Genetic regulation of gene expression and splicing during a 10-year period of human aging.
2019; 20 (1): 230
BACKGROUND: Molecular and cellular changes are intrinsic to aging and age-related diseases. Prior cross-sectional studies have investigated the combined effects of age and genetics on gene expression and alternative splicing; however, there has been no long-term, longitudinal characterization of these molecular changes, especially in older age.RESULTS: We perform RNA sequencing in whole blood from the same individuals at ages 70 and 80 to quantify how gene expression, alternative splicing, and their genetic regulation are altered during this 10-year period of advanced aging at a population and individual level. We observe that individuals are more similar to their own expression profiles later in life than profiles of other individuals their own age. We identify 1291 and 294 genes differentially expressed and alternatively spliced with age, as well as 529 genes with outlying individual trajectories. Further, we observe a strong correlation of genetic effects on expression and splicing between the two ages, with a small subset of tested genes showing a reduction in genetic associations with expression and splicing in older age.CONCLUSIONS: These findings demonstrate that, although the transcriptome and its genetic regulation is mostly stable late in life, a small subset of genes is dynamic and is characterized by a reduction in genetic regulation, most likely due to increasing environmental variance with age.
View details for DOI 10.1186/s13059-019-1840-y
View details for PubMedID 31684996
BCDIN3D regulates tRNAHis 3' fragment processing.
2019; 15 (7): e1008273
5' ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5' monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3' end of tRNAHis. Moreover, we were able to generate this 'miRNA' in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5'P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3'-fragment/'miR-4454' levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3'-fragment processing without negatively affecting tRNAHis's canonical function of aminoacylation.
View details for DOI 10.1371/journal.pgen.1008273
View details for PubMedID 31329584
Transcriptional and Position Effect Contributions to rAAV-Mediated Gene Targeting
CELL PRESS. 2019: 294
View details for Web of Science ID 000464381003086
Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms.
2019; 2: 186
The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs). Co-localizations of fRPE e/sQTLs with age-related macular degeneration (AMD) and myopia GWAS data suggest new candidate genes, and mechanisms by which a common RDH5 allele contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTLs in a critical ocular cell type to monogenic and complex eye disorders.
View details for DOI 10.1038/s42003-019-0430-6
View details for PubMedID 31123710
Crosstalk between the RNA Methylation and Histone-Binding Activities of MePCE Regulates P-TEFb Activation on Chromatin
2018; 22 (6): 1374?83
RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol. Here, we report that MePCE also binds chromatin through the histone H4 tail to serve as a P-TEFb activator at specific genes important for cellular identity. Notably, this histone binding abolishes MePCE's RNA methyltransferase activity toward 7SK, which explains why MePCE-bound P-TEFb on chromatin may not be associated with the full 7SK snRNP and is competent for RNAP II activation. Overall, our results suggest that crosstalk between the histone-binding and RNA methylation activities of MePCE regulates P-TEFb activation on chromatin in a 7SK- and Brd4-independent manner.
View details for PubMedID 29425494
Click Quantitative Mass Spectrometry Identifies PIWIL3 as a Mechanistic Target of RNA Interference Activator Enoxacin in Cancer Cells
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2017; 139 (4): 1400-1403
Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs. Our results suggest that cancer cells re-express PIWIL3 to repress RNAi through miRNAs and thus open a new opportunity for cancer-specific targeting.
View details for DOI 10.1021/jacs.6b11751
View details for PubMedID 28094937
Small RNA Sequencing in Cells and Exosomes Identifies eQTLs and 14q32 as a Region of Active Export
G3-GENES GENOMES GENETICS
2017; 7 (1): 31-39
Exosomes are small extracellular vesicles that carry heterogeneous cargo, including RNA, between cells. Increasing evidence suggests that exosomes are important mediators of intercellular communication and biomarkers of disease. Despite this, the variability of exosomal RNA between individuals has not been well quantified. To assess this variability, we sequenced the small RNA of cells and exosomes from a 17-member family. Across individuals, we show that selective export of miRNAs occurs not only at the level of specific transcripts, but that a cluster of 74 mature miRNAs on chromosome 14q32 is massively exported in exosomes while mostly absent from cells. We also observe more interindividual variability between exosomal samples than between cellular ones and identify four miRNA expression quantitative trait loci shared between cells and exosomes. Our findings indicate that genomically colocated miRNAs can be exported together and highlight the variability in exosomal miRNA levels between individuals as relevant for exosome use as diagnostics.
View details for DOI 10.1534/g3.116.036137
View details for Web of Science ID 000392200800003
View details for PubMedCentralID PMC5217120
Chromatin Regulates Genome Targeting with Cisplatin.
Angewandte Chemie (International ed. in English)
2017; 56 (23): 6483?87
Cisplatin derivatives can form various types of DNA lesions (DNA-Pt) and trigger pleiotropic DNA damage responses. Here, we report a strategy to visualize DNA-Pt with high resolution, taking advantage of a novel azide-containing derivative of cisplatin we named APPA, a cellular pre-extraction protocol and the labeling of DNA-Pt by means of click chemistry in cells. Our investigation revealed that pretreating cells with the histone deacetylase (HDAC) inhibitor SAHA led to detectable clusters of DNA-Pt that colocalized with the ubiquitin ligase RAD18 and the replication protein PCNA. Consistent with activation of translesion synthesis (TLS) under these conditions, SAHA and cisplatin cotreatment promoted focal accumulation of the low-fidelity polymerase Pol? that also colocalized with PCNA. Remarkably, these cotreatments synergistically triggered mono-ubiquitination of PCNA and apoptosis in a RAD18-dependent manner. Our data provide evidence for a role of chromatin in regulating genome targeting with cisplatin derivatives and associated cellular responses.
View details for PubMedID 28474855
Genetic effects on gene expression across human tissues.
2017; 550 (7675): 204?13
Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease.
View details for PubMedID 29022597
MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes
2015; 10 (5)
miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.
View details for DOI 10.1371/journal.pone.0126535
View details for PubMedID 25992613
miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing
The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation.Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5' seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis.Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2.
View details for DOI 10.1186/s12864-015-1279-9
View details for Web of Science ID 000349254600001
View details for PubMedID 25653011
View details for PubMedCentralID PMC4326481
A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes
NUCLEIC ACIDS RESEARCH
2013; 41 (4): 2239-2254
The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. However, the interplay between transcription factors and miRNAs in modulating gene regulatory networks involved in human cell proliferation is largely unknown. Here we show that the miRNA miR-22 promotes proliferation in primary human cells, and through a combination of Argonaute-2 immunoprecipitation and reporter assays, we identified multiple novel targets of miR-22, including several cell-cycle arrest genes that mediate the effects of the tumor-suppressor p53. In addition, we found that miR-22 suppresses interferon gene expression by directly targeting high mobility group box-1 and interferon regulatory factor (IRF)-5, preventing activation of IRF3 and NF-?B, which are activators of interferon genes. The expression of interferon genes is elevated in quiescent cells and their expression is inhibitory for cell proliferation. In addition, we find that miR-22 is activated by the transcription factor Myc when quiescent cells enter proliferation and that miR-22 inhibits the Myc transcriptional repressor MXD4, mediating a feed-forward loop to elevate Myc expression levels. Our results implicate miR-22 in downregulating the anti-proliferative p53 and interferon pathways and reveal a new transcription factor-miRNA network that regulates the transition of primary human cells from quiescence to proliferation.
View details for DOI 10.1093/nar/gks1452
View details for Web of Science ID 000318062000021
View details for PubMedID 23303785
View details for PubMedCentralID PMC3575845