicing within human transcripts. The observed order of splicing cannot be explained by a simple kinetic model. Simulations reveal a bias toward a particular, transcript-specific order of intron removal in human genes. We validate an extreme class of intron that can only splice in a multi-intron context. Special categories of splicing such as exon circularization, first and last intron processing, alternative 5 and 3′ss usage and exon skipping are marked by distinct patterns of ordered intron removal. Excessive intronic length and silencer density tend to delay splicing. Shorter introns that contain enhancers splice early.

DMD transcripts in CRL-2061 rhabdomyosarcoma cells show high levels of intron retention by intron-specific PCR amplification

BackgroundThe DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease. DMD has also been shown to act as a tumor suppressor gene. Rhabdomyosarcoma (RMS) is a mesodermal sarcoma that shares characteristics of skeletal muscle precursors. Products of the DMD gene in RMS have not yet been fully clarified. Here, DMD products were analyzed in CRL-2061 cells established from alveolar RMS.MethodsThe 14-kb long DMD cDNA was PCR amplified as 20 separated fragments, as were nine short intron regions. Dystrophin was analyzed by Western blotting using an antibody against the C-terminal region of dystrophin.ResultsSixteen of the 20 DMD cDNA fragments could be amplified from CRL-2061 cells as muscle cDNA. Three fragments included aberrant gene products, including one in which exon 71 was omitted and one each with retention of introns 40 and 58. In one fragment, extending from exon 70 to 79, no normally spliced product was obtained. Rather, six alternatively spliced products were identified, including a new product deleting exon 73, with the most abundant product showing deletion of exon 78. Although dystrophin expression was expected in CRL-2061 cells, western blotting of cell lysates showed no evidence of dystrophin, suggesting that translation of full-length DMD mRNA was inhibited by intron retention that generated a premature stop codon. Intron specific PCR amplification of nine short introns, showed retention of introns 40, 58, and 70, which constituted about 60, 25 and 9%, respectively, of the total PCR amplified products. The most abundant DMD transcript contained two abnormalities, intron 40 retention and exon 78 skipping.ConclusionsIntron-specific PCR amplification showed that DMD transcripts contained high levels of introns 40, 58 and 70. Retention of these introns may have been responsible for the lack of dystrophin expression by CRL-2061 cells, thereby abolishing the tumor suppressor activity of dystrophin.

Identification of human short introns.

Canonical pre-mRNA splicing requires snRNPs and associated splicing factors to excise conserved intronic sequences, with a minimum intron length required for efficient splicing. Non-canonical splicing–intron excision without the spliceosome–has been documented; most notably, some tRNAs and the XBP1 mRNA contain short introns that are not removed by the spliceosome. There have been some efforts to identify additional short introns, but little is known about how many short introns are processed from mRNAs. Here, we report an approach to identify RNA short introns from RNA-Seq data, discriminating against small genomic deletions. We identify hundreds of short introns conserved among multiple human cell lines. These short introns are often alternatively spliced and are found in a variety of RNAs–both mRNAs and lncRNAs. Short intron splicing efficiency is increased by secondary structure, and we detect both canonical and non-canonical short introns. In many cases, splicing of these short introns from mRNAs is predicted to alter the reading frame and change protein output. Our findings imply that standard gene prediction models which often assume a lower limit for intron size fail to predict short introns effectively. We conclude that short introns are abundant in the human transcriptome, and short intron splicing represents an added layer to mRNA regulation.

Variation in the Intensity of Selection on Codon Bias over Time Causes Contrasting Patterns of Base Composition Evolution in Drosophila.

Four-fold degenerate coding sites form a major component of the genome, and are often used to make inferences about selection and demography, so that understanding their evolution is important. Despite previous efforts, many questions regarding the causes of base composition changes at these sites in Drosophila remain unanswered. To shed further light on this issue, we obtained a new whole-genome polymorphism data set from D. simulans. We analyzed samples from the putatively ancestral range of D. simulans, as well as an existing polymorphism data set from an African population of D. melanogaster. By using D. yakuba as an outgroup, we found clear evidence for selection on 4-fold sites along both lineages over a substantial period, with the intensity of selection increasing with GC content. Based on an explicit model of base composition evolution, we suggest that the observed AT-biased substitution pattern in both lineages is probably due to an ancestral reduction in selection intensity, and is unlikely to be the result of an increase in mutational bias towards AT alone. By using two polymorphism-based methods for estimating selection coefficients over different timescales, we show that the selection intensity on codon usage has been rather stable in D. simulans in the recent past, but the long-term estimates in D. melanogaster are much higher than the short-term ones, indicating a continuing decline in selection intensity, to such an extent that the short-term estimates suggest that selection is only active in the most GC-rich parts of the genome. Finally, we provide evidence for complex evolutionary patterns in the putatively neutral short introns, which cannot be explained by the standard GC-biased gene conversion model. These results reveal a dynamic picture of base composition evolution.

Molecular cloning, characterization and expression analysis of a novel PDRG1 gene from black tiger shrimp (Penaeus monodon).

P53 And DNA Damage-Regulated Gene 1 (PDRG1) is a novel gene which plays an important role in chaperone-mediated protein folding. In the present study, the full-length complementary DNA (cDNA) sequence of the PDRG1 gene from Penaeus monodon (PmPDRG1) was cloned by the rapid amplification of cDNA ends (RACE) method. The cDNA of PmPDRG1 spans 1,613 bp, interrupted by only one short intron, and encodes a protein of 136 amino acids with calculated molecular weight of 15.49 kDa. The temporal expression profile of PmPDRG1 in different tissues and in different developmental stages of the ovary was investigated by real-time quantitative PCR (RT-qPCR). An RNA interference (RNAi) experiment was performed to study the relationship between P. monodon p53 (Pmp53) and PmPDRG1, and the results showed that the relative expression level of PmPDRG1 mRNA was notably up-regulated from 12 h to 96 h after Pmp53 was silenced both in ovary and hepatopancreas. To further explore the role of PmPDRG1 in ovarian development, dopamine (DA) and 5-hydroxytryptamine (5-HT)-injected shrimps were analyzed by RT-qPCR, indicating that PmPDRG1 may be involved in the regulation of ovarian development of P. monodon.

Relationships Among Powered Flight, Metabolic Rate, Body Mass, Genome Size, and the Retrotransposon Complement of Volant Birds

Avian genomes are of interest because the rapid metabolic rate associated with powered flight requires small cells which constrain genome size. Consequently, flying birds tend to have small genomes relative to other vertebrates such as mammals. It thus stands to reason that flying birds should have smaller genomes than ground-dwelling birds with lower metabolic rates. Small genomes could be condensed but uncompromised in a number of ways, including smaller intergenic intervals, shorter introns, and/or a reduced transposable element (TE) complement. We evaluated genome size in light of the orthologous TE complement among 41 flying (FY) and seven ground-dwelling (GD) bird species to determine if a preponderance of deletions in orthologous TEs might explain the compact genomes of flying birds with high metabolic rates. We measured, across multiple loci in all 48 species, the lengths of 50 contemporary orthologous chicken repeat 1 (CR1, a non-LTR retrotransposon) copies relative to inferred ancestral CR1 sequences. We found genome sizes in GD birds were not different than those in FY birds, but the mean lengths of orthologous CR1 loci were significantly shorter in FY birds than in GD birds. Moreover, we observed a negative correlation between basal metabolic rate and length of orthologous CR1 loci. Finally, we observed positive correlations between body mass and both genome sizes as well as length of orthologous CR1 loci, which we expected given that body mass correlates negatively with metabolic rates. Our results support the contention that metabolism helps shape the avian TE complement and thus indirectly contributes to the compact genomes of birds.

Establishing species-specific sexing markers suitable for non-invasive samples of species lacking genomic resources: an example using the highly endangered common hamster Cricetus cricetus

Here we present an approach to establish species-specific genetic markers for sex identification suitable for non-invasive samples. Such markers are not yet available for the endangered common hamster (Cricetus cricetus) because of the lack of genomic resources. Using Y chromosome conserved anchored tagged sequences (YCATS) exonic primers, we obtained Y-chromosomal sequences from hamsters and sympatric rodent species. From this, we designed hamster-specific primers targeting two short Y-chromosomal intron fragments and included them in microsatellite multiplex reactions, using autosomal loci also as amplification controls. The method yielded highly consistent results. The approach can be easily applied to development of sex markers in species for which there are no genome sequences available and thus aid conservation genetics efforts.

The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression

The fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition. Here we perform a domain analysis of the evolutionarily conserved Schizosaccharomyces pombe pre-mRNA-processing factor, SpPrp18. Our mutational and biophysical analyses of the C-terminal α-helical bundle reveal critical roles for the conserved region as well as helix five. We generate a novel conditional missense mutant, spprp18-5 To assess the role of SpPrp18, we performed global splicing analyses on cells depleted of prp18+ and the conditional spprp18-5 mutant, which show widespread but intron-specific defects. In the absence of functional SpPrp18, primer extension analyses on a tfIId+ intron 1-containing minitranscript show accumulated pre-mRNA, whereas the lariat intron-exon 2 splicing intermediate was undetectable. These phenotypes also occurred in cells lacking both SpPrp18 and SpDbr1 (lariat debranching enzyme), a genetic background suitable for detection of lariat RNAs. These data indicate a major precatalytic splicing arrest that is corroborated by the genetic interaction between spprp18-5 and spprp2-1, a mutant in the early acting U2AF59 protein. Interestingly, SpPrp18 depletion caused cell cycle arrest before S phase. The compromised splicing of transcripts coding for G1-S regulators, such as Res2, a transcription factor, and Skp1, a regulated proteolysis factor, are shown. The cumulative effects of SpPrp18-dependent intron splicing partly explain the G1 arrest upon the loss of SpPrp18. Our study using conditional depletion of spprp18+ and the spprp18-5 mutant uncovers an intron-specific splicing function and early spliceosomal interactions and suggests links with cell cycle progression.