UTR Ends Research Articles

EIF3 engages with 3'-UTR termini of highly translated mRNAs.

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3' untranslated region (3'-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3'-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3'-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. The results presented here show that eIF3 engages with 3'-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

CLCN5 5’UTR isoforms in human kidneys: differential expression analysis between controls and patients with glomerulonephritis

ClC-5, the electrogenic chloride/proton exchanger strongly expressed in renal proximal tubules, belongs to the endocytic macromolecular complex responsible for albumin and low-molecular-weight protein uptake. ClC-5 was found to be overexpressed...

BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq

BackgroundThe time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants.ResultsA high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts – BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427–433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20–28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5′ and 3′ UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage.ConclusionA high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

BackgroundDent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5’UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the CLCN5 5’UTR region has not yet been fully characterized. To date 6 different 5’ alternatively used exons - 1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb) - have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5’UTR region in the human kidney and other tissues.MethodsTo clone more of the 5’ end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5’ ends was applied.The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real -Time RT/PCR.ResultsEleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5’UTR isoforms were confirmed, but their ends were extended. Six additional 5’UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein.ConclusionsOur results confirm the structural complexity of the CLCN5 5’UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.

Resequencing and association study of the NFKB activating protein-like gene (NKAPL) in schizophrenia

ObjectivesSchizophrenia is a highly inheritable disorder, but many aspects of its etiology and pathophysiology remain poorly understood. Recently, in the Han Chinese population, a SNP rs1635 located within the exon of the NKAPL gene (encoding NFKB activating protein-like) achieved genome-wide significance in schizophrenia. MethodsIn order to find the causal variants of the NKAPL gene in schizophrenia, we searched for genetic variants in the promoter region, and exons (including both UTR ends) using direct sequencing in a sample of patients with schizophrenia (n=515) and non-psychotic controls (n=456), all Han Chinese from Taiwan, and conducted an association and rudimentary functional study. ResultsWe identified 5 common SNPs (defined as minor allele frequency (MAF)>0.01) in the NKAPL gene. In a case–control association analysis, the minor allele (A) of rs1635 was significantly more common among patients than controls (P=0.0003, OR=1.41, 95% CI=1.17–1.71). A haplotype analysis of the 5 common SNPs indicated a risk haplotype (rs12000C–rs1635A–rs9461446C–rs3734564G–rs1679709G) associated with schizophrenia (P=2.77e-005, OR=1.53, 95% CI=1.25–1.87). In addition, we identified 4 patient-specific rare SNPs (MAF<0.01) (c.137G>A, c.213G>A, c.752C>T (rs370337122), and c.844G>A (rs147161729)) within the NKAPL gene. In silico analysis demonstrated their functional impact on the protein; however, there was also 1 control-specific rare SNP (c.119G>A) detected within the NKAPL gene, indicating that the clinical relevance of these putatively pathological rare SNPs is not straightforward. ConclusionsThis study suggested that rs1635 in the NKAPL gene appeared to play a role in conferring susceptibility to schizophrenia. In addition, some rare SNPs in the NKAPL gene with possibly damaging effects may be important in our patients. Our study provides genetic clues to indicate the involvement of NKAPL in schizophrenia.

Measurements of the Impact of 3′ End Sequences on Gene Expression Reveal Wide Range and Sequence Dependent Effects

A full understanding of gene regulation requires an understanding of the contributions that the various regulatory regions have on gene expression. Although it is well established that sequences downstream of the main promoter can affect expression, our understanding of the scale of this effect and how it is encoded in the DNA is limited. Here, to measure the effect of native S. cerevisiae 3′ end sequences on expression, we constructed a library of 85 fluorescent reporter strains that differ only in their 3′ end region. Notably, despite being driven by the same strong promoter, our library spans a continuous twelve-fold range of expression values. These measurements correlate with endogenous mRNA levels, suggesting that the 3′ end contributes to constitutive differences in mRNA levels. We used deep sequencing to map the 3′UTR ends of our strains and show that determination of polyadenylation sites is intrinsic to the local 3′ end sequence. Polyadenylation mapping was followed by sequence analysis, we found that increased A/T content upstream of the main polyadenylation site correlates with higher expression, both in the library and genome-wide, suggesting that native genes differ by the encoded efficiency of 3′ end processing. Finally, we use single cells fluorescence measurements, in different promoter activation levels, to show that 3′ end sequences modulate protein expression dynamics differently than promoters, by predominantly affecting the size of protein production bursts as opposed to the frequency at which these bursts occur. Altogether, our results lead to a more complete understanding of gene regulation by demonstrating that 3′ end regions have a unique and sequence dependent effect on gene expression.

Genetic and functional analyses of the gene encoding synaptophysin in schizophrenia

ObjectivesSynaptophysin (SYP) has been shown to be critical for regulating neurotransmitter release and synaptic plasticity, a process thought to be disrupted in schizophrenia. In addition, abnormal SYP expression in different brain regions has been linked to this disorder in postmortem brain studies. We investigated the involvement of the SYP gene in the susceptibility to schizophrenia. MethodsWe searched for genetic variants in the promoter region, all exons, and both UTR ends of the SYP gene using direct sequencing in a sample of patients with schizophrenia (n=586) and non-psychotic controls (n=576), both being Han Chinese from Taiwan, and conducted an association and functional study. ResultsWe identified 2 common SNPs (c.*4+271A>G and c.*4+565T>C) in the SYP gene. SNP and haplotype-based analyses displayed no associations with schizophrenia. In addition, we identified 6 rare variants in 7 out of 586 patients, including 1 variant (g.-511T>C) located at the promoter region, 1 synonymous (A104A) and 2 missense variants (G293A and A324T) located at the exonic regions, and 2 variants (c.*31G>A and c.*1001G>T) located at the 3′UTR. No rare variants were found in the control subjects. The results of the reporter gene assay demonstrated the influence of g.-511T>C and c.*1001G>T on the regulatory function of the SYP gene, while that the influence of c.*31G>A may be tolerated. In silico analysis demonstrated the functional relevance of other rare variants. ConclusionOur study lends support to the hypothesis of multiple rare mutations in schizophrenia, and provides genetic clues that indicate the involvement of SYP in this disorder.

Genetic and functional analysis of the gene encoding neurogranin in schizophrenia

ObjectivesSchizophrenia is a highly heritable disorder, but many aspects of its etiology and pathophysiology remain poorly understood. Recently, a SNP rs12807809 located upstream of the neurogranin (NRGN) gene achieved genome-wide significance in this disorder. MethodsIn order to find the causal variants of NRGN gene in schizophrenia, we searched for genetic variants in the promoter region and all the exons (including both UTR ends and rs12807809) using direct sequencing in a sample of patients with schizophrenia (n=346) and non-psychotic controls (n=345), both being Han Chinese from Taiwan, and conducted an association and functional study. ResultsWe identified 7 common polymorphisms in the NRGN gene. SNP and haplotype-based analyses displayed no associations with schizophrenia. Additionally, we identified 5 rare variants in 6 out of 346 patients, including 3 rare variants located at the promoter region (g.-620A>G, g.-578C>G, and g.-344G>A) and 2 rare variants located at 5′ UTR (c.-74C>G, and c.-41G>A). No rare variants were found in the control subjects. The results of the reporter gene assay demonstrated that the regulatory activity of construct containing g.-620G, g.-578G, g.-344A, c.-74G, and c.-41A was significantly lower as compared to the wild type construct (P<0.01 for g.-578G; P<0.001 for the other constructs). In silico analysis also demonstrated their influences on the regulatory function of NRGN gene. ConclusionsOur study lends support to the hypothesis of multiple rare mutations in schizophrenia, and provides genetic clues that indicate the involvement of NRGN in this disorder.

Genetic and functional analysis of the gene encoding GAP-43 in schizophrenia

ObjectivesIn earlier reports, growth-associated protein 43 (GAP-43) has been shown to be critical for initial establishment or reorganization of synaptic connections, a process thought to be disrupted in schizophrenia. Additionally, abnormal GAP-43 expression in different brain regions has been linked to this disorder in postmortem brain studies. In this study, we investigated the involvement of the gene encoding GAP-43 in the susceptibility to schizophrenia. MethodsWe searched for genetic variants in the promoter region and 3 exons (including both UTR ends) of the GAP-43 gene using direct sequencing in a sample of patients with schizophrenia (n=586) and non-psychotic controls (n=576), both being Han Chinese from Taiwan, and conducted an association and functional study. ResultsWe identified 11 common polymorphisms in the GAP-43 gene. SNP and haplotype-based analyses displayed no associations with schizophrenia. Additionally, we identified 4 rare variants in 5 out of 586 patients, including 1 variant located at the promoter region (c.-258-4722G>T) and 1 synonymous (V110V) and 2 missense (G150R and P188L) variants located at exon 2. No rare variants were found in the control subjects. The results of the reporter gene assay demonstrated that the regulatory activity of construct containing c.-258-4722T was significantly lower as compared to the wild type construct (c.-258-4722G; p<0.001). In silico analysis also demonstrated the functional relevance of other rare variants. ConclusionsOur study lends support to the hypothesis of multiple rare mutations in schizophrenia, and it provides genetic clues that indicate the involvement of GAP-43 in this disorder.

P02-218 - Resequencing of the GAP-43 gene reveals some rare genetic variants that may increase the genetic burden in schizophrenia

ObjectivesGrowth-associated protein 43 (GAP-43) was critical for initial establishment or reorganization of synaptic connections, a process thought to be disrupted in schizophrenia. Abnormal GAP-43 expression has been linked to this disorder in numerous postmortem brain studies. The purpose of this study was to investigate the involvement of the gene encoding GAP-43 in the susceptibility to schizophrenia.MethodsWe searched for genetic variants in the promoter region and 3 exons (including both UTR ends) of the GAP-43 gene using direct sequencing in a sample of Han Chinese schizophrenic patients (n = 354) and non-psychotic controls (n = 338) from Taiwan, and conducted a case-control association study.ResultsWe identified 11 common SNPs in the GAP-43 gene. SNP and haplotype-based analyses showed no association with schizophrenia. Besides, we identified 4 rare variants in 4 out of 354 patients, including 1 variant located at the promoter region, 1 synonymous and 2 missense variants located at exon 2. No rare variants were found in the control subjects. Collectively, these rare variants were significantly overrepresented in the patient group (1.1% v.s 0; p value of Fisher’s exact test = 0.02), suggesting they may increase the genetic burden in schizophrenia.ConclusionAlthough the functional significance of these rare variants remained to be characterized, our study lent support to the hypothesis of multiple rare mutations in schizophrenia, and provided genetic clues to indicate the involvement of neurodevelopment defect in this disorder.

Resequencing of the vesicular glutamate transporter 2 gene (VGLUT2) reveals some rare genetic variants that may increase the genetic burden in schizophrenia

Objectives Vesicular glutamate transporters (VGLUT1-3) package glutamate into vesicles in the presynaptic terminal and regulate the release of glutamate. In mesencephalic dopamine neuron culture, the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3, have been demonstrated. As related to the dysregulated glutamatergic hypothesis of schizophrenia, the gene encoding VGLUT2 is the most plausible candidate involved in the pathogenesis of this illness. Methods We searched for genetic variants in the promoter region and 12 exons (including UTR ends) of the VGLUT2 gene using direct sequencing in a sample of Han Chinese schizophrenic patients ( n = 375) and non-psychotic controls ( n = 366) from Taiwan, and conducted a case-control association study. Results We identified 8 common SNPs in the VGLUT2 gene. SNP and haplotype-based analyses showed no association with schizophrenia. Besides, we identified 9 rare variants in 13 out of 375 patients, including 3 variants located at the promoter region, 2 synonymous variants located at protein coding regions, and 4 variants located at UTR ends. No rare variants were found in the control subjects. Collectively, these rare variants were significantly overrepresented in the patient group (3.5% versus 0, p value of Fisher's exact test = 2.3 × 10 − 5), suggesting they may contribute to the pathogenesis of schizophrenia. Conclusion Although the functional significance of these rare variants remains to be characterized, our study may lend support to the multiple rare mutations hypothesis of schizophrenia, and may provide genetic clues to indicate the involvement of the glutamate transmission pathway in the pathogenesis of schizophrenia.

Resequencing and association study of vesicular glutamate transporter 1 gene (VGLUT1) with schizophrenia

Dysregulation of glutamate neurotransmission is implicated in the pathphysiology of schizophrenia. Vesicular glutamate transporters (VGLUTs) package glutamate into vesicles in the presynaptic terminal and regulate the release of glutamate. Abnormal VGLUT1 expression has been linked to schizophrenia in postmortem brain studies. The purpose of this study was to investigate the involvement of the human VGLUT1 in the susceptibility to schizophrenia. In this study, we searched for genetic variants in the putative core promoter region and 12 exons (including UTR ends) of the VGLUT1 gene using direct sequencing in a sample of Han Chinese schizophrenic patients (n=376) and non-psychotic controls (n=368) from Taiwan, and conducted a case-control association study. We identified two common SNPs (g.-248G>C (ss159695612) and c.2697C>A (rs1043558)) in the VGLUT1 gene. No differences in the allele and genotype frequencies were detected between the patients and control subjects. Besides, we identified eight patient-specific rare variants in 16 out of 376 patients, including two variants (g.-296A>G (ss159695611) and g.-32Cv>T (ss159695613)) at the core promoter region and 5′UTR, two missense variants (L516M (ss159695617) and P551S (ss159695618)) and three silent variants (E24E (ss159695614), L118L (ss159695615), and P133P (ss159695616)) at protein-coding regions, and one variant (c.2201G>A (ss159695619)) at the 3′UTR. No rare variants were found in 368 control subjects (4.3% versus 0, P=1.5×10−5). Although the functional significance of these rare variants remains to be characterized, our study may lend support to the multiple rare mutation hypothesis of schizophrenia, and may provide genetic clues to indicate the involvement of the glutamate transmission pathway in the pathogenesis of schizophrenia.

Exomic sequencing of the glutamate receptor, ionotropic, N-methyl- d-aspartate 3A gene (GRIN3A) reveals no association with schizophrenia

Background Growing evidence suggests that dysregulation of N-methyl- d-aspartate receptor (NMDAR)-mediated glutamate neurotransmission may be involved in the pathophysiology of schizophrenia. The NMDAR is a heteromeric protein complex consisting of subunits from three subfamilies (NR1, NR2A, 2B, 2C, 2D and NR3A, 3B). The unique ability of NR3A to modulate the NMDAR function makes it an attractive candidate gene of schizophrenia. The purpose of this study was to investigate the involvement of the gene encoding the human NR3A subunit (GRIN3A) in the liability to schizophrenia. Methods We searched for genetic variants in the putative core promoter region and all the exons (including UTR ends) of the GRIN3A gene in 333 Han Chinese patients with schizophrenia and 369 control subjects from Taiwan using direct polymerase chain reaction (PCR) autosequencing, and assessed their association with schizophrenia. Results We identified 22 single nucleotide polymorphisms (SNPs) in the GRIN3A gene in this sample. SNP- and haplotype-based analyses showed no association of these 22 SNPs with schizophrenia. Nevertheless, we identified two missense mutations (D133N and Q1091H), one nonsense mutation (R1024X), and two synonymous mutations (Y873Y and E889E) of the GRIN3A gene in 6 out of 333 (1.8%) patients, while no rare mutations were found in 369 control subjects ( p = 0.011, Fisher's exact test, one-tailed). In silico analysis showed that the R1024X and Q1091H mutations are possibly damaging. Conclusions Although the functional significance of these mutations remains to be characterized, our study indicates that rare mutations in the GRIN3A gene may contribute to the pathogenesis of schizophrenia in certain patients.

Application of single-strand conformation polymorphism to the study of bovine viral diarrhea virus isolates.

Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products is a genetic screening technique for rapid detection of nucleotide substitutions in PCR-amplified genomic DNA or cDNA. It is based on the observation that partially formamide-denatured double-stranded DNA migrates as 2 single-stranded DNA molecules when electrophoresed in nondenaturing polyacrylamide gels. The mobility depends on the 3-dimensional conformation of the strand under the conditions used. It is possible to discriminate between DNA strands differing in only 1 nucleotide. The method was applied to the analysis of Bovine Viral Diarrhea Virus (BVDV) isolates. Reference and Argentinian strains were assessed for variations in their 5' untranslated region (5'-UTR). The PCR products of the 5'-UTR ends were formamide denatured and compared by SSCP analysis in nondenaturing 15% polyacrylamide and 15% polyacrilamide-5% glycerol gels. The reference strains SD-1, Singer, and Oregon C24V had differences in electrophoretic patterns. Despite the high conservation among the 5'-UTR of pestiviruses, the method allowed discrimination among all 9 Argentinian isolates. The 5'-UTR of a fetal kidney-derived isolate (1R93) was PCR amplified and cloned in a plasmid vector; the SSCP analysis of 30 PCR products obtained by direct amplification over randomly selected clones produced 5 different banding patterns, indicating the existence of viral quasispecies. The results show that SSCP may be used to identify and differentiate among BVDV isolates.

Identification by Denaturing High-Performance Liquid Chromatography of Numerous Polymorphisms in a Candidate Region for Multiple Sclerosis Susceptibility

Genetic association analysis of candidate regions where evidence of linkage has accumulated is becoming a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently available single nucleotide polymorphism (SNP) map designed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approximately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chromatography (DHPLC). A systematic search for SNPs was performed in the region 5ptel–q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymorphisms was performed on 124 sequence tagged sites (STSs) in the 3′UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on expressed gene sequences tagged by a set of standard commercially available primers.