Boris Guennewig

Systematic screens of proteins binding to synthetic microRNA precursors

We describe a new, broadly applicable methodology for screening in parallel interactions of RNA-binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre-miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts. Detection of bound RBPs is achieved by protein or tag-specific antibodies allowing crude cell lysates to be used as a source of RBP. We selected 70 pre-miRNAs with phylogenetically conserved loop regions and 25 precursors of other well-characterized miRNAs for chemical synthesis in 3′-biotinylated form. An equivalent set in unmodified form served as inhibitors in affinity determinations. By testing three RBPs known to regulate miRNA biogenesis on this set of pre-miRNAs, we demonstrate that Lin28 and hnRNP A1 from cell lysates or as recombinant protein domains recognize preferentially precursors of the let-7 family, and that KSRP binds strongly to pre-miR-1-2.

Activity-Dependent Changes in Gene Expression in Schizophrenia Human-Induced Pluripotent Stem Cell Neurons.

Importance Schizophrenia candidate genes participate in common molecular pathways that are regulated by activity-dependent changes in neurons. One important next step is to further our understanding on the role of activity-dependent changes of gene expression in the etiopathogenesis of schizophrenia. Objective To examine whether neuronal activity-dependent changes of gene expression are dysregulated in schizophrenia. Design, Setting, and Participants Neurons differentiated from human-induced pluripotent stem cells derived from 4 individuals with schizophrenia and 4 unaffected control individuals were depolarized using potassium chloride. RNA was extracted followed by genome-wide profiling of the transcriptome. Neurons were planted on June 21, 2013, and harvested on August 2, 2013. Main Outcomes and Measures We performed differential expression analysis and gene coexpression analysis to identify activity-dependent or disease-specific changes of the transcriptome. Gene expression differences were assessed with linear models. Furthermore, we used gene set analyses to identify coexpressed modules that are enriched for schizophrenia risk genes. Results We identified 1669 genes that were significantly different in schizophrenia-associated vs control human-induced pluripotent stem cell-derived neurons and 1199 genes that are altered in these cells in response to depolarization (linear models at false discovery rate ≤0.05). The effect of activity-dependent changes of gene expression in schizophrenia-associated neurons (59 significant genes at false discovery rate ≤0.05) was attenuated compared with control samples (594 significant genes at false discovery rate ≤0.05). Using gene coexpression analysis, we identified 2 modules (turquoise and brown) that were associated with diagnosis status and 2 modules (yellow and green) that were associated with depolarization at a false discovery rate of ≤0.05. For 3 of the 4 modules, we found enrichment with schizophrenia-associated variants: brown (χ2 = 20.68; P = .002), turquoise (χ2 = 12.95; P = .04), and yellow (χ2 = 15.34; P = .02). Conclusions and Relevance In this analysis, candidate genes clustered within gene networks that were associated with a blunted effect of activity-dependent changes of gene expression in schizophrenia-associated neurons. Overall, these findings link schizophrenia candidate genes with specific molecular functions in neurons, which could be used to examine underlying mechanisms and therapeutic interventions related to schizophrenia.

Long non-coding RNA expression during aging in the human subependymal zone

The human subependymal zone (SEZ) is debatably a source of newly born neurons throughout life and neurogenesis is a multi-step process requiring distinct transcripts during cell proliferation and early neuronal maturation, along with orchestrated changes in gene expression during cell state/fate transitions. Furthermore, it is becoming increasingly clear that the majority of our genome that results in production of non-protein-coding RNAs plays vital roles in the evolution, development, adaptation, and region-specific function of the human brain. We predicted that some transcripts expressed in the SEZ may be unique to this specialized brain region, and that a comprehensive transcriptomic analysis of this region would aid in defining expression changes during neuronal birth and growth in adult humans. Here, we used deep RNA sequencing of human SEZ tissue during adulthood and aging to characterize the transcriptional landscape with a particular emphasis on long non-coding RNAs (lncRNAs). The data show predicted age-related changes in mRNAs encoding proliferation, progenitor, and inflammatory proteins as well as a unique subset of lncRNAs that are highly expressed in the human SEZ, many of which have unknown functions. Our results suggest the existence of robust proliferative and neuronal differentiation potential in the adult human SEZ and lay the foundation for understanding the involvement of lncRNAs in postnatal neurogenesis and potentially associated neurodevelopmental diseases that emerge after birth.

Properties of N4-Methylated Cytidines in miRNA Mimics

Experiments conducted with micro RNA (miRNA) mimics often result in subtle phenotypic changes and hence require careful controls. A commonly used type of control reagent in the antisense/RNA interference fields is the mismatched sequence. However, it is difficult to use mismatch controls for miRNAs, mainly because base permutation in the seed region may generate a new miRNA seed with its own associated target transcripts. We incorporated N(4)-methylcytidine and N(4),N(4)-dimethylcytidine into a series of RNAs using the convertible nucleoside approach and measured their effects on hybridization affinity with complementary RNAs, and on miRNA-mediated and small interfering RNA (SiRNA)-mediated silencing. We report here that incorporation of a single N(4),N(4)-dimethylcytidine into the seed region of miRNAs can be used as a new class of negative miRNA control which (1) does not constitute a new seed sequence; (2) is accepted by the RNA-induced silencing complex (RISC); (3) causes a significant loss of binding affinity to target RNAs; and (4) is synthesized conveniently into oligoribonucleotides.

THC exposure of human iPSC neurons impacts genes associated with neuropsychiatric disorders

There is a strong association between cannabis use and schizophrenia but the underlying cellular links are poorly understood. Neurons derived from human-induced pluripotent stem cells (hiPSCs) offer a platform for investigating both baseline and dynamic changes in human neural cells. Here, we exposed neurons derived from hiPSCs to Δ9-tetrahydrocannabinol (THC), and identified diagnosis-specific differences not detectable in vehicle-controls. RNA transcriptomic analyses revealed that THC administration, either by acute or chronic exposure, dampened the neuronal transcriptional response following potassium chloride (KCl)-induced neuronal depolarization. THC-treated neurons displayed significant synaptic, mitochondrial, and glutamate signaling alterations that may underlie their failure to activate appropriately; this blunted response resembles effects previously observed in schizophrenia hiPSC- derived neurons. Furthermore, we show a significant alteration in THC-related genes associated with autism and intellectual disability, suggesting shared molecular pathways perturbed in neuropsychiatric disorders that are exacerbated by THC.

Enhancers active in dopamine neurons are a primary link between genetic variation and neuropsychiatric disease

Enhancers function as DNA logic gates and may control specialized functions of billions of neurons. Here we show a tailored program of noncoding genome elements active in situ in physiologically distinct dopamine neurons of the human brain. We found 71,022 transcribed noncoding elements, many of which were consistent with active enhancers and with regulatory mechanisms in zebrafish and mouse brains. Genetic variants associated with schizophrenia, addiction, and Parkinson’s disease were enriched in these elements. Expression quantitative trait locus analysis revealed that Parkinson’s disease-associated variants on chromosome 17q21 cis-regulate the expression of an enhancer RNA in dopamine neurons. This study shows that enhancers in dopamine neurons link genetic variation to neuropsychiatric traits.The BRAINcode consortium found that tens of thousands of transcribed noncoding elements (TNEs) from the ‘dark matter’ of our genome are active in dopamine neurons. They may be linked to schizophrenia, Parkinson’s disease, and addiction.

Adar3 Is Involved in Learning and Memory in Mice

The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3-deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.

blkbox: Integration Of Multiple Machine Learning Approaches To Identify Disease Biomarkers

Motivation Machine learning (ML) is a powerful tool to create supervised models that can distinguish between classes and facilitate biomarker selection in high-dimensional datasets, including RNA Sequencing (RNA-Seq). However, it is variable as to which is the best performing ML algorithm(s) for a specific dataset, and identifying the optimal match is time consuming. blkbox is a software package including a shiny frontend, that integrates nine ML algorithms to select the best performing classifier for a specific dataset. blkbox accepts a simple abundance matrix as input, includes extensive visualization, and also provides an easy to use feature selection step to enable convenient and rapid potential biomarker selection, all without requiring parameter optimization. Results Feature selection makes blkbox computationally inexpensive while multi-functionality, including nested cross-fold validation (NCV), ensures robust results. blkbox identified algorithms that outperformed prior published ML results. Applying NCV identifies features, which are utilized to gain high accuracy. Availability The software is available as a CRAN R package and as a developer version with extended functionality on github (https://github.com/gboris/blkbox). Contact b.guennewig@garvan.org.au

Schizophrenia hiPSC neurons display expression changes that are enriched for disease risk variants and a blunted activity-dependent response

IMPORTANCE Schizophrenia (SCZ) is a common illness with complex genetic architecture where both common genetic variation and rare mutations have been implicated. SCZ candidate genes participate in common molecular pathways that are regulated by activity-dependent changes in neurons, including the signaling network that modulates synaptic strength and the network of genes that are targets of fragile X mental retardation protein. One important next step is to further our understanding on the role of activity-dependent changes of genes expression in the etiopathogenesis of SCZ. OBJECTIVE To examine whether neuronal activity-dependent changes of gene expression is dysregulated in SCZ. DESIGN, SETTING, AND PARTICIPANTS Neurons differentiated from human induced pluripotent stem cells (hiPSCs) derived from 4 cases with SCZ and 4 unaffected controls were depolarized using potassium chloride. RNA was extracted followed by genome-wide profiling of the transcriptome. MAIN OUTCOMES AND MEASURES We performed differential expression analysis and gene co-expression analysis to identify activity-dependent or disease-specific changes of the transcriptome. Further, we used gene set analyses to identify co-expressed modules that are enriched for SCZ risk genes. RESULTS We identified 1,669 genes that are significantly different in SCZ-associated vs. control hiPSC-derived neurons and 1,199 genes that are altered in these cells in response to depolarization. We show that the effect of activity-dependent changes of gene expression in SCZ-associated neurons is attenuated compared to controls. Furthermore, these differentially expressed genes are co-expressed in modules that are highly enriched for genes affected by genetic risk variants in SCZ and other neurodevelopmental disorders. CONCLUSIONS AND RELEVANCE Our results show that SCZ candidate genes converge to gene networks that are associated with a blunted effect of activity-dependent changes of gene expression in SCZ-associated neurons. Overall, these findings show that hiPSC neurons demonstrate activity-dependent transcriptional changes that can be utilized to examine underlying mechanisms and therapeutic interventions related to SCZ.