Intron Removal Research Articles

A Common Regulator Couples Nuclear and Chloroplast mRNA Splicing Mechanisms in Plants

Eukaryotic organisms accomplish the removal of introns to produce mature mRNAs through splicing. Nuclear and organelle splicing mechanisms are distinctively executed by spliceosome and group II intron complex, respectively. Here, we show that LEFKOTHEA a nuclear encoded RNA-binding protein participates in chloroplast group II intron and nuclear pre-mRNA splicing. Transiently optimized LEFKOTHEA nuclear activity is fundamental for plant growth, whereas the loss-of-function abruptly arrests embryogenesis. Nucleocytoplasmic partitioning and chloroplast allocation are efficiently balanced via functional motifs in LEFKOTHEA polypeptide. In the context of nuclear-chloroplast coevolution, our results provide a strong paradigm of the convergence of RNA maturation mechanisms in the nucleus and chloroplasts to coordinately regulate gene expression and effectively control plant growth.

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.

Genetic engineering of Theileria parva lactate dehydrogenase gene: a new anti-theilerial target

ABSTRACT: Theileria parva is the causative agent of East Coast Fever (ECF), a tick borne disease, which results in major economic losses in cattle. Major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. Thus, exploiting new targets for cost effective and higher therapeutic value drugs are imperative. Glycolysis is the main pathway for generation of ATP in T. parva, given its development inside erythrocytes. Thus, the enzymes of this pathway may prove potential targets for designing new-generation anti-theilerials. Lactate dehydrogenase of T. parva (TpLDH) has the highest activity of all glycolytic enzymes and thus we selected this enzyme as the potential therapeutic target. Our study is the first to report the isolation, removal of introns through directed mutagenesis, and cloning of TpLDH and showing that amino acid insertions or deletions most notably corresponded to a 5-amino acid sequence (Asn-91A, Glu-91B, Glu-91C, Trp-91D, Asn-91E) between Ser-91 ve Arg-92 of the enzyme. This region is also present in other apicomplexan such as Babesia bovis, a pathogen of cattle and Plasmodium falciparum, a human pathogen. Providing as the attachment site for the enzyme inhibitors and not being present in LDH of respective hosts, we propose this site as an attractive drug target. The work here is expected to lead new studies on detailed structural and kinetic aspects of apicomplexan LDHs and development of new inhibitors.

Myeloperoxidase as an Active Disease Biomarker: Recent Biochemical and Pathological Perspectives.

Myeloperoxidase (MPO) belongs to the family of heme-containing peroxidases, produced mostly from polymorphonuclear neutrophils. The active enzyme (150 kDa) is the product of the MPO gene located on long arm of chromosome 17. The primary gene product undergoes several modifications, such as the removal of introns and signal peptides, and leads to the formation of enzymatically inactive glycosylated apoproMPO which complexes with chaperons, producing inactive proMPO by the insertion of a heme moiety. The active enzyme is a homodimer of heavy and light chain protomers. This enzyme is released into the extracellular fluid after oxidative stress and different inflammatory responses. Myeloperoxidase is the only type of peroxidase that uses H2O2 to oxidize several halides and pseudohalides to form different hypohalous acids. So, the antibacterial activities of MPO involve the production of reactive oxygen and reactive nitrogen species. Controlled MPO release at the site of infection is of prime importance for its efficient activities. Any uncontrolled degranulation exaggerates the inflammation and can also lead to tissue damage even in absence of inflammation. Several types of tissue injuries and the pathogenesis of several other major chronic diseases such as rheumatoid arthritis, cardiovascular diseases, liver diseases, diabetes, and cancer have been reported to be linked with MPO-derived oxidants. Thus, the enhanced level of MPO activity is one of the best diagnostic tools of inflammatory and oxidative stress biomarkers among these commonly-occurring diseases.

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.

Short cryptic exons mediate recursive splicing in Drosophila.

Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. We used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.

A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype in Arabidopsis thaliana.

Splicing of pre-mRNA involves two consecutive trans-esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the Arabidopsis thaliana genome encodes around 430 predicted splicing factors, functional information about these proteins is limited. In a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana, we identified a mutant impaired in putative step II factor PRP18a, which has not yet been investigated for its role in pre-mRNA splicing in plants. Step II entails cleavage at the 3′ splice site accompanied by ligation of the 5′ and 3′ exons and intron removal. In the prp18 mutant, splicing of a U2-type intron with non-canonical AT-AC splice sites in GFP pre-mRNA is reduced while splicing of a canonical GT-AG intron is enhanced, resulting in decreased levels of translatable GFP mRNA and GFP protein. These findings suggest that wild-type PRP18a may in some cases promote splicing at weak, non-canonical splice sites. Analysis of genome-wide changes in alternative splicing in the prp18a mutant identified numerous cases of intron retention and a preponderance of altered 3′ splice sites, suggesting an influence of PRP18a on 3′ splice site selection. The prp18a mutant featured short roots on synthetic medium and small siliques, illustrating that wild-type PRP18a function is needed for a normal phenotype. Our study expands knowledge of plant splicing factors and provides foundational information and resources for further functional studies of PRP18 proteins in plants.

Coordinated Control of mRNA and rRNA Processing Controls Embryonic Stem Cell Pluripotency and Differentiation

Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis.

Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization.

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

Coupling between Transcription & Splicing Tunes Gene Expression

RNA is synthesized by RNA polymerases that travel along template DNA. The growing RNA polymers are referred to as nascent. As transcription proceeds, nascent RNA grows in length and sequence complexity, increasing opportunity for folding and interacting with RNA binding proteins. In eukaryotes, the capping enzymes modify the mRNA 5’ end, the spliceosome assembles and removes introns, and the polyadenylation machinery cleaves 3’ ends to initiate polyadenylation of mRNA and terminate transcription. All of this takes place in a nascent RNP, which lies close to the DNA axis. I will illustrate how recent single molecule RNA-Seq strategies have revealed the in vivo kinetics of pre-mRNA splicing relative to the progress of RNA polymerase II (Pol II). In budding and fission yeast, the spliceosome can act on introns as soon as they emerge from Pol II, showing that splicing and transcription rates are matched. This predicts that introns present in multi-intron transcripts may be removed in the order of their transcription. Instead, sequencing of full-length nascent RNA molecules in S. pombe revealed that intron removal is equally likely to occur “not in order”, showing regulation. Surprisingly, partially spliced transcripts were rare; most nascent RNA detected was either fully spliced or unspliced, indicating that splicing of any given intron may depend on the splicing status of the other introns in the transcript. Fully unspliced transcripts failed to cleave at the polyA site, underwent transcriptional read-through at gene 3’ends, and were degraded by the exosome. These observations suggest crosstalk among spliceosomes and 3’end processing machineries assembling on the same nascent transcript. I will discuss an example of regulated gene expression in which reduced splicing leads to lower reduced mRNA levels. These findings highlight coordination between transcription and RNA processing steps along the pathway of gene expression.

The life of U6 small nuclear RNA, from cradle to grave.

Removal of introns from precursor messenger RNA (pre-mRNA) and some noncoding transcripts is an essential step in eukaryotic gene expression. In the nucleus, this process of RNA splicing is carried out by the spliceosome, a multi-megaDalton macromolecular machine whose core components are conserved from yeast to humans. In addition to many proteins, the spliceosome contains five uridine-rich small nuclear RNAs (snRNAs) that undergo an elaborate series of conformational changes to correctly recognize the splice sites and catalyze intron removal. Decades of biochemical and genetic data, along with recent cryo-EM structures, unequivocally demonstrate that U6 snRNA forms much of the catalytic core of the spliceosome and is highly dynamic, interacting with three snRNAs, the pre-mRNA substrate, and >25 protein partners throughout the splicing cycle. This review summarizes the current state of knowledge on how U6 snRNA is synthesized, modified, incorporated into snRNPs and spliceosomes, recycled, and degraded.

Flow cytometric analysis identifies changes in S and M phases as novel cell cycle alterations induced by the splicing inhibitor isoginkgetin.

The spliceosome is a large ribonucleoprotein complex that catalyzes the removal of introns from RNA polymerase II-transcribed RNAs. Spliceosome assembly occurs in a stepwise manner through specific intermediates referred to as pre-spliceosome complexes E, A, B, B* and C. It has been reported that small molecule inhibitors of the spliceosome that target the SF3B1 protein component of complex A lead to the accumulation of cells in the G1 and G2/M phases of the cell cycle. Here we performed a comprehensive flow cytometry analysis of the effects of isoginkgetin (IGG), a natural compound that interferes with spliceosome assembly at a later step, complex B formation. We found that IGG slowed cell cycle progression in multiple phases of the cell cycle (G1, S and G2) but not M phase. This pattern was somewhat similar to but distinguishable from changes associated with an SF3B1 inhibitor, pladienolide B (PB). Both drugs led to a significant decrease in nascent DNA synthesis in S phase, indicative of an S phase arrest. However, IGG led to a much more prominent S phase arrest than PB while PB exhibited a more pronounced G1 arrest that decreased the proportion of cells in S phase as well. We also found that both drugs led to a comparable decrease in the proportion of cells in M phase. This work indicates that spliceosome inhibitors affect multiple phases of the cell cycle and that some of these effects vary in an agent-specific manner despite the fact that they target splicing at similar stages of spliceosome assembly.

Splicing of platelet resident pre-mRNAs upon activation by physiological stimuli results in functionally relevant proteome modifications

Platelet activation triggers thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies still fail to prevent thrombotic events in numerous patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how megakaryocyte-derived mRNAs are regulated in these anucleate cell fragments. Proteogenomics was applied here to investigate this phenomeon in platelets activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining proteomics and transcriptomics allowed in depth platelet proteome characterization, revealing a significant effect of either stimulus on proteome composition. In silico analysis revealed the presence of resident immature RNAs in resting platelets, characterized by retained introns, while unbiased proteogenomics correlated intron removal by RNA splicing with changes on proteome composition upon activation. This allowed identification of a set of transcripts undergoing maturation by intron removal during activation and resulting in accumulation of the corresponding peptides at exon-exon junctions. These results indicate that RNA splicing events occur in platelets during activation and that maturation of specific pre-mRNAs is part of the activation cascade, contributing to a dynamic fine-tuning of the transcriptome.

Introns provide a platform for intergenic regulatory feedback of RPL22 paralogs in yeast.

Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs.Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role.The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.

Pre-mRNA Splicing Modulation by Antisense Oligonucleotides.

Pre-mRNA splicing, a dynamic process of intron removal and exon joining, is governed by a combinatorial control exerted by overlapping cis-elements that are unique to each exon and its flanking intronic sequences. Splicing cis-elements are usually 4-to-8-nucleotide-long linear motifs that provide binding sites for specific proteins. Pre-mRNA splicing is also influenced by secondary and higher order RNA structures that affect accessibility of splicing cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate splicing in vivo. Therefore, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of splicing in pathological conditions. Here we describe an ASO-based approach to increase the production of the full-length SMN2 mRNA in spinal muscular atrophy patient cells.

Parallel loss of introns in the ABCB1 gene in angiosperms

BackgroundThe presence of non-coding introns is a characteristic feature of most eukaryotic genes. While the size of the introns, number of introns per gene and the number of intron-containing genes can vary greatly between sequenced eukaryotic genomes, the structure of a gene with reference to intron presence and positions is typically conserved in closely related species. Unexpectedly, the ABCB1 (ATP-Binding Cassette Subfamily B Member 1) gene which encodes a P-glycoprotein and underlies dwarfing traits in maize (br2), sorghum (dw3) and pearl millet (d2) displayed considerable variation in intron composition.ResultsAn analysis of the ABCB1 gene structure in 80 angiosperms revealed that the number of introns ranged from one to nine. All introns in ABCB1 underwent either a one-time loss (single loss in one lineage/species) or multiple independent losses (parallel loss in two or more lineages/species) with the majority of losses occurring within the grass family. In contrast, the structure of the closest homolog to ABCB1, ABCB19, remained constant in the majority of angiosperms analyzed. Using known phylogenetic relationships within the grasses, we determined the ancestral branch-points where the losses occurred. Intron 7, the longest intron, was lost in only a single species, Mimulus guttatus, following duplication of ABCB1. Semiquantitative PCR showed that the M. guttatus ABCB1 gene copy without intron 7 had significantly lower transcript levels than the gene copy with intron 7. We further demonstrated that intron 7 carried two motifs that were highly conserved across the monocot-dicot divide.ConclusionsThe ABCB1 gene structure is highly dynamic, while the structure of ABCB19 remained largely conserved through evolution. Precise removal of introns, preferential removal of smaller introns and presence of at least 2 bp of microhomology flanking most introns indicated that intron loss may have predominantly occurred through non-homologous end-joining (NHEJ) repair of double strand breaks. Lack of microhomology in the exon upstream of lost phase I introns was likely due to release of the selective constraint on the penultimate base (3rd base in codon) of the terminal codon by the splicing machinery. In addition to size, the presence of regulatory motifs will make introns recalcitrant to loss.

Analysis of the Roles of the Arabidopsis nMAT2 and PMH2 Proteins Provided with New Insights into the Regulation of Group II Intron Splicing in Land-Plant Mitochondria.

Plant mitochondria are remarkable with respect to the presence of numerous group II introns which reside in many essential genes. The removal of the organellar introns from the coding genes they interrupt is essential for respiratory functions, and is facilitated by different enzymes that belong to a diverse set of protein families. These include maturases and RNA helicases related proteins that function in group II intron splicing in different organisms. Previous studies indicate a role for the nMAT2 maturase and the RNA helicase PMH2 in the maturation of different pre-RNAs in Arabidopsis mitochondria. However, the specific roles of these proteins in the splicing activity still need to be resolved. Using transcriptome analyses of Arabidopsis mitochondria, we show that nMAT2 and PMH2 function in the splicing of similar subsets of group II introns. Fractionation of native organellar extracts and pulldown experiments indicate that nMAT2 and PMH2 are associated together with their intron-RNA targets in large ribonucleoprotein particle in vivo. Moreover, the splicing efficiencies of the joint intron targets of nMAT2 and PMH2 are more strongly affected in a double nmat2/pmh2 mutant-line. These results are significant as they may imply that these proteins serve as components of a proto-spliceosomal complex in plant mitochondria.

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription

SummarySmall RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.

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.

The emerging role of alternative splicing in senescence and aging.

SummaryDeregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age‐related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF‐1, SIRT1, and ING‐1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype.