Half Of Protein-coding Genes Research Articles

Abstract 1712: Identifying a novel alternative promoter usage in HDAC9: Implications for LUAD prognosis and therapy

Abstract Promoters are key elements in gene regulation. It has been estimated that more than half of protein-coding genes in the human genome possess multiple promoters. Aberrant expression and dysregulated activity of alternative promoters have been correlated with numerous diseases, especially cancer. In this study, we utilized RNA-seq data from 608 lung adenocarcinoma (LUAD) patients and systematically analyzed promoter activities using the proActiv algorithm. Of the expressed genes in LUAD, approximately 57% have at least two distinct promoters. In addition, we identified 1852 tumor-specific alternative promoters that showed a significant shift (2-fold changes) in promoter activity without any notable changes in the expression of corresponding 1603 genes between tumor and adjacent non-tumor tissues. Functional enrichment analysis showed that these genes were associated with key cellular functions such as signaling pathways, cell cycle, and cell junctions. Unsupervised clustering of patients based on altered promoters revealed a significant prognostic value. Among them, we observed a major promoter switch in HDAC9, which mainly utilizes three promoters: prmt3280, prmt3283, and prmt3287 in our LUAD dataset. Notably, prmt3287 was exclusively active in tumor samples and functioned as the dominant promoter, while prmt3283 served as the main promoter in adjacent non-tumor samples. Intriguingly, even though the overall expression of HDAC9 mRNA lacked prognostic relevance both in our dataset and in the TCGA dataset, higher expression of the alternative promoter prmt3287 (using ≥50% activity level as a cutoff) was associated with adverse outcomes in LUAD patients. We further compared the DNA methylation level around the HDAC9 domain between paired tumor and non-tumor samples and found that positions with the most significant differences (p<0.001) overlapped with the prmt3287 region. Moreover, DNA methylation levels around prmt3287 significantly decreased in prmt3287 high-activity patient groups. Next, we investigated the ChIP-seq levels over the HDAC9 region, and ChIPseeker successfully annotated the alternative promoters of HDAC9. In addition, the alternative promoter prmt3287 exhibited the highest active enhancer signals. Furthermore, we performed PCR to determine the expression of six distinct transcript isoforms from alternative promoters in four different LUAD cell lines, and we observed high expression of isoform 10, which is possibly generated by prmt3287 in all cell lines. The impact of prmt3287 on cell proliferation, cell cycle, and cell migration will be further addressed. Our results indicate that alternative promoter usage can capture insights that might be missed at the overall gene expression level, and alternative promoter usage in HDAC9 could be a novel biomarker and a promising therapy target. Citation Format: Amina Bolatkan, Ken Asada, Syuzo Kaneko, Masaaki Komatsu, Ryuji Hamamoto. Identifying a novel alternative promoter usage in HDAC9: Implications for LUAD prognosis and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1712.

Abstract 3711: Regulation of oncogenic targets by miR-99a-3p (the passenger strand of the miR-99a-duplex) in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with approximately 650,000 new cases diagnosed annually and 400,000 HNSCC-related deaths worldwide each year. HNSCC is typically diagnosed when already at an advanced stage. Despite advancements in surgery, radiation therapy, and chemotherapy, patients with advanced HNSCC have a poor prognosis. The median overall survival time for patients with recurrence and metastasis is 10-13 months in the setting of first-line chemotherapy and 6 months in the second-line setting. Many studies have shown that noncoding RNAs encoded by the human genome are functional and play critical roles in various cellular processes, e.g., cell growth, migration, invasion, and apoptosis. Among noncoding RNAs, microRNAs (miRNAs) are endogenous single-stranded RNA molecules comprising 19-22 nucleotides that function as fine-tuners of RNA expression. A single miRNA regulates many RNA transcripts, and bioinformatics studies have shown that more than half of protein-coding genes are controlled by miRNAs. Aberrantly expressed miRNAs are closely associated with cancer pathogenesis via the disruption of RNA networks within cancer cells. To identify novel oncogenic targets in head and neck squamous cell carcinoma (HNSCC), we have analyzed antitumor microRNAs (miRNAs) and their controlled molecular networks in HNSCC cells. Based on our miRNA signature in HNSCC, both strands of the miR-99a-duplex (miR-99a-5p: the guide strand, and miR-99a-3p: the passenger strand) are downregulated in cancer tissues. Moreover, low expression of miR-99a-5p and miR-99a-3p significantly predicts poor prognosis in HNSCC, and these miRNAs regulate cancer cell migration and invasion. We previously showed that passenger strands of miRNAs have antitumor functions. Here, we screened miR-99a-3p-controlled oncogenes involved in HNSCC pathogenesis. Thirty-two genes were identified as miR-99a-3p-regulated genes, and 10 genes (STAMBP, TIMP4, TMEM14C, CANX, SUV420H1, HSP90B1, PDIA3, MTHFD2, BCAT1, and SLC22A15) significantly predicted 5-year overall survival. Notably, among these genes, STAMBP, TIMP4, TMEM14C, CANX, and SUV420H1 were independent prognostic markers of HNSCC by multivariate analyses. We further investigated the oncogenic function of STAMBP in HNSCC cells using knockdown assays. Our data demonstrated that the aggressiveness of phenotypes in HNSCC cells was attenuated by siSTAMBP transfection. Moreover, aberrant STAMBP expression was detected in HNSCC clinical specimens by immunochemistry. Our strategy, i.e., identification of antitumor miRNAs and their targets, maybe an attractive tool to reveal novel prognostic and therapeutic targets in HNSCC. Citation Format: Reona Okada, Keiichi Koshizuka, Yasutaka Yamada, Shogo Moriya, Naoko Kikkawa, Takashi Kinoshita, Toyoyuki Hanazawa, Naohiko Seki. Regulation of oncogenic targets by miR-99a-3p (the passenger strand of the miR-99a-duplex) in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3711.

Retrotransposons evolution and impact on lncRNA and protein coding genes in pigs

BackgroundRetrotransposons are the major determinants of genome sizes and they have shaped both genes and genomes in mammalian organisms, but their overall activity, diversity, and evolution dynamics, particularly their impact on protein coding and lncRNA genes in pigs remain largely unknown.ResultsIn the present study, we performed de novo detection of retrotransposons in pigs by using multiple pipelines, four distinct families of pig-specific L1 s classified into 51 distinct subfamilies and representing four evolution models and three expansion waves of pig-specific SINEs represented by three distinct families were identified. ERVs were classified into 18 families and found two most “modern” subfamilies in the pig genome. The transposition activity of pig L1 was verified by experiment, the sense and antisense promoter activities of young L1 5′UTRs and ERV LTRs and expression profiles of young retrotransposons in multiple tissues and cell lines were also validated. Furthermore, retrotransposons had an extensive impact on lncRNA and protein coding genes at both the genomic and transcriptomic levels. Most protein coding and lncRNA (> 80%) genes contained retrotransposon insertions, and about half of protein coding genes (44.30%) and one-fourth (24.13%) of lncRNA genes contained the youngest retrotransposon insertions. Nearly half of protein coding genes (43.78%) could generate chimeric transcripts with retrotransposons. Significant distribution bias of retrotransposon composition, location, and orientation in lncRNA and protein coding genes, and their transcripts, were observed.ConclusionsIn the current study, we characterized the classification and evolution profile of retrotransposons in pigs, experimentally proved the transposition activity of the young pig L1 subfamily, characterized the sense and antisense expression profiles and promoter activities of young retrotransposons, and investigated their impact on lncRNA and protein coding genes by defining the mobilome landscapes at the genomic and transcriptomic levels. These findings help provide a better understanding of retrotransposon evolution in mammal and their impact on the genome and transcriptome.

Target mRNA-Driven Biogenesis of Cognate MicroRNAs In Vitro.

miRNAs are 20-22 nucleotide long noncoding RNAs that act as post-transcriptional regulators of gene expression controlling more than half of protein coding genes in humans. Being the critical modulators of the mRNA translation process, biogenesis, function, and turnover of these small RNAs are tightly regulated in cells. We have reported that target mRNAs induce increased biogenesis of cognate miRNAs from pre-miRNAs by increased activity of Ago-associated Dicer endonuclease that processes precursor miRNAs to their mature form. In the current chapter, we discuss how target mRNA-driven RISC loading can be monitored in vitro using affinity-purified miRISC or recombinant AGO2 and DICER1 proteins and scoring the processivity of AGO2-associated DICER1 in vitro.

Spatiotemporal Uncoupling of MicroRNA-Mediated Translational Repression and Target RNA Degradation Controls MicroRNP Recycling in Mammalian Cells.

MicroRNA (miRNA)-mediated repression controls expression of more than half of protein-coding genes in metazoan animals. Translation repression is associated with target mRNA degradation initiated by decapping and deadenylation of the repressed mRNAs. Earlier evidence suggests the endoplasmic reticulum (ER) as the site where microRNPs (miRNPs) interact with their targets before translation repression sets in, but the subcellular location of subsequent degradation of miRNA-repressed messages is largely unidentified. Here, we explore the subcellular distribution of essential components of degradation machineries of miRNA-targeted mRNAs. We have noted that interaction of target mRNAs with AGO2 protein on the ER precedes the relocalization of repressed messages to multivesicular bodies (MVBs). The repressed messages subsequently get deadenylated, lose their interaction with AGO2, and become decapped. Blocking maturation of endosomes to late endosome and MVBs by targeting the endosomal protein HRS uncouples miRNA-mediated translation repression from target RNA degradation. HRS is also targeted by the intracellular parasite Leishmania donovani, which curtails the HRS level in infected cells to prevent uncoupling of mRNA-AGO2 interaction, preventing degradation of translationally repressed messages, and thus stops recycling of miRNPs preengaged in repression.

The pervasiveness and plasticity of circadian oscillations: the coupled circadian-oscillators framework

Circadian oscillations have been observed in animals, plants, fungi and cyanobacteria and play a fundamental role in coordinating the homeostasis and behavior of biological systems. Genetically encoded molecular clocks found in nearly every cell, based on negative transcription/translation feedback loops and involving only a dozen genes, play a central role in maintaining these oscillations. However, high-throughput gene expression experiments reveal that in a typical tissue, a much larger fraction ([Formula: see text]) of all transcripts oscillate with the day-night cycle and the oscillating species vary with tissue type suggesting that perhaps a much larger fraction of all transcripts, and perhaps also other molecular species, may bear the potential for circadian oscillations. To better quantify the pervasiveness and plasticity of circadian oscillations, we conduct the first large-scale analysis aggregating the results of 18 circadian transcriptomic studies and 10 circadian metabolomic studies conducted in mice using different tissues and under different conditions. We find that over half of protein coding genes in the cell can produce transcripts that are circadian in at least one set of conditions and similarly for measured metabolites. Genetic or environmental perturbations can disrupt existing oscillations by changing their amplitudes and phases, suppressing them or giving rise to novel circadian oscillations. The oscillating species and their oscillations provide a characteristic signature of the physiological state of the corresponding cell/tissue. Molecular networks comprise many oscillator loops that have been sculpted by evolution over two trillion day-night cycles to have intrinsic circadian frequency. These oscillating loops are coupled by shared nodes in a large network of coupled circadian oscillators where the clock genes form a major hub. Cells can program and re-program their circadian repertoire through epigenetic and other mechanisms. High-resolution and tissue/condition specific circadian data and networks available at http://circadiomics.igb.uci.edu. pfbaldi@ics.uci.edu Supplementary data are available at Bioinformatics online.

High-Resolution Mapping of Epigenetic Modifications of the Rice Genome Uncovers Interplay between DNA Methylation, Histone Methylation, and Gene Expression

We present high-resolution maps of DNA methylation and H3K4 di- and trimethylation of two entire chromosomes and two fully sequenced centromeres in rice (Oryza sativa) shoots and cultured cells. This analysis reveals combinatorial interactions between these epigenetic modifications and chromatin structure and gene expression. Cytologically densely stained heterochromatin had less H3K4me2 and H3K4me3 and more methylated DNA than the less densely stained euchromatin, whereas centromeres had a unique epigenetic composition. Most transposable elements had highly methylated DNA but no H3K4 methylation, whereas more than half of protein-coding genes had both methylated DNA and di- and/or trimethylated H3K4. Methylation of DNA but not H3K4 was correlated with suppressed transcription. By contrast, when both DNA and H3K4 were methylated, transcription was only slightly reduced. Transcriptional activity was positively correlated with the ratio of H3K4me3/H3K4me2: genes with predominantly H3K4me3 were actively transcribed, whereas genes with predominantly H3K4me2 were transcribed at moderate levels. More protein-coding genes contained all three modifications, and more transposons contained DNA methylation in shoots than cultured cells. Differential epigenetic modifications correlated to tissue-specific expression between shoots and cultured cells. Collectively, this study provides insights into the rice epigenomes and their effect on gene expression and plant development.