Targeting and function of mammalian microRNAs

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2009.Cataloged from PDF version of thesis.Includes bibliographical references.In the span of a few short years, animal microRNAs have become recognized as broad regulators of gene expression, largely in part due to our improved understanding of how animal microRNAs recognize their targets. Crucial to microRNA targeting are the ~7-nt seed sites complementary to nucleotides 2-8 at the 5' end of the microRNA. We show that protein-coding genes preferentially expressed at the same time and place as a highly expressed microRNA have evolved their 3' UTR sequence to specifically avoid seed sites matching that microRNA. In contrast, conserved sites appear to be preferentially expressed in developmental states prior to microRNA expression, and are downregulated upon induction of that microRNA. Combined with the result that both conserved and nonconserved seed sites are generally functional, our findings extend the direct and indirect influence of mammalian microRNAs to the majority of protein-coding genes. Although seed sites account for much of the specificity of microRNA regulation, they are not always sufficient for repression, suggesting the contribution of additional specificity determinants. Combining independent computational and experimental approaches, we found five general features associated with site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed microRNAs, pairing outside of the seed region at microRNA nucleotides 13-16, and positioning within the 3' UTR at least 15nt from the stop codon and away from the center of long UTRs. By incorporating these five features, we are able to explain much of the differences in site efficacy for both exogenously added microRNAs and for endogenous microRNA-message interactions. We further refined the seed site motif involved in microRNA repression, by demonstrating experimentally an Adenosine preference across from the unpaired first nucleotide of the microRNA and ranking the relative effectiveness of different classes of seed sites. Although sites lacking perfect seed pairing were generally ineffective, a fraction of these sites were supplemented by detectable compensatory 3' pairing. In addition, by extending our conservation analysis to 11 genomes, we show that the confidence with which conserved target sites can be predicted is a function of the conservation of the seed site itself relative to the conservation of surrounding sequence. This allows individual conserved sites to be assigned a confidence score reflecting the likelihood that the site is being conserved due to selection rather than by chance.by Kyle Kai-How Farh.Ph. D

Clinical impact of splicing in neurodevelopmental disorders.

Clinical exome sequencing is frequently used to identify gene-disrupting variants in individuals with neurodevelopmental disorders. While splice-disrupting variants are known to contribute to these disorders, clinical interpretation of cryptic splice variants outside of the canonical splice site has been challenging. Here, we discuss papers that improve such detection

Expanding the MicroRNA Targeting Code: Functional Sites with Centered Pairing

Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5′ end. Although pairing to the 3′ region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present “centered sites,” a class of miRNA target sites that lack both perfect seed pairing and 3′-compensatory pairing and instead have 11–12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg[superscript 2+]), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved.National Institutes of Health (U.S.)Damon Runyon Cancer Research Foundation. Fellowship Awar

Phylogenomic analyses provide insights into primate evolution

Comparative analysis of primate genomes within a phylogenetic context is essential for understanding the evolution of human genetic architecture and primate diversity. We present such a study of 50 primate species spanning 38 genera and 14 families, including 27 genomes first reported here, with many from previously less well represented groups, the New World monkeys and the Strepsirrhini. Our analyses reveal heterogeneous rates of genomic rearrangement and gene evolution across primate lineages. Thousands of genes under positive selection in different lineages play roles in the nervous, skeletal, and digestive systems and may have contributed to primate innovations and adaptations. Our study reveals that many key genomic innovations occurred in the Simiiformes ancestral node and may have had an impact on the adaptive radiation of the Simiiformes and human evolution

Identification of constrained sequence elements across 239 primate genomes

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3–9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals

Genetic and Epigenetic Fine-Mapping of Causal Autoimmune Disease Variants

Summary Genome-wide association studies have identified loci underlying human diseases, but the causal nucleotide changes and mechanisms remain largely unknown. Here we developed a fine-mapping algorithm to identify candidate causal variants for 21 autoimmune diseases from genotyping data. We integrated these predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4+ T-cell subsets, regulatory T-cells, CD8+ T-cells, B-cells, and monocytes. We find that ~90% of causal variants are noncoding, with ~60% mapping to immune-cell enhancers, many of which gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation. Causal variants tend to occur near binding sites for master regulators of immune differentiation and stimulus-dependent gene activation, but only 10–20% directly alter recognizable transcription factor binding motifs. Rather, most noncoding risk variants, including those that alter gene expression, affect non-canonical sequence determinants not well-explained by current gene regulatory models

A global catalog of whole-genome diversity from 233 primate species.

The rich diversity of morphology and behavior displayed across primate species provides an informative context in which to study the impact of genomic diversity on fundamental biological processes. Analysis of that diversity provides insight into long-standing questions in evolutionary and conservation biology and is urgent given severe threats these species are facing. Here, we present high-coverage whole-genome data from 233 primate species representing 86% of genera and all 16 families. This dataset was used, together with fossil calibration, to create a nuclear DNA phylogeny and to reassess evolutionary divergence times among primate clades. We found within-species genetic diversity across families and geographic regions to be associated with climate and sociality, but not with extinction risk. Furthermore, mutation rates differ across species, potentially influenced by effective population sizes. Lastly, we identified extensive recurrence of missense mutations previously thought to be human specific. This study will open a wide range of research avenues for future primate genomic research

The landscape of tolerated genetic variation in humans and primates.

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole-genome sequencing data for 809 individuals from 233 primate species and identified 4.3 million common protein-altering variants with orthologs in humans. We show that these variants can be inferred to have nondeleterious effects in humans based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases