Ionotropic glutamate receptors (iGluRs) play key roles for signaling in the central nervous system. Alternative splicing and RNA editing are well-known mechanisms to increase iGluR diversity and to provide context-dependent regulation. Earlier work on isoform identification has focused on the analysis of cloned transcripts, mostly from rodents. We here set out to obtain a systematic overview of iGluR splicing and editing in human brain based on RNA-Seq data. Using data from two large-scale transcriptome studies, we established a workflow for the de novo identification and quantification of alternative splice and editing events. We detected all canonical iGluR splice junctions, assessed the abundance of alternative events described in the literature, and identified new splice events in AMPA, kainate, delta, and NMDA receptor subunits. Notable events include an abundant transcript encoding the GluA4 amino-terminal domain, GluA4-ATD, a novel C-terminal GluD1 (delta receptor 1) isoform, GluD1-b, and potentially new GluK4 and GluN2C isoforms. C-terminal GluN1 splicing may be controlled by inclusion of a cassette exon, which shows preference for one of the two acceptor sites in the last exon. Moreover, we identified alternative untranslated regions (UTRs) and species-specific differences in splicing. In contrast, editing in exonic iGluR regions appears to be mostly limited to ten previously described sites, two of which result in silent amino acid changes. Coupling of proximal editing/editing and editing/splice events occurs to variable degree. Overall, this analysis provides the first inventory of alternative splicing and editing in human brain iGluRs and provides the impetus for further transcriptome-based and functional investigations.

A promoter can be regulated by various cis-acting elements so that delineation of the regulatory modes among them may help understand developmental, environmental and genetic mechanisms in gene activity. Here we report that the human dopamine transporter gene SLC6A3 carries a 5\' distal 5-kb super enhancer (5KSE) which upregulated the promoter by 5-fold. Interestingly, 5KSE is able to prevent 3\' downstream variable number tandem repeats (3\'VNTRs) from silencing the promoter. This new enhancer consists of a 5\'VNTR and three repetitive sub-elements that are conserved in primates. Two of 5KSE\'s sub-elements, E-9.7 and E-8.7, upregulate the promoter, but only the later could continue doing so in the presence of 3\'VNTRs. Finally, E-8.7 is activated by novel dopaminergic transcription factors including SRP54 and Nfe2l1. Together, these results reveal a multimodal regulatory mechanism in SLC6A3.

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

OBJECTIVE: The role of short tandem repeats (STRs) in the control of gene expression among species is being increasingly understood following the identification of several instances in which certain STRs occur identically, or expand differentially, in primates versus nonprimates. These STRs may regulate genes that participate in characteristics that are associated with the divergence of primates from sibling orders (e.g., brain higher order functions). The CYTH4 gene contains the longest tetranucleotide STR in its core promoter, at 7-repeats, and links to the evolution of human and nonhuman primates. Allele and genotype distribution of this STR were studied in patients affected by schizophrenia (SCZ) and controls.
METHODS: High-resolution data were obtained on the allele and genotype distribution of the CYTH4 STR and a novel C > T single-nucleotide polymorphism (SNP) at its immediate upstream sequence in 255 patients with SCZ and 249 controls. Each sample was sequenced twice using the fluorescent dye termination method.
RESULTS: Novel alleles were detected at the long extreme of the GTTT-repeat, at 10- and 11-repeats, in the SCZ cases and controls. Excess of homozygosity was observed for the entire range of alleles across the GTTT-repeat and the C > T SNP in the SCZ patients in comparison with the controls (Yates corrected p < 0.011). Three genotypes consisting of the 11-repeat allele (i.e., 11/11, 10/11, and 7/11) were detected only in the SCZ patients (i.e., disease-only genotypes), and contributed to 2.3% of the SCZ genotypes (Mid p exact <0.007). The frequency of the 11-repeat allele was estimated at 0.02 and 0.006 in the SCZ patients and controls, respectively (Mid p exact <0.006).
CONCLUSIONS: This indicates that STR genotypes that are absent in the control group may be risk factors for SCZ. Future studies are warranted to test the significance of our findings.

Non-coding RNAs (ncRNAs) are critical regulators of gene expression in essentially all life forms. Long ncRNAs (lncRNAs) and microRNAs (miRNAs) are two important RNA classes possessing regulatory functions. Up to date, many primate-specific ncRNAs have been identified and investigated. Their expression specificity to primate lineage suggests primate-specific roles. It is thus critical to elucidate the biological significance of primate or even human-specific ncRNAs, and to develop potential ncRNA-based therapeutics. Here, we have summarized the studies regarding regulatory roles of some key primate-specific lncRNAs and miRNAs.

Barbara McClintock discovered the existence of transposable elements (TEs) in the late 1940s and initially proposed that they contributed to the gene regulatory program of higher organisms. This controversial idea gained acceptance only much later in the 1990s, when the first examples of TE-derived promoter sequences were uncovered. It is now known that half of the human genome is recognizably derived from TEs. It is thus important to understand the scope and nature of their contribution to gene regulation. Here, we provide a timeline of major discoveries in this area and discuss how transposons have revolutionized our understanding of mammalian genomes, with a special emphasis on the massive contribution of TEs to primate evolution. Our analysis of primate-specific functional elements supports a simple model for the rate at which new functional elements arise in unique and TE-derived DNA. Finally, we discuss some of the challenges and unresolved questions in the field, which need to be addressed in order to fully characterize the impact of TEs on gene regulation, evolution and disease processes.

Recent reports of a link between human- and primate-specific genetic factors and human/primate-specific characteristics and diseases necessitate genome-wide identification of those factors. We have previously reported core promoter short tandem repeats (STRs) of extreme length (≥6-repeats) that have expanded exceptionally in primates vs. non-primates, and may have a function in adaptive evolution. In the study reported here, we extended our study to the human STRs of ≥3-repeats in the category of penta and hexaucleotide STRs, across the entire human protein coding gene core promoters, and analyzed their status in several superorders and orders of vertebrates, using the Ensembl database. The ConSite software was used to identify the transcription factor (TF) sets binding to those STRs. STR specificity was observed at different levels of human and non-human primate (NHP) evolution. 73% of the pentanucleotide STRs and 68% of the hexanucleotide STRs were found to be specific to human and NHPs. AP-2alpha, Sp1, and MZF were the predominantly selected TFs (90%) binding to the human-specific STRs. Furthermore, the number of TF sets binding to a given STR was found to be a selection factor for that STR. Our findings indicate that selected STRs, the cognate binding TFs, and the number of TF set binding to those STRs function as switch codes at different levels of human and NHP evolution and speciation.

We have previously reported genome-scale short tandem repeats (STRs) in the core promoter interval (i.e. -120 to +1 to the transcription start site) of protein-coding genes that have evolved identically in primates vs. non-primates. Those STRs may function as evolutionary switch codes for primate speciation. In the current study, we used the Ensembl database to analyze the 5\' untranslated region (5\' UTR) between +1 and +60 of the transcription start site of the entire human protein-coding genes annotated in the GeneCards database, in order to identify \"exceptionally long\" STRs (≥5-repeats), which may be of selective/adaptive advantage. The importance of this critical interval is its function as core promoter, and its effect on transcription and translation. In order to minimize ascertainment bias, we analyzed the evolutionary status of the human 5\' UTR STRs of ≥5-repeats in several species encompassing six major orders and superorders across mammals, including primates, rodents, Scandentia, Laurasiatheria, Afrotheria, and Xenarthra. We introduce primate-specific STRs, and STRs which have expanded from mouse to primates. Identical co-occurrence of the identified STRs of rare average frequency between 0.006 and 0.0001 in primates supports a role for those motifs in processes that diverged primates from other mammals, such as neuronal differentiation (e.g. APOD and FGF4), and craniofacial development (e.g. FILIP1L). A number of the identified STRs of ≥5-repeats may be human-specific (e.g. ZMYM3 and DAZAP1). Future work is warranted to examine the importance of the listed genes in primate/human evolution, development, and disease.

Evidence of primate-specific genes and gene regulatory mechanisms linked to bipolar disorder (BD) lend support to evolutionary/adaptive processes in the pathogenesis of this disorder. Following a genome-scale analysis of the entire protein coding genes annotated in the GeneCards database, we have recently reported that cytohesin-4 (CYTH4) contains the longest tetra-nucleotide short tandem repeat (STR) identified in a human protein-coding gene core promoter, which may be of adaptive advantage to this species. In the current study, we analyzed the evolutionary trend of this STR across evolution. We also analyzed the functional implication and distribution of this STR in a group of patients with type 1 BD (n=233) and controls (n=262). We found that this STR is exceptionally expanded in primates (Fisher exact p<0.00003). Association was observed between type I BD and the 6-repeat allele of this STR, (GTTT)₆ (Yates corrected Χ(2)=12.68, p<0.0001, OR: 1.68). This allele is the shortest length of the GTTT-repeat identified in the human subjects studied. Consistent with that finding, excess homozygosity was observed for the shorter alleles, (GTTT)₆ and (GTTT)₇, vs. the longer alleles, (GTTT)₈ and (GTTT)₉ in the BD group (Yates corrected Χ(2)=5.18, p<0.01, 1 df, OR: 1.96). Using Dual Glo luciferase system in HEK-293 cells, a trend for gene expression repression was observed from the 6- to the 9-repeat allele (p<0.003), and the GTTT-repeat significantly down-regulated gene expression, per se (p<0.0006). This is the first evidence of a link between a primate-specific STR and a major psychiatric disorder in human. It may be speculated that the CYTH4 GTTT-repeat in primates may have conferred selective advantage to this order, reflected in neural function and neurophenotypes. The role of the CYTH4 gene in the pathogenesis of type I BD remains to be clarified in the future studies.

Type-2 diabetes (T2D) is a complex disease characterized by insulin resistance in target tissues and impaired insulin release from pancreatic beta cells. As central tissue of glucose homeostasis, the pancreatic islet continues to be an important focus of research to understand the pathophysiology of the disease. The increased access to human pancreatic islets has resulted in improved knowledge of islet function, and together with advances in RNA sequencing and related technologies, revealed the transcriptional and epigenetic landscape of human islet cells. The discovery of thousands of long non-coding RNA (lncRNA) transcripts highly enriched in the pancreatic islet and/or specifically expressed in the beta-cells, points to yet another layer of gene regulation of many hitherto unknown mechanistic principles governing islet cell functions. Here we review fundamental islet physiology and propose functional implications of the lncRNAs in islet development and endocrine cell functions. We also take into account important differences between rodent and human islets in terms of morphology and function, and suggest how species-specific lncRNAs may partly influence gene regulation to define the unique phenotypic identity of an organism and the functions of its constituent cells. The implication of primate-specific lncRNAs will be far-reaching in all aspects of diabetes research, but most importantly in the identification and development of novel targets to improve pancreatic islet cell functions as a therapeutic approach to treat T2D.