Human Intron Research Articles

HERV-K(HML7) Integrations in the Human Genome: Comprehensive Characterization and Comparative Analysis in Non-Human Primates.

Simple SummaryThe human genome is not human at all, but it includes a multitude of sequences inherited from ancient viral infections that affected primates’ germ line. These elements can be seen as the fossils of now-extinct retroviruses, and are called Human Endogenous Retroviruses (HERVs). View as “junk DNA” for a long time, HERVs constitute 4 times the amount of DNA needed to produce all cellular proteins, and growing evidence indicates their crucial role in primate brain evolution, placenta development, and innate immunity shaping. HERVs are also intensively studied for a pathological role, even if the incomplete knowledge about their exact number and genomic position has thus far prevented any causal association. Among possible relevant HERVs, the HERV-K supergroup is of particular interest, including some of the oldest (HML5) as well as youngest (HML2) integrations. Among HERV-Ks, the HML7 group still lack a detailed description, and the present work thus aimed to identify and characterize all HML7 elements in the human genome. Results showed that the HML7 group includes 23 elements and an additional 160 “scars” of past infection that invaded in primates mostly between 20 and 30 million years ago, providing an exhaustive background to study their impact on human pathophysiology.Endogenous Retroviruses (ERVs) are ancient relics of infections that affected the primate germ line and constitute about 8% of our genome. Growing evidence indicates that ERVs had a major role in vertebrate evolution, being occasionally domesticated by the host physiology. In addition, human ERV (HERV) expression is highly investigated for a possible pathological role, even if no clear associations have been reported yet. In fact, on the one side, the study of HERV expression in high-throughput data is a powerful and promising tool to assess their actual dysregulation in diseased conditions; but, on the other side, the poor knowledge about the various HERV group genomic diversity and individual members somehow prevented the association between specific HERV loci and a given molecular mechanism of pathogenesis. The present study is focused on the HERV-K(HML7) group that—differently from the other HERV-K members—still remains poorly characterized. Starting from an initial identification performed with the software RetroTector, we collected 23 HML7 proviral insertions and about 160 HML7 solitary LTRs that were analyzed in terms of genomic distribution, revealing a significant enrichment in chromosome X and the frequent localization within human gene introns as well as in pericentromeric and centromeric regions. Phylogenetic analyses showed that HML7 members form a monophyletic group, which based on age estimation and comparative localization in non-human primates had its major diffusion between 20 and 30 million years ago. Structural characterization revealed that besides 3 complete HML7 proviruses, the other group members shared a highly defective structure that, however, still presents recognizable functional domains, making it worth further investigation in the human population to assess the presence of residual coding potential.

Reconstitution of Human tRNA Intron Cleavage Leads to an Updated Model of tRNA Splicing

Transfer RNAs (tRNAs) are critical for translation. tRNAs require extensive post-transcriptional maturation, including the removal of an intron for a subset of tRNAs. tRNA splicing is a multi-step process that involves the cleavage of introns and the ligation of tRNA exons back together. In eukaryotes, tRNA introns are removed by the heterotetrametric tRNA splicing endonuclease (TSEN) complex. In metazoans, ligation is carried out by the tRNA ligase, RTCB, which also ligates introns into tRNA intronic circular RNAs (tricRNAs). Furthermore, the metazoan TSEN complex associates with an accessory protein 5’-hydroxyl polyribonucleotide kinase CLP1. Until recently, the function of CLP1 in tRNA splicing was thought to be in supporting TSEN function/stability as mutations in CLP1 and the TSEN proteins are known to lead to a family of neurological and neurodegenerative diseases, pontocerebellar hypoplasia (PCH). Despite these connections, non-metazoan eukaryotes do not utilize CLP1 in tRNA splicing. To address the role of CLP1 in TSEN function and tRNA splicing, we developed the first system for the production of recombinant active TSEN complex from E. coli and complemented this approach with immunoprecipitations from HEK cells. We showed for the first time that CLP1 is not required for the TSEN complex to remove tRNA introns, uniting how eukaryotes cleave tRNA introns. We then utilized a reporter tRNA to monitor tRNA splicing in cells. We overexpressed CLP1 and kinase-dead CLP1 in HEK cells and monitored splicing of our reporter tRNA. We found that kinase-active but not kinase-dead CLP1 reduced the production of tricRNAs, suggesting that CLP1 might negatively regulate the ligation step of tRNA splicing. In parallel, we knocked down CLP1's homolog, cbc, in Drosophila cells and looked at the maturation of a Drosophila-specific tRNA reporter. We found that depletion of cbc led to a significant increase in both mature tRNA as well as tricRNAs for our reporter construct. Together, our data support a new model of tRNA splicing in which CLP1 is a negative regulator of tRNA and tricRNAs maturation. Hayne, et al. Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage (2020), Nucleic Acids Research.

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

Detection of Tandem Repeats in DNA Sequences Using Short-Time Ramanujan Fourier Transform.

Tandem repeats in genomic sequences are characterized by two or more contiguous copies of a pattern of nucleotides. The role of these repeats as molecular markers is well established in various genetic disorders, human evolution studies, DNA forensics and intron retention. In this work a computational method has been developed for the extraction of both exact and approximate tandem repeats. The proposed algorithm uses Ramanujan Fourier Transform (RFT) to identify periodicities in the DNA sequences. Since RFT estimates the period directly, rather than inferring it from the signal's spectrum, it provides a more sensitive and rapid detection of tandem repeats as compared to other available popular computational methods.

Reduced translation efficiency due to novel splicing variants in 5\u2032 untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

BackgroundBreast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination.ResultsIn this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1.ConclusionsThis study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations.

Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human β-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

Minor intron splicing revisited: identification of new minor intron-containing genes and tissue-dependent retention and alternative splicing of minor introns

BackgroundMutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA (microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1)) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues.ResultsWe employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns.ConclusionHere we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at https://midb.pnb.uconn.edu.

Abstract 772: The Highly Prevalent 25bp Intronic Deletion in MYBPC3 is Benign Under Baseline Conditions

Background: Hypertrophic cardiomyopathy (HCM) affects at least 1 in 500 people worldwide, and results in the thickening of the ventricular walls and reduced cardiac function. Mutations in MYBPC3 , encoding cardiac myosin binding protein-C, are the most common cause of HCM. Previously, a highly prevalent 25bp deletion within intron 32 of MYBPC3 was described in the South Asian population. The MYBPC3 d25bp variant is present in approximately 100 million people, and encompasses a splicing branch point predicted to result in abnormal splicing of exon 33. Thus, there is a critical need to understand the mechanism by which MYBPC3 d25bp may cause cardiomyopathy. Methods: To determine the role of the 25bp deletion in vivo , knock-in humanized mice were created in which intron 32 was replaced with the human intron 32, with or without the MYBPC3 d25bp mutation. Mice were characterized at 3- and 6-months of age by echocardiography, histological, and protein analysis. The presence of aberrant exon splicing was also determined in mice carrying the MYBPC3 d25bp variant through RT-PCR and mini-gene assays. Finally, exon trapping experiments were performed to understand the mechanism behind exon skipping. Results: Under baseline conditions, MYBPC3 d25bp displayed no changes in cardiac function or morphology as measured by echocardiography (FS (%): NTG 35.3%, WT 32.8%, Het 33.7%), heart weight to body weight ratio, or histology. While exon 33 skipping was not detected by RT-PCR, the presence of an alternative splice site within exon 33 was identified in MYBPC3 d25bp mice. However, this did not affect the protein levels of cMyBP-C. Furthermore, mini-gene experiments demonstrated that the MYBPC3 d25bp mutation significantly reduced the percentage of correctly spliced transcripts (86.2% vs. 77.5%). Conclusions: These data demonstrate that the presence of the highly prevalent 25bp deletion is not sufficient to cause disease under baseline conditions. However, it is possible that the increased levels of aberrant splicing may increase the risk for developing HCM.

Increasing transgenic expression in recombinant Chinese hamster ovary cells using introns in different directions

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.

Restoration of correct \u03b2IVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in \u03b2-thalassaemia/HbE erythroid cells

A cytosine to thymine mutation at nucleotide 654 of human β-globin intron 2 (βIVS2-654) is one of the most common mutations causing β-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant β-globin pre-mRNA splicing and prevents synthesis of β-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the β-globin protein, but to maintain therapeutically relevant levels of β-globin it would require lifelong administration. Here, we demonstrate long-term splicing correction using U7 snRNA lentiviral vectors engineered to target several pre-mRNA splicing elements on the βIVS2-654-globin pre-mRNA such as cryptic 3′ splice site, aberrant 5′ splice site, cryptic branch point and an exonic splicing enhancer. A double-target engineered U7 snRNAs targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP + 623, was the most effective in a model cell line, HeLa IVS2-654. Moreover, the therapeutic potential of the vector was demonstrated in erythroid progenitor cells derived from βIVS2-654-thalassaemia/HbE patients, which showed restoration of correctly spliced β-globin mRNA and led to haemoglobin A synthesis, and consequently improved thalassaemic erythroid cell pathology. These results demonstrate proof of concept of using the engineered U7 snRNA lentiviral vector for treatment of β-thalassaemia.

Regulation of GLI1 by cis DNA elements and epigenetic marks

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

Structures of the human spliceosomes before and after release of the ligated exon

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3′-splice site is recognized by the junction between the 5′-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

TMEM173 Alternative Spliced Isoforms Modulate Viral Replication through the STING Pathway.

The innate immune system provides a primary line of defense against pathogens. Stimulator of IFN genes (STING), encoded by the TMEM173 gene, is a critical protein involved in IFN-β induction in response to infection by different pathogens. In this study, we describe the expression of three different alternative-spliced human (h) TMEM173 mRNAs producing STING truncated isoforms 1, 2, and 3 in addition to the full-length wild-type (wt) hSTING. All of the truncated isoforms lack exon 7 and share the N-terminal transmembrane region with wt hSTING. Overexpression of the three STING truncated isoforms failed to induce IFN-β, and they acted as selective pathway inhibitors of wt hSTING even in combination with upstream inducer cyclic-di-GMP-AMP synthase. Truncated isoforms alter the stability of wt hSTING, reducing protein t 1/2 to some extent by the induction of proteasome-dependent degradation. Knocking down expression of truncated isoforms increased production of IFN-β by THP1 monocytes in response to intracellular cytosolic DNA or HSV-1 infection. At early stages of infection, viruses like HSV-1 or vesicular stomatitis virus reduced the ratio of full-length wt hSTING/truncated STING isoforms, suggesting the skewing of alternative splicing of STING toward truncated forms as a tactic to evade antiviral responses. Finally, in silico analysis revealed that the human intron-exon gene architecture of TMEM173 (splice sites included) is preserved in other mammal species, predominantly primates, stressing the relevance of alternative splicing in regulating STING antiviral biology.

Abstract 373: The Molecular Consequence of a Polymorphic 25bp Deletion in Intron 32 of MYBPC3, Specific to South Asians

Background: MYBPC3 encodes cardiac myosin binding protein-C, which regulates sarcomeric stability and contractility. Previous work described a prevalent 25bp deletion in intron 32, MYBPC3 Δ 25bp , in the South Asian population. Occurring in an estimated 100 million carriers, this variant may be associated with cardiomyopathy and heart failure. MYBPC3 Δ25bp encompasses a splicing branch point that could affect splicing of exon 32 to exon 33, potentially resulting in an aberrantly spliced MYBPC3 transcript where exon 33 is skipped entirely. However, the mechanism by which MYBPC3 Δ 25bp associates with disease remains unclear. Methods: To determine the consequence of the 25bp deletion in vivo , mice were engineered that replaced the endogenous Mybpc3 intron 32 with either the normal human intron 32 or human intron 32 containing the 25bp deletion ( Mybpc3 Δ 25bp ) . We characterized Mybpc3 splicing in mice carrying human intron-32 and human iPSC derived cardiomyocytes (hiPSC-CM) from patients heterozygous and homozygous for MYBPC3 Δ 25bp by RT-PCR and using mini-gene assays (across 5 different mouse and human gene segments encompassing exon 32 through exon 34). Results: Echocardiography of mice 3 months of age revealed no changes in cardiac function by fractional shortening (WT: 32.8%, Het: 34.2%, Homo: 36.7%). However, homozygous Mybpc3 Δ 25bp mice had significantly smaller hearts compared to heterozygous and wildtype (3.4 vs 3.71 and 4.25 mg/g p=0.001). Mybpc3 Δ 25bp heterozygous and homozygous mice displayed minimal alternative splicing in exon 33 at baseline. Similarly, transcripts from hiPSC-CM from heterozygous and homozygous MYBPC3 Δ 25bp carriers also show minimal alternative splicing. Mini gene assays suggest that the 25bp deletion does reduce the proportion (77.5% v. 86.2%) of normally spliced transcripts. Conclusions: Taken together, these data suggest that under baseline conditions, MYBPC3 Δ 25bp results in minimal alternative splicing in both human iPSC-CMs and humanized mice harboring the 25bp deletion. While increase aberrant splicing may increase risk of hypertrophic cardiomyopathy in variant-carrying individuals, ongoing studies will determine whether other factors or gene mutations modulate the pathogenicity of the MYBPC3 Δ25bp variant.

Most human introns are recognized via multiple and tissue-specific branchpoints

Although branchpoint recognition is an essential component of intron excision during the RNA splicing process, the branchpoint itself is frequently assumed to be a basal, rather than regulatory, sequence feature. However, this assumption has not been systematically tested due to the technical difficulty of identifying branchpoints and quantifying their usage. Here, we analyzed ∼1.31 trillion reads from 17,164 RNA sequencing data sets to demonstrate that almost all human introns contain multiple branchpoints. This complexity holds even for constitutive introns, 95% of which contain multiple branchpoints, with an estimated five to six branchpoints per intron. Introns upstream of the highly regulated ultraconserved poison exons of SR genes contain twice as many branchpoints as the genomic average. Approximately three-quarters of constitutive introns exhibit tissue-specific branchpoint usage. In an extreme example, we observed a complete switch in branchpoint usage in the well-studied first intron of HBB (β-globin) in normal bone marrow versus metastatic prostate cancer samples. Our results indicate that the recognition of most introns is unexpectedly complex and tissue-specific and suggest that alternative splicing catalysis typifies the majority of introns even in the absence of differences in the mature mRNA.

The emerging role of minor intron splicing in neurological disorders.

Pre-mRNA splicing is an essential step in eukaryotic gene expression. Mutations in cis-acting sequence elements within pre-mRNA molecules or trans-acting factors involved in pre-mRNA processing have both been linked to splicing dysfunction that give rise to a large number of human diseases. These mutations typically affect the major splicing pathway, which excises more than 99% of all introns in humans. However, approximately 700-800 human introns feature divergent intron consensus sequences at their 5' and 3' ends and are recognized by a separate pre-mRNA processing machinery denoted as the minor spliceosome. This spliceosome has been studied less than its major counterpart, but has received increasing attention during the last few years as a novel pathomechanistic player on the stage in neurodevelopmental and neurodegenerative diseases. Here, we review the current knowledge on minor spliceosome function and discuss its potential pathomechanistic role and impact in neurodegeneration.

Identification of functional single nucleotide polymorphisms in the branchpoint site

BackgroundThe human genome contains millions of single nucleotide polymorphisms (SNPs); many of these SNPs are intronic and have unknown functional significance. SNPs occurring within intron branchpoint sites, especially at the adenine (A), would presumably affect splicing; however, this has not been systematically studied. We employed a splicing prediction tool to identify human intron branchpoint sites and screened dbSNP for identifying SNPs located in the predicted sites to generate a genome-wide branchpoint site SNP database.ResultsWe identified 600 SNPs located within branchpoint sites; among which, 216 showed a change in A. After scoring the SNPs by counting the As in the ± 10 nucleotide region, only four SNPs were identified without additional As (rs13296170, rs12769205, rs75434223, and rs67785924). Using minigene constructs, we examined the effects of these SNPs on splicing. The three SNPs (rs13296170, rs12769205, and rs75434223) with nucleotide substitution at the A position resulted in abnormal splicing (exon skipping and/or intron inclusion). However, rs67785924, a 5-bp deletion that abolished the branchpoint A nucleotide, exhibited normal RNA splicing pattern, presumably using two of the downstream As as alternative branchpoints. The influence of additional As on splicing was further confirmed by studying rs2733532, which contains three additional As in the ± 10 nucleotide region.ConclusionsWe generated a high-confidence genome-wide branchpoint site SNP database, experimentally verified the importance of A in the branchpoint, and suggested that other nearby As can protect branchpoint A substitution from abnormal splicing.

Machine learning annotation of human branchpoints.

The branchpoint element is required for the first lariat-forming reaction in splicing. However current catalogues of human branchpoints remain incomplete due to the difficulty in experimentally identifying these splicing elements. To address this limitation, we have developed a machine-learning algorithm-branchpointer-to identify branchpoint elements solely from gene annotations and genomic sequence. Using branchpointer, we annotate branchpoint elements in 85% of human gene introns with sensitivity (61.8%) and specificity (97.8%). In addition to annotation, branchpointer can evaluate the impact of SNPs on branchpoint architecture to inform functional interpretation of genetic variants. Branchpointer identifies all published deleterious branchpoint mutations annotated in clinical variant databases, and finds thousands of additional clinical and common genetic variants with similar predicted effects. This genome-wide annotation of branchpoints provides a reference for the genetic analysis of splicing, and the interpretation of noncoding variation. Branchpointer is written and implemented in the statistical programming language R and is freely available under a BSD license as a package through Bioconductor. b.signal@garvan.org.au or t.mercer@garvan.org. Supplementary data are available at Bioinformatics online.

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells.

Low‐level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR‐6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR‐6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP‐B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP‐B, DHFR intron MAR element and MAR‐6. Additionally, as expected, the three MAR‐containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non‐MAR‐containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP‐B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.

Identification of introns harboring functional sequence elements through positional conservation

Many human introns carry out a function, in the sense that they are critical to maintain normal cellular activity. Their identification is fundamental to understanding cellular processes and disease. However, being noncoding elements, such functional introns are poorly predicted based on traditional approaches of sequence and structure conservation. Here, we generated a dataset of human functional introns that carry out different types of functions. We showed that functional introns share common characteristics, such as higher positional conservation along the coding sequence and reduced loss rates, regardless of their specific function. A unique property of the data is that if an intron is unknown to be functional, it still does not mean that it is indeed non-functional. We developed a probabilistic framework that explicitly accounts for this unique property, and predicts which specific human introns are functional. We show that we successfully predict function even when the algorithm is trained on introns with a different type of function. This ability has many implications in studying regulatory networks, gene regulation, the effect of mutations outside exons on human disease, and on our general understanding of intron evolution and their functional exaptation in mammals.