Validation Of Functional Impact Of UORF-altering Mutations On The Expression Of Its Host Gene By Dual-luciferase Assays

Background and ObjectivesIn eukaryotic organism, the process which DNA is as a template to synthesize RNA is called transcription. In vast majority of cells, RNA including three categories: messenger RNA (mRNA)、ribosomeal RNA(rRNA)、and transfer RNA(tRNA). As the translational template, mRNA guides the protein biosynthesis. Ribosome consists of rRNA and ribosomal proteins scanning on mRNA, and becomes the place where it translates into protein while tRNA as an adapter carrying the corresponding amino acids into ribosome to assemble polypeptide chains, according to the codons in the mRNA. In addition, some small nuclear RNA (snRNA) and micro RNA (mi RNA) associate with mRNA splicing and regulation of gene expression, respectively. Thus, mRNA occupies a pivotal position in the protein biosynthesis.Gene expression is a very complex process. How to express or how much can express for a gene to a large extent depend on many levels of regulation, such as DNA replication、RNA transcription、mRNA processing、protein translation and the posttranslational modification. Post-transcriptional regulation mechanism is investigated more clearly. After transcription, precursor mRNA must be porcessing and splicing to become mature mRNA for template guiding the protein biosynthesis. As a mature mRNA, it has several sections, including 5 ’end of the 7-methyl guanine cap structure, 5’untranslated region, coding sequence, 3’untranslated region and PloyA tail. There are some cis-acting elements whitch could impact on the efficiency of translation of dowmstream main open reading frame (mORF) in the 5’leader sequence. These elements include the secondary structure that is able to inhibit scanning ribosome to recogntion AUG initiation condon. internal ribosome entry sites (IRESs) that associated with cap-independent translation, hairpin structure, some protein binding sites and upstream open reading frames(uORFs). To date, uORFs were generally investigsted by many reseachers. There was reported that about 49% of human and 44% of mouse transcripts contain at least 1 uORF. It showed that the presence of uORFs is a very commonly phenomenon in mammalian. These references also showed that uORFs can reduce the efficiency of translation initiation of downstream mORF through many regulation mechanisms or trigger mRNA decay to repress gene expression. Furthermore, these mechanisms involved in the length of uORF, the number of uORFs, conservation among species, distance between 5’cap and the uORF, length of the intercistronic region and context in which the uORF AUG is located.The studies also shown that single nucleotide polymorphisms(SNPs) in the uORFs could affect gene expression which may have some relevance to human disease phenotype. In this case, an SNP consists of a common C to T polymorphism in the uORF of the human clotting factor Ⅻ(FⅫ), reseachers have experimentally confrimed that containing T allele could reduce protein levels by about 50%, it is likely to change downstream gene expression in the naturally occurring uORF polymorphism. Besides, having references reported, genetics and bioinformatic studies shown that some human diseases have closely associated with mutations occurred in the uORF. These mutations could create or disrupt uORF and result in significantly affecting gene expression. Created uORF by polymorphisms or mutations leading to human diseases have 14. On the contrary, disrupted uORF by polymorphisms or mutations leading to human diseases have only 2. In addition, some indirect factors including peptide coded by uORF, phosphorylation of the translation initiation factor may be around uORF and have dramaticly impact on gene expression.To date, there has been no systematic investigation into the relationship between variations in human diseases and uORF status. Therefore, we took the advantage of bioinformatics to study uORFs in human transcripts using several datasets. After data filtering, screening, we have had GO annotation, enrichment and analysis of sequence features, as well as ClinVar, TCGA, COSMIC disease databases. Later, we randomly selected 4 target genes from patients with glioblastoma multiforme, uterine corpus endometrial carcinoma and head and neck squamous cell carcinoma, which have highly suscepted that the change of protein expression closely associated with uORF and took a experimental verification. We hope that our study could make researchers pay more attention to the relationship between human diseases and uORF.This help to understand genotype-phenotype correlation of human disorders, which this will provide a new idea for the mechanism of diseases, as well as a new direction for the clinical treatment.Materials and Method1. Data analysis of bioinformaticsThrough analysing ClinVar、TCGA、COSMIC, we randomly selected 4 target genes:GCSAM, PSTPIP1、HIS1H2BD、CEBPB,、VAT1、ERP29, which they were highly suspected that mutations in the uORFs associated with genes expression. Protein expression and mRNA transcription were experimentally verified in the funtional tests.2. Experiment verificationpsiCHECKTM-2 is a dual luciferase reporter vector as our experiment verification vector. But the vector can not meet our requirment because of only insertion sites in the 3’end. So, we must construct the vector, multiple cloning sites(MCS) were introduced to it so that the 5’UTR leading sequences can be inserted between the Synthetic Renilla luciferase gene(hRluc) and the T7 Promoter by a 2 step PCR-amplified strategy.. The 5’UTR leading sequences were amplified from human genomic DNA. To construct the wildtype plasmid, the 5’UTR leading sequences were subcloned into the psiCHECKTM-2 vector using double restriction sites located in 5’end. DpnI site-directed mutagenesis assay was introduced to construct the mutant plasmid. In the functional tests, the wildtype and mutant plasmid DNA were transfected into HEK293T cells using LipofectamineTM 2000, respectively., and then, The firefly and renilla luciferase signals were generated using the Dual-Luciferase(?) Reporter 1000 Assay System to detect the protein expression while QPCR was performed to detect the transcription level.ResultsScanning ClinVar, COSMIC and TCGA databases. The mutations found in the 5’UTR were annotated whether it can alter the number of the uORFs. Whether the uORFs creation, disruption or the mutations in different positions of the uORFs they can result in alteration. According to this, alteration in the uORFs were divided into six categories. Containing-uORF genes have significantly impact on the protein expression, compared to the non-uORF genes. Furthermore, with the increase in uORF numbers, the impact on the protein translation also have additive effects. We randomly chosed GCSAM, PSTPIP1、HIS1H2BD、VAT1、ERP29、CEBPB, from filtered TCGA, which the first five genes don’t contain uORF in the 5’UTR and the last one has only uORF. Construction of psiCHECKTM-2 plasmid vector, after comparing the sequences of gene 5’UTR and the psiCHECKTM-2, we selected 6 restriction sites of NdeI、AscI、 AgeI、PacI、SalI and SaCI and inserted between T7 promoter and hRluc using a 2 step PCR-amplified strategy. Construction of gene 5’UTR-psiCHECKTM-2 plasmid, the amplified products of 5’UTR gene were then double digested. and linked into the psiCHECKTM-2 containing 5’UTR MCS. DpnI site-directed mutagenesis assay was introduced to construct the mutanttions plasmid so that all of the GCSAM、PSTPIP1、 HIS1H2BD and ERP29 generated a ATG in the 5’UTR, which it created a uORF, because of the 3 multiple with the downstream termination condon. VAT1 generated a TGA in the 5’UTR, which it also created a uORF. On the contrary, CEBPB was disrupted in the 5’UTR after site-directed mutagenesis resulting in the uORF disappearance. The luciferase activities were measured in the Dual-Luciferase(?) Reporter Assay System after the wild type and the muttant plamid DNA transfecting into HEK293T cells. The protein expression of the first three genes 5’UTR reduced about 50%, especially the PSTPIP1, was about 90%, while compareed with wild type, the protein expression of VAT1 and ERP29 haven’t significant difference. But the CEBPB mostly increased 50%. The transcription levels of the target genes were little changes using qPCR to detection, only the PSTPIP1 did has modest decrease.DiscussionResearch of our bioinformatics data indicated that uORFs occur widely in human transcripts and a common mechanrism for post-transcriptional regulation of gene expression. only a limited number of uORF-altering mutations have been reported to be associated with human diseases, this study analyzed the abundant sequence variants in patients with various diseases and their potential relationships with alterations in uORF status. Four selected genes were detected in patients with glioblastoma multiforme, uterine corpus endometrial carcinoma and head and neck squamous cell carcinoma, respectively. The references reported that the relationship between GCSAM and the development of the human lymphoma is very closely, GCSAM protein is expressed in human GC B-cells and in lymphomas of GC B-cell derivation and it can inhibit the lymphocyte migration. For this, it exhibit low-stage disease and a better overall clinical outcome. Many reseachers have further investigated for the regulatory mechanism from signaling pathway、transgenic mice and the inhibitory effect of the transcription repressor. As an adaptor protein, PSTPIP1 associated with the cytoskeleton that is mainly expressed in hematopoietic cells. Some rare autosomal dominant inherited autoinflammatory syndrome may be related to it, for example, pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. It is reported that it is a negative regulator of T-cell receptor (TCR)-dependent T-cell activation, and expression of PSTPIP1 inhibited the activation of different transcription factors that are relevant for immune cell fuction the protein. PSTPIP1 encoded an SH3 domain at the C-terminal end that is very important for its function. Many researchers focus on it which the coding region often have different mutations. HIST1H2BD is one of the histones that they are the major protein component of the eukaryotic chromatin. To date, it is not so much investigation about it, and little is known about their regulation, only some studies focus on the 3’end polyadenylation.. In physiological condition, CEBPB regulate the proliferation and differentiation of multiple cell types including granulocytes, macrophages adipocytes, osteoclasts, osteoblasts, keratinocytes, mammary epithelial cells, and hepatocytes. C/EBP transcription factors are implicated in the regulation of various (patho)phy-siological processes including metabolism, inflammation, and malignant transformation. uORF inactivation resulted into truncated isoform encoded by CEBPB increase, which associated with hodgkin lymphoma, anaplastic large cell lymphoma and aggressive forms of breast cancer.All of the four target genes, the relationship between the first three genes and human diseases haven’t any investigated on the uORF-regulated protein expression, except for the CEBPB. But our data intensive suggested that created an uORF after mutation in the 5’UTR indeed generated a significantly inhibitory effects. It is necessary to conduct careful annotations of uORF status upon high-throughput sequencing data to find out new genomic alterations that can potentially change protein expression. Except for uORF, other cis-elements in the 5’UTR that co-regulate genes experssion with uORF is unkown, it will need further to mining data and more experimental data to support. For VAT1 and ERP29 negative interpretation of the results, there may be several reasons:① all uORF are not deterring effect, ribosome can translate again through the leaky scanning or regulated reinitiation.② there may be some other mechanisms to offset uORF repression. ③ not all uORF-containing sequence features have uORF-mediated gene regulation mechanism of uORF. for VAT1 and ERP29, mutant uORF may be not function. For these analyses is reasonable or not, it needs to be studied further to VTA1, ERP29 sequences characteristic. Finally, we hope that our study could provid a new idea for other reseachers exploring the ralationship between gene experssion and hunman diseases, and try to unravel the implications of uORF-mediated translational control in a multitude of as-yet-unexplained human diseases.