Introns control stochastic allele expression bias

Monoallelic expression (MAE) or extreme allele bias can account for incomplete penetrance, missing heritability and non-Mendelian diseases. In cancer, MAE is associated with shorter patient survival times and higher tumor grade. Prior studies showed that stochastic MAE is caused by stochastic epigenetic silencing, in a gene and tissue-specific manner. Here, we used C. elegans to study stochastic MAE in vivo. We found allele bias/MAE to be widespread within C. elegans tissues, presenting as a continuum from fully biallelic to MAE. We discovered that the presence of introns within alleles robustly decreases MAE. We determined that introns control MAE at distinct loci, in distinct cell types, with distinct promoters, and within distinct coding sequences, using a 5’-intron position-dependent mechanism. Bioinformatic analysis showed human intronless genes are significantly enriched for MAE. Our experimental evidence demonstrates a role for introns in regulating MAE, possibly explaining why some mutations within introns result in disease.

The spread of the first introns in proto-eukaryotic paralogs

Spliceosomal introns are a unique feature of eukaryotic genes. Previous studies have established that many introns were present in the protein-coding genes of the last eukaryotic common ancestor (LECA). Intron positions shared between genes that duplicated before LECA could in principle provide insight into the emergence of the first introns. In this study we use ancestral intron position reconstructions in two large sets of duplicated families to systematically identify these ancient paralogous intron positions. We found that 20-35% of introns inferred to have been present in LECA were shared between paralogs. These shared introns, which likely preceded ancient duplications, were widespread across different functions, with the notable exception of nuclear transport. Since we observed a clear signal of pervasive intron loss prior to LECA, it is likely that substantially more introns were shared at the time of duplication than we can detect in LECA. The large extent of shared introns indicates an early origin of introns during eukaryogenesis and suggests an early origin of a nuclear structure, before most of the other complex eukaryotic features were established.

SMAD3 Hypomethylation as a Biomarker for Early Prediction of Colorectal Cancer

The incidence and mortality rates of colorectal cancer (CRC) have been high in recent years. Prevention and early detection are crucial for decreasing the death rate. Therefore, this study aims to characterize the alteration patterns of mothers against decapentaplegic homolog 3 (SMAD3) in patients with CRC and its applications in early detection by using a genome-wide methylation array to identify an aberrant hypomethylation site in the intron position of the SMAD3 gene. Quantitative methylation-specific polymerase chain reaction showed that hypomethylated SMAD3 occurred in 91.4% (501/548) of Taiwanese CRC tissues and 66.6% of benign tubular adenoma polyps. In addition, SMAD3 hypomethylation was observed in 94.7% of patients with CRC from The Cancer Genome Atlas dataset. A decrease in circulating cell-free methylation SMAD3 was detected in 70% of CRC patients but in only 20% of healthy individuals. SMAD3 mRNA expression was low in 42.9% of Taiwanese CRC tumor tissues but high in 29.4% of tumors compared with paired adjacent normal tissues. Hypomethylated SMAD3 was found in cancers of the digestive system, such as liver cancer, gastric cancer, and colorectal cancer, but not in breast cancer, endometrial cancer, and lung cancer. In conclusion, SMAD3 hypomethylation is a potential diagnostic marker for CRC in Western and Asian populations.

Complex intron generation in the yeast genus Lipomyces

In primary transcripts of eukaryotic nuclear genes, coding sequences are often interrupted by U2-type introns. Such intervening sequences can constitute complex introns excised by consecutive splicing reactions. The origin of spliceosomal introns is a vexing problem. Sequence variation existent across fungal taxa provides means to study their structure and evolution. In one class of complex introns called [D] stwintrons, an (internal) U2 intron is nested within the 5'-donor element of another (external) U2 intron. In the gene for a reticulon-like protein in species of the ascomycete yeast genus Lipomyces, the most 5' terminal intron position is occupied by one of three complex intervening sequences consistent of differently nested U2 intron units, as demonstrated in L. lipofer, L. suomiensis, and L. starkeyi. In L. starkeyi, the donor elements of the constituent introns are abutting and the complex intervening sequence can be excised alternatively either with one standard splicing reaction or, as a [D] stwintron, by two consecutive reactions. Our work suggests how [D] stwintrons could emerge by the appearance of new functional splice sites within an extant intron. The stepwise stwintronisation mechanism may involve duplication of the functional intron donor element of the ancestor intron.

Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi

MotivationGenome annotation is of key importance in many research questions. The identification of protein-coding genes is often based on transcriptome sequencing data, ab-initio or homology-based prediction. Recently, it was demonstrated that intron position conservation improves homology-based gene prediction, and that experimental data improves ab-initio gene prediction.ResultsHere, we present an extension of the gene prediction tool GeMoMa that utilizes amino acid sequence conservation, intron position conservation and optionally RNA-seq data for homology-based gene prediction. We show on published benchmark data for plants, animals and fungi that GeMoMa performs better than the gene prediction programs BRAKER1, MAKER2, and CodingQuarry, and purely RNA-seq-based pipelines for transcript identification. In addition, we demonstrate that using multiple reference organisms may help to further improve the performance of GeMoMa. Finally, we apply GeMoMa to four nematode species and to the recently published barley reference genome indicating that current annotations of protein-coding genes may be refined using GeMoMa predictions.AvailabilityGeMoMa has been published under GNU GPL3 and is freely available at http://www.jstacs.de/index.php/[email protected]

Intron Sliding and Length Variability of Genes Enriched of Phase 1 Long Introns

Due to high mutagenesis of intron sequences, intron evolution is usually considered in terms of evolution of exon-intron structures (EIS). The shifting of intron over short distances (rare evolutionary event called intron sliding) could lead to the change of intron phase, i.e. the intron position relative to the open reading frame. Here we analyze the EIS from four datasets of eukaryotic orthologues in order to find out the preferable choice of intron phase during sliding and to study the correlation between orthologous intron lengths. To identify the orthologous introns we have constructed the alignments of EIS of orthologous genes. Several sliding events with intron phase change were revealed from the analysis; however, our initial hypothesis that in the process of sliding introns prefer to change its phase to 0 more frequently, was not been confirmed. Nevertheless, it is necessary to expand the analysis on a larger dataset for making a proper conclusions. Despite high variability of intron length, some taxonomic groups share the similar length values. Moreover, some length conservation could be observed if instead of intron length L we consider a normalized length N = (L-A)/A, where A is an average length within an orthologous intron group. E.g. for ptprd genes of birds (28 species) the normalized value is in the interval (-0.15, 0.15) for 85.2 % of introns what is significantly higher than the values for random lengths set in accordance with the intron lengths distribution. That length “conservation” leads us to the question what intron length was in the ancient introns.

Intron-driven gene expression in the absence of a core promoter in Arabidopsis thaliana

In diverse eukaryotes, certain introns increase mRNA accumulation through the poorly understood mechanism of intron-mediated enhancement (IME). A distinguishing feature of IME is that these introns have no effect from upstream or more than 1 Kb downstream of the transcription start site (TSS). To more precisely define the intron position requirements for IME in Arabidopsis, we tested the effect of the UBQ10 intron on gene expression from 6 different positions surrounding the TSS of a TRP1:GUS fusion. The intron strongly increased expression from all transcribed positions, but had no effect when 204 nt or more upstream of the 5’-most TSS. When the intron was located in the 5’ UTR, the TSS unexpectedly changed, resulting in longer transcripts. Remarkably, deleting 303 nt of the core promoter, including all known TSS’s and all but 18 nt of the 5’ UTR, had virtually no effect on the level of gene expression as long as a stimulating intron was included in the gene. When the core promoter was deleted, transcription initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. Together, these results suggest that certain introns play unexpectedly large roles in directing transcription initiation and represent a previously unrecognized type of downstream regulatory elements for genes transcribed by RNA polymerase II. This study also demonstrates considerable flexibility in the sequences surrounding the TSS, indicating that the TSS is not determined by promoter sequences alone. These findings are relevant in practical applications where introns are used to increase gene expression and contribute to our general understanding of gene structure and regulation in eukaryotes.

Differences in 5-hydroxytryptamine-3B haplotype frequencies between Asians and Caucasians

Background The 5-hydroxytryptamine (5-HT3) receptor is a ligand-operated ion channel with five different receptor subunits (5-HT3A, B, C, D, and E) found in humans. Activation of 5-HT3 receptors influences various effects such as drug-induced emesis and causes behavioral problems such as anxiety, depression and cognitive disorders. To explore interethnic differences in the response to 5-HT3 antagonists, we studied haplotype frequencies in the gene encoding the 5-HT3B receptor in Asians and Caucasians. Methods Three single nucleotide polymorphisms (SNPs) of the 5-HT3B receptor gene, i.e., deletion AAG at the 5′-UTR position, 18792A>G at the intron position, and 46698G>A at the 3′ near gene position, were selected and genotyped in 165 Indonesian cancer patients and 188 Caucasian healthy volunteers. Haplotypes were set with gPlink, whereas the differences in haplotype frequencies between Indonesians and Caucasians were compared using multivariate analysis. Results The haplotype profiles based on the deletion AAG, 18792A>G and 46698G>A were AAGAA, AAGAG, AAGGG, and deletion AG in both Indonesians and Caucasians. The frequency of the AAGAG haplotype was 54.8% in Indonesians and 39.9% in Caucasians (p<0.05). The frequency of the AAGGG haplotype was 14.3% in Indonesians and 29.3% in Caucasians. Moreover, there were significant differences in the frequencies of haplotype pairs between Indonesians and Caucasians (p<0.001). Conclusion Indonesian cancer patients had significantly different AAGAG and AAGGG haplotype frequencies of the gene encoding the 5-HT3B receptor compared to healthy Caucasians. This finding could be useful for understanding interethnic differences in the response to drugs targeting the 5-HT3B receptor in cancer-treatment-related emesis.