End Of Intron Research Articles

A mutation of the β-domain in POU1F1 causes pituitary deficiency due to dominant PIT-1β expression.

POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1β, which contains an insertion of 26-amino acids (β-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1β is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with β-domain mutations have been reported. Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1β (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1β transcript without other transcripts. The lymphocyte PIT-1β mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1β-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells. We describe, for the first time, that the PIT-1β mutation can cause CPHD through a novel genetic mechanism, such as PIT-1β overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1β mutations.

TG12-T5-V470 haplotype in the CFTR gene is associated with non-obstructive azoospermia in Iranian infertile men

IntroductionRecent studies have shown that some variations and polymorphisms of CFTR gene can be involved in other types of infertility apart from CBAVD. In this study, we decided to survey the poly-T, TG repeats and M470V of the CFTR gene in infertile men with non-obstructive azoospermia in Iran. Material and methodThis case-control study was performed on 203 NOA patients and a control group (n = 200), the peripheral blood samples were collected in EDTA vacuum tubes. Then genomic DNA was extracted and PCR-sequencing was carried out on the 3′ ends of intron 8 CFTR gene to identify poly-T and TG repeats. Genotyping of M470V was carried out using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). ResultThe mean age of the infertile men and in control group was 31 ± 3.7, 30 ± 2.6 years, respectively. Frequency of T5 allele in males with non-obstructive azoospermia was found higher than that of the control group (OR 2.5798, 95% CI 1.3013–5.1143, P < 0.001). The allelic frequency of TG12-5T in males with non-obstructive azoospermia was significantly higher than that observed in the control group (OR 15.7143, 95% CI 2.7576–89.5497, P < 0.001) and also the TG12-5T-V470 haplotype was significantly associated with NOA as compared to normal controls, (OR 19.2857, 95% CI 1.0458–355.6390, P < 0.0001). ConclusionIt seems that T5, TG12-T5 and also TG12-T5-V470 haplotype can be associated with the risk of NOA in men, but further researches are necessary to clarify accurate role CFTR gene in NOA.

Does Saccharomyces cerevisiae Require Specific Post-Translational Silencing against Leaky Translation of Hac1up?

HAC1 encodes a key transcription factor that transmits the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus and regulates downstream UPR genes in Saccharomyces cerevisiae. In response to the accumulation of unfolded proteins in the ER, Ire1p oligomers splice HAC1 pre-mRNA (HAC1u) via a non-conventional process and allow the spliced HAC1 (HAC1i) to be translated efficiently. However, leaky splicing and translation of HAC1u may occur in non-UPR cells to induce undesirable UPR. To control accidental UPR activation, multiple fail-safe mechanisms have been proposed to prevent leaky HAC1 splicing and translation and to facilitate rapid degradation of translated Hac1up and Hac1ip. Among proposed regulatory mechanisms is a degron sequence encoded at the 5′ end of the HAC1 intron that silences Hac1up expression. To investigate the necessity of an intron-encoded degron sequence that specifically targets Hac1up for degradation, we employed publicly available transcriptomic data to quantify leaky HAC1 splicing and translation in UPR-induced and non-UPR cells. As expected, we found that HAC1u is only efficiently spliced into HAC1i and efficiently translated into Hac1ip in UPR-induced cells. However, our analysis of ribosome profiling data confirmed frequent occurrence of leaky translation of HAC1u regardless of UPR induction, demonstrating the inability of translation fail-safe to completely inhibit Hac1up production. Additionally, among 32 yeast HAC1 surveyed, the degron sequence is highly conserved by Saccharomyces yeast but is poorly conserved by all other yeast species. Nevertheless, the degron sequence is the most conserved HAC1 intron segment in yeasts. These results suggest that the degron sequence may indeed play an important role in mitigating the accumulation of Hac1up to prevent accidental UPR activation in the Saccharomyces yeast.

Structure of a transcribing RNA polymerase II-U1 snRNP complex.

To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.

Study of carotenoid content in durum wheat

The purpose of the study is to determine the variability and stability of traits related to the amount of yellow pigments in the grain, semolina and the finished pasta product. In addition the polymorphism in one of the phytoene synthase gene on chromosome 7A related to carotenoids synthesis was detected. In the investigation were included 10 durum wheat genotypes - cultivars and breeding lines of different origin - Bulgaria - Field Crops Institute - Chirpan and Dobroudja Agricultural Institute - General Toshevo, Europe, CYMIT-Mexico. All genotypes were grown in field conditions in competitive variety trials in three replications in harvesting years 2014/2016. The results obtained were processed statistically via basic statistics (TIBCO statistica 13.3.0 software package). Great variability was found between genotypes on the study traits. The breeding lines: M-431 (10.71 ppm in grain, 10.42 ppm in semolina and 8.96 ppm in pasta) and TD-97 (9.83 ppm in grain, 9.58 ppm in semolina and 8.52 ppm in pasta) and the Bulgarian standard cultivar Predel (9.25 ppm in grain, 8.87 ppm in semolina and 7.27 ppm in pasta) are characterized by the highest yellow pigment content in grain, semolina and pasta. Both lines are also distinguished by the lowest total pigment losses (2.6%) in milling grain to semolina and in processing to pasta product (11 - 11.4 %), while line TD-97 has the lowest total pigment losses (13.6%). Furthermore the genotypes characterized by the highest yellow pigment content (M-431 and TD-97) have an acceptable stability and can be used in the hybridization program to improve the color potential of Bulgarian durum wheat varieties. The Psy-A1 codominant marker, based on a 37 bp insertion at the 5' end of intron 2 of the PsyA1 gene amplifies two fragments. The Psy-A1a allele with a 194 bp fragment in length was observed in all 7 studied cultivars and lines of durum wheat. The 231-bp Psy-A1b allele is found in 3 genotypes. The obtained results could not be associated with phenotypic differences in the yellow pigment content in the durum wheat genotypes studied.

A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids

Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.

Distant coupling between RNA editing and alternative splicing of the osmosensitive cation channel Tmem63b

Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5′ end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.

A Seven-Transmembrane Protein -TM7SF3, Resides in Nuclear Speckles and Regulates Alternative Splicing

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates the development of cellular stress and unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This rather unexpected location of a membranal protein enables it to form stable complexes with regulators of pre-mRNA splicing including DHX15, LARP7, HNRNPU and RBM14 that co-precipitate with TM7SF3. Indeed, TM7SF3 modulates alternative splicing of >400 genes, mainly splicing at the 3’end of introns. These effects are observed both in primary human pancreatic islets and in a number of cell lines, under basal conditions and under stress. Accordingly, silencing of TM7SF3 resulted in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

Identification of a novel splicing variant of IDS gene in a pedigree affected with type II glycosaminoglycan product storage disease

To analyze variant of IDS gene in a pedigree affected with mucopolysaccharidosis type II (MPS II). The proband was subjected to next generation sequencing and Sanger sequencing to identify potential variants. Suspected variant was analyzed by its co-segregation with the disease in the pedigree. Its impact on mRNA splicing was analyzed by using reverse transcription PCR (RT-PCR). A hemizygous IVS1-3T>G variant was found in the IDS gene in the proband. RT-PCR results revealed two abnormal cDNA fragments of 600 bp and 300 bp. The 600 bp fragment had inserted 216 nucleotides at the 3' end of intron 1, while the 300 bp fragment had lost 109 nucleotides at the 5' end of exon 2, which resulted in two truncated proteins comprising 38 and 92 amino acids, respectively, instead of the normal product (550 amino acids). The proband and his mother were respectively hemizygous and heterozygous for the variant. The same variant was not found among 100 normal controls. The IVS1-3T>G variant of the IDS gene probably underlies the MPS II in this pedigree by causing reduction or elimination of the IDS protein.

The novel gene BrMYB2, located on chromosome A07, with a short intron 1 controls the purple-head trait of Chinese cabbage (Brassica rapa L.)

Anthocyanins are important secondary metabolites in plants, but information on anthocyanin biosynthesis mechanisms in Chinese cabbage is limited. The new purple head Chinese cabbage cultivar 11S91 was analyzed, and an R2R3-MYB regulatory gene BrMYB2, located on chromosome A07, controlling the dominant purple-head trait was isolated. High expression of BrMYB2 generated a large accumulation of anthocyanins in 11S91, accompanied by highly upregulated BrTT8, BrF3′H, BrDFR1, BrANS1, BrUGTs, BrATs, and BrGSTs. 11S91 inherited the purple locus from purple trait donor 95T2-5, and they shared consensus CDSs and gDNAs with those of BrMYB2 (cBrMYB2 and gBrMYB2). Two SNPs in cBrMYB2 in 11S91 did not cause loss of function; in addition to several SNPs at both ends of intron 1, a large deletion had occurred in intron 1 of gBrMYB2 in 11S91. Genetic transformation of Arabidopsis showed that gBrMYB2 overexpression lines presented deeper purple color and higher expression than did the cBrMYB2 and cBrmyb2 lines, whereas gBrmyb2 with a long intron 1 did not cause the purple phenotype. We first show that BrMYB2 promotes anthocyanin biosynthesis under the control of the short intron 1 of gBrMYB2 in purple head Chinese cabbage, and gBrmyb2 with a long intron 1 represses anthocyanin production in white head Chinese cabbage. This evidence provides a new understanding of anthocyanin biosynthesis and purple germplasm generation in Brassica vegetables.

Analysis of Putative Epigenetic Regulatory Elements in the FXN Genomic Locus.

Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an abnormally expanded Guanine-Adenine-Adenine (GAA) repeat sequence within the first intron of the frataxin gene (FXN). The molecular mechanisms associated with FRDA are still poorly understood and most studies on FXN gene regulation have been focused on the region around the minimal promoter and the region in which triplet expansion occurs. Nevertheless, since there could be more epigenetic changes involved in the reduced levels of FXN transcripts, the aim of this study was to obtain a more detailed view of the possible regulatory elements by analyzing data from ENCODE and Roadmap consortia databases. This bioinformatic analysis indicated new putative regulatory regions within the FXN genomic locus, including exons, introns, and upstream and downstream regions. Moreover, the region next to the end of intron 4 is of special interest, since the enhancer signals in FRDA-affected tissues are weak or absent in this region, whilst they are strong in the rest of the analyzed tissues. Therefore, these results suggest that there could be a direct relationship between the absence of enhancer sequences in this specific region and their predisposition to be affected in this pathology.

ATP2A2 SINE Insertion in an Irish Terrier with Darier Disease and Associated Infundibular Cyst Formation.

A 4-month-old female Irish Terrier presented with a well demarcated ulcerative and crusting lesion in the right ear canal. Histological analysis revealed epidermal hyperplasia with severe acantholysis affecting all suprabasal layers of the epidermis, which prompted a presumptive diagnosis of canine Darier disease. The lesion was successfully treated by repeated laser ablation of the affected epidermis. Over the course of three years, the dog additionally developed three dermal nodules of up to 4 cm in diameter that were excised and healed without complications. Histology of the excised tissue revealed multiple infundibular cysts extending from the upper dermis to the subcutis. The cysts were lined by squamous epithelium, which presented with abundant acantholysis of suprabasal keratinocytes. Infundibular cysts represent a novel finding not previously reported in Darier patients. Whole genome sequencing of the affected dog was performed, and the functional candidate genes for Darier disease (ATP2A2) and Hailey-Hailey disease (ATP2C1) were investigated. The analysis revealed a heterozygous SINE insertion into the ATP2A2 gene, at the end of intron 14, close to the boundary of exon 15. Analysis of the ATP2A2 mRNA from skin of the affected dog demonstrated a splicing defect and marked allelic imbalance, suggesting nonsense-mediated decay of the resulting aberrant transcripts. As Darier disease in humans is caused by haploinsufficiency of ATP2A2, our genetic findings are in agreement with the clinical and histopathological data and support the diagnosis of canine Darier disease.

TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1.

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.

Genome-Wide Characterization of Alternative Splicing Events and Their Responses to Cold Stress in Tilapia.

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism for cells to generate transcript variability and proteome diversity. No systematic investigation of AS events among different tissues in response to stressors is available for tilapia currently. In this study, AS among different tissues was identified and the cold stress-related AS events were explored in a Nile tilapia (Oreochromis niloticus) line based on 42 RNA-seq datasets using a bioinformatics pipeline. 14,796 (82.76%; SD = 2,840) of the expression genes showed AS events. The two most abundant AS types were alternative transcription start site (TSS) and terminal site (TTS) in tilapia. Testis, brain and kidney possess the most abundant AS gene number, while the blood, muscle and liver possess the least number in each tissue. Furthermore, 208 differentially alternative splicing (DAS) genes in heart and 483 DAS in brain in response to cold stress. The number of AS types for alternative exon end, exon skipping and retention of single intron increased significantly under cold stress. GO enrichment and pathway overrepresentation analysis indicated that many DAS genes, e.g., genes in circadian clock pathway, may influence expression of downstream genes under cold stress. Our study revealed that AS exists extensively in tilapia and plays an important role in cold adaption.

Crispr/Cas9 Gene Editing Targeted to an Intron of a Novel Isoform of the β-Subunit of Thyroid Stimulating Hormone in Peripheral Leukocytes

Abstract Hashimoto’s disease and Graves’ disease are two forms of autoimmune thyroiditis (AIT). Although various treatments protocols are available for each disease, the specific events involved in the induction of AIT remain largely unknown. We identified a novel splice variant of the β-subunit of thyroid stimulating hormone (TSHβ) in humans and mice, referred to as TSHβv, that is produced almost exclusively by bone marrow cells and peripheral leukocytes. In mice, pituitary-derived TSHβ uses exon 4 and exon 5 to code for the TSHβ polypeptide, whereas TSHβv uses exon 5 and the 3′ end of intron 4. We recently demonstrated that during viral and bacterial infection, peripheral leukocytes traffic to the thyroid and produce large amounts of TSHβv intrathyroidally. We have speculated that although this may have beneficial physiological effects for the host, it also may be a factor in the initiation and/or perpetuation of AIT. To explore this, we made Crispr/Cas9 knockout mice in which the production of immune system TSHβv but not pituitary TSHβ is disrupted by insertion of a stop codon in the terminal portion of intron 4. Of 24 founder mice, 3 had gene editing in intron 4 as determined by PCR and sequence analyses. Those animals were back-crossed with wildtype mice to get F1 heterozygous colonies and clean genomic DNA with deletions at the stop codon insertion site. F1 offspring from two of the founder mice lacked the gene-edited site in intron 4. However, 6 offspring from the third founder animal had the expected editing in intron 4. Those animals are being bred to obtain homozygous mice for experimental use. To our knowledge, this is one of the few examples of gene disruption targeted to an intron with potential functional deletion of a novel isoform.

Complete nucleotide sequence characterization of DRB5 alleles reveals a homogeneous allele group that is distinct from other DRB genes

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A better and comprehensive sequence assessment can be achieved by the inclusion of full gene sequences of all the common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of two alleles available. In the present study the DRB5 genes from 18 subjects alleles were cloned and sequenced; haplotype analysis showed that 17 of them had a single copy of DRB5 and one consanguineous subject was homozygous at all HLA loci. Methodological approaches including robust and efficient long-range PCR amplification, molecular cloning, nucleotide sequencing and de novo sequence assembly were combined to characterize DRB5 alleles. DRB5 sequences covering from 5′UTR to the end of intron 5 were obtained for DRB5*01:01, 01:02 and 02:02; partial coverage including a segment spanning exon 2 to exon 6 was obtained for DRB5*01:03, 01:08N and 02:03. Phylogenetic analysis of the generated sequences showed that the DRB5 alleles group together and have distinctive differences with other DRB loci. Novel intron variants of DRB5*01:01:01, 01:02 and 02:02 were identified. The newly characterized DRB5 intron variants of each DRB5 allele were found in subjects harboring distinct associations with alleles of DRB1, B and/or ethnicity. The new information provided by this study provides reference sequences for HLA typing methodologies. Extending sequence coverage may lead to identify the disease susceptibility factors of DRB5 containing haplotypes while the unexpected intron variations may shed light on understanding of the evolution of the DRB region.

RNA-Seq approach for accurate characterization of splicing efficiency of yeast introns.

Introns in different genes, or even different introns within the same gene, often have different splice sites and differ in splicing efficiency (SE). One expects mass-transcribed genes to have introns with higher SE than weakly transcribed genes. However, such a simple expectation cannot be tested directly because variable SE for these genes is often not measured. Mechanistically, SE should depend on signal strength at key splice sites (SS) such as 5'SS, 3'SS and branchpoint site (BPS), i.e., SE = F(5'SS, 3'SS, BPS). However, without SE, we again cannot model how these splice sites contribute to SE. Here I present an RNA-Seq approach to quantify SE for each of the 304 introns in yeast (Saccharomyces cerevisiae) genes, including 24 in the 5'UTR, by measuring 1) number of reads mapped to exon-exon junctions (NEE) as a proxy for the abundance of spliced form, and 2) number of reads mapped to exon-intron junction (NEI5 and NEI3 at 5' and 3' ends of intron) as a proxy for the abundance of unspliced form. The total mRNA is NTotal = NEE + p * NEI5 + (1-p) * NEI3, with the simplest p = 0.5 but statistical methods were presented to estimate p from data. An estimated p is needed because NEI5 is expected to be smaller than NEI3 due to 1) step 1 splicing occurs before step 2 so EI5 is broken before EI3, 2) enrichment of poly(A) mRNA by oligo-dT, and 3) 5' degradation. SE is defined as the proportion (NEE/NTotal). Application of the method shows that ribosomal protein messages are efficiently and mostly cotranscriptionally spliced. Yeast genes with long introns are also spliced efficiently. HAC1/YFL031W is poorly spliced partly because its splicing involves a nonspliceosome mechanism and partly because Ire1p, which participate in splicing HAC1, is hardly expressed. Many putative yeast genes have low SE, and some splice sites are incorrectly annotated.

Novel Splicing of Immune System Thyroid Stimulating Hormone β-Subunit-Genetic Regulation and Biological Importance.

Thyroid stimulating hormone (TSH), a glycoprotein hormone produced by the anterior pituitary, controls the production of thyroxine (T4) and triiodothyronine (T3) in the thyroid. TSH is also known to be produced by the cells of the immune system; however, the physiological importance of that to the organism is unclear. We identified an alternatively-spliced form of TSHβ that is present in both humans and mice. The TSHβ splice variant (TSHβv), although produced at low levels by the pituitary, is the primary form made by hematopoietic cells in the bone marrow, and by peripheral leukocytes. Recent studies have linked TSHβv functionally to a number of health-related conditions, including enhanced host responses to infection and protection against osteoporosis. However, TSHβv also has been associated with autoimmune thyroiditis in humans. Yet to be identified is the process by which the TSHβv isoform is produced. Here, a set of genetic steps is laid out through which human TSHβv is generated using splicing events that result in a novel transcript in which exon 2 is deleted, exon 3 is retained, and the 3′ end of intron 2 codes for a signal peptide of the TSHβv polypeptide.

Widespread Separation of the Polypyrimidine Tract From 3' AG by G Tracts in Association With Alternative Exons in Metazoa and Plants.

At the end of introns, the polypyrimidine tract (Py) is often close to the 3′ AG in a consensus (Y)20NCAGgt in humans. Interestingly, we have found that they could also be separated by purine-rich elements including G tracts in thousands of human genes. These regulatory elements between the Py and 3′ AG (REPA) mainly regulate alternative 3′ splice sites (3′ SS) and intron retention. Here we show their widespread distribution and special properties across kingdoms. The purine-rich 3′ SS are found in up to about 60% of the introns among more than 1,000 species/lineages by whole genome analysis, and up to 18% of these introns contain the REPA G-tracts (REPAG) in about 0.6 million of 3′ SS in total. In particular, they are significantly enriched over their 3′ SS and genome backgrounds in metazoa and plants, and highly associated with alternative splicing of genes in diverse functional clusters. Cryptic splice sites harboring such G- and the other purine-triplets tend to be enriched (2–9 folds over the disrupted canonical 3′ SS) and aberrantly used in cancer patients carrying mutations of the SF3B1 or U2AF35, factors critical for branch point (BP) or 3′ AG recognition, respectively. Moreover, the REPAGs are significantly associated with reduced occurrences of BP motifs between the −24 and −4 positions, in particular absent between the −7 and −5 positions in several model organisms examined. The more distant BPs are associated with increased occurrences of alternative splicing in humans and zebrafish. The REPAGs appear to have evolved in a species- or phylum-specific way. Thus, there is widespread separation of the Py and 3′ AG by REPAGs that have evolved differentially. This special 3′ SS arrangement likely contributes to the generation of diverse transcript or protein isoforms in biological functions or diseases through alternative or aberrant splicing.

Understanding human DNA variants affecting pre-mRNA splicing in the NGS era.

Pre-mRNA splicing, an essential step in eukaryotic gene expression, relies on recognition of short sequences on the primary transcript intron ends and takes place along transcription by RNA polymerase II. Exonic and intronic auxiliary elements may modify the strength of exon definition and intron recognition. Splicing DNA variants (SV) have been associated with human genetic diseases at canonical intron sites, as well as exonic substitutions putatively classified as nonsense, missense or synonymous variants. Their effects on mRNA may be modulated by cryptic splice sites associated to the SV allele, comprehending exon skipping or shortening, and partial or complete intron retention. As splicing mRNA outputs result from combinatorial effects of both intrinsic and extrinsic factors, in vitro functional assays supported by computational analyses are recommended to assist SV pathogenicity assessment for human Mendelian inheritance diseases. The increasing use of next-generating sequencing (NGS) targeting full genomic gene sequence has raised awareness of the relevance of deep intronic SV in genetic diseases and inclusion of pseudo-exons into mRNA. Finally, we take advantage of recent advances in sequencing and computational technologies to analyze alternative splicing in cancer. We explore the Catalog of Somatic Mutations in Cancer (COSMIC) to describe the proportion of splice-site mutations in cis and trans regulatory elements. Genomic data from large cohorts of different cancer types are increasingly available, in addition to repositories of normal and somatic genetic variations. These are likely to bring new insights to understanding the genetic control of alternative splicing by mapping splicing quantitative trait loci in tumors.