Identification, improved modeling and integration of signals to predict constitutive and altering splicing

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2004.Includes bibliographical references.(cont.) manipulation of intronic elements that enables fish genes to be spliced properly in mammalian cells; (iii) A computational analysis using EST data, genome sequence data, and microarray expression data of tissue- specific alternative splicing is conducted, which distinguishes human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-factors likely to play important roles in tissue-specific AS in human cells; (iv) The identification of a set of discriminatory sequence features and their integration into a statistical machine-learning algorithm, ACEScan, which distinguishes exons subject to evolutionarily conserved alternative splicing from constitutively spliced or lineage-specifically-spliced exons is described; (v) The genome-wide search for and experimental validation of exon-skipping events using the combination of two silencing cis-elements, UAGG and GGGG.The regulation of pre-messenger RNA splicing by the spliceosomal machinery via interactions between cis-regulatory elements and splicing trans-factors to generate a specific mRNA i.e. constitutive splicing, or sometimes many distinct mRNA isoforms i.e. alternative splicing, is still a poorly understood process. Progress into illuminating this process is further exacerbated by the variation of splicing in the multitude of tissues and cell types present, as well as the variation of cis and trans elements in different organisms, and the possibility that some alternative splicing events present in expressed sequence tag (EST) databases may constitute biochemical 'noise' or transient evolutionary fluctuations. Several studies, mainly computational in nature, addressing different questions regarding constitutive and alternative splicing are described here, ranging from improved modeling of splicing signals, studying the variation of alternative splicing in various tissues, analyzing evolutionary differences of cis and trans elements of splicing in various vertebrates, and utilizing attributes indicative of alternative splicing events conserved in human and mouse to identify novel alternatively spliced exons. In particular: (i) A general approach for improved modeling of short sequence motifs, based on the Maximum Entropy principle, that incorporates local adjacent and non-adjacent position dependencies is introduced, and applied to understanding splice site signals. The splice site recognition algorithm, MaxENTScan, performs better than previous models that utilize as input similar length sequences; (ii) The first large-scale bioinformatics study is conducted that identifies similarities and differences in candidate cis-regulatory elements and trans-acting splicingby Gene W. Yeo.Ph.D

Conserved metabolite regulation of stress granule assembly via AdoMet

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Begovich, K., Vu, A. Q., Yeo, G., & Wilhelm, J. E. Conserved metabolite regulation of stress granule assembly via AdoMet. Journal of Cell Biology, 219(8), (2020): e201904141, doi:10.1083/jcb.201904141.Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5′ end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43–positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.Work from the Wilhelm laboratory was supported by a grant to J.E. Wilhelm from the Collaborative Innovation Awards program of Howard Hughes Medical Institute and the James Wilhelm Memorial Fund. K. Begovich is a Howard Hughes Medical Institute Gilliam Fellow. Work from the Yeo laboratory was supported by grants to G. Yeo from the National Institutes of Health (HG004659), Target ALS (20193440), and the ALS Association (272 and 438)

Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events

A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes

Circadian Oscillations of Protein-Coding and Regulatory RNAs in a Highly Dynamic Mammalian Liver Epigenome

SummaryIn the mouse liver, circadian transcriptional rhythms are necessary for metabolic homeostasis. Whether dynamic epigenomic modifications are associated with transcript oscillations has not been systematically investigated. We found that several antisense RNA, lincRNA, and microRNA transcripts also showed circadian oscillations in adult mouse livers. Robust transcript oscillations often correlated with rhythmic histone modifications in promoters, gene bodies, or enhancers, although promoter DNA methylation levels were relatively stable. Such integrative analyses identified oscillating expression of an antisense transcript (asPer2) to the gene encoding the circadian oscillator component Per2. Robust transcript oscillations often accompanied rhythms in multiple histone modifications and recruitment of multiple chromatin-associated clock components. Coupling of cycling histone modifications with nearby oscillating transcripts thus established a temporal relationship between enhancers, genes, and transcripts on a genome-wide scale in a mammalian liver. The results offer a framework for understanding the dynamics of metabolism, circadian clock, and chromatin modifications involved in metabolic homeostasis

Inference of Splicing Regulatory Activities by Sequence Neighborhood Analysis

Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences

Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently, advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method, referred to as Patch-seq, enables a thorough, multimodal profiling of neurons and permits us to expose the links between functional properties, morphology, and gene expression. Here, we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects, but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost