Predicting Functional Alternative Splicing by Measuring RNA Selection Pressure from Multigenome Alignments

High-throughput methods such as EST sequencing, microarrays and deep sequencing have identified large numbers of alternative splicing (AS) events, but studies have shown that only a subset of these may be functional. Here we report a sensitive bioinformatics approach that identifies exons with evidence of a strong RNA selection pressure ratio (RSPR) —i.e., evolutionary selection against mutations that change only the mRNA sequence while leaving the protein sequence unchanged—measured across an entire evolutionary family, which greatly amplifies its predictive power. Using the UCSC 28 vertebrate genome alignment, this approach correctly predicted half to three-quarters of AS exons that are known binding targets of the NOVA splicing regulatory factor, and predicted 345 strongly selected alternative splicing events in human, and 262 in mouse. These predictions were strongly validated by several experimental criteria of functional AS such as independent detection of the same AS event in other species, reading frame-preservation, and experimental evidence of tissue-specific regulation: 75% (15/20) of a sample of high-RSPR exons displayed tissue specific regulation in a panel of ten tissues, vs. only 20% (4/20) among a sample of low-RSPR exons. These data suggest that RSPR can identify exons with functionally important splicing regulation, and provides biologists with a dataset of over 600 such exons. We present several case studies, including both well-studied examples (GRIN1) and novel examples (EXOC7). These data also show that RSPR strongly outperforms other approaches such as standard sequence conservation (which fails to distinguish amino acid selection pressure from RNA selection pressure), or pairwise genome comparison (which lacks adequate statistical power for predicting individual exons)

Unraveling Patterns of Site-to-Site Synonymous Rates Variation and Associated Gene Properties of Protein Domains and Families

In protein-coding genes, synonymous mutations are often thought not to affect fitness and therefore are not subject to natural selection. Yet increasingly, cases of non-neutral evolution at certain synonymous sites were reported over the last decade. To evaluate the extent and the nature of site-specific selection on synonymous codons, we computed the site-to-site synonymous rate variation (SRV) and identified gene properties that make SRV more likely in a large database of protein-coding gene families and protein domains. To our knowledge, this is the first study that explores the determinants and patterns of the SRV in real data. We show that the SRV is widespread in the evolution of protein-coding sequences, putting in doubt the validity of the synonymous rate as a standard neutral proxy. While protein domains rarely undergo adaptive evolution, the SRV appears to play important role in optimizing the domain function at the level of DNA. In contrast, protein families are more likely to evolve by positive selection, but are less likely to exhibit SRV. Stronger SRV was detected in genes with stronger codon bias and tRNA reusage, those coding for proteins with larger number of interactions or forming larger number of structures, located in intracellular components and those involved in typically conserved complex processes and functions. Genes with extreme SRV show higher expression levels in nearly all tissues. This indicates that codon bias in a gene, which often correlates with gene expression, may often be a site-specific phenomenon regulating the speed of translation along the sequence, consistent with the co-translational folding hypothesis. Strikingly, genes with SRV were strongly overrepresented for metabolic pathways and those associated with several genetic diseases, particularly cancers and diabetes

Regulation of splicing networks in neurodevelopment

Alternative splicing of pre-mRNA is a critical mechanism for enabling genetic diversity, and is a carefully regulated process in neuronal differentiation. RNA binding proteins (RBPs) are developmentally expressed and physically interact with RNA to drive specific splicing changes. This work tests the hypothesis that RBP-RNA interactions are critical for regulating timed and coordinated alternative splicing changes during neurodevelopment and that these splicing changes are in turn part of major regulatory mechanisms that underlie morphological and functional maturation of neurons. I describe our efforts to identify functional RBP-RNA interactions, including the identification of previously unobserved splicing events, and explore the combinatorial roles of multiple brain-specific RBPs during development. Using integrative modeling that combines multiple sources of data, we find hundreds of regulated splicing events for each of RBFOX, NOVA, PTBP, and MBNL. In the neurodevelopmental context, we find that the proteins control different sets of exons, with RBFOX, NOVA, and PTBP regulating early splicing changes and MBNL largely regulating later splicing changes. These findings additionally led to the observation that CNS and sensory neurons express a variety of different RBP programs, with many sensory neurons expressing a less mature splicing pattern than CNS neurons. We also establish a foundation for further exploration of neurodevelopmental splicing, by investigating the regulation of previously unobserved splicing events

Genetic and immunological characterization of new subtype G envelope expressing HIV-1 pseudoviruses

Includes bibliographical references.One of the greatest challenges for the development of an effective HIV-1 vaccine is the high genetic diversity of this pathogen and the complex escape mechanisms employed by the envelope gp120 and gp41 glycoprotein that form the envelope spike. An ideal vaccine would elicit the production of broadly neutralizing antibodies (nAb), capable of having potent activity against worldwide viral isolates. This thesis encompasses a series of studies on characterizing the genetic diversity of envelope genes of subtype G and the neutralization antibody responses to these viruses. HIV-1 subtype G accounts for 5% of HIV-1 infection worldwide, centered on West Africa, with spread to European countries, primarily Spain and Portugal, presumably with immigration of infected individuals. Small satellite subtype G epidemics have been documented in Cuba and among Russian intravenous drug users. In addition, 80% of the recombinant strains circulating in Cameroon contain segments attributed to subtype G. However, until recently, little research has been done on the neutralization sensitivity and vulnerabilities of subtype G viruses, particularly those that circulate in its main reservoir, Central and West Africa. We studied plasma derived viruses from eight HIV-1 subtype G infected individuals. Five samples were collected for donation by the Yaoundé Central Hospital Blood Service from individuals who were subsequently found to be HIV-infected. Presumably, these donors were unaware of their HIV status. The remaining three samples were collected at the CIRCB Research Institute in Yaounde during testing for antiretroviral resistance. These donors were failing therapy, and had sufficiently high viral loads that HIV-1 envelope clones could be isolated. The objectives of my thesis were: 1) to examine the molecular and functional characteristics of the HIV-1 envelope glycoproteins of subtype G viral variants which is crucial to improving strategies to prevent transmission; 2) to evaluate the neutralization sensitivity subtype G viruses and the neutralizing capacities of antibodies induced by the viruses by determining the neutralization antibodies titers against autologous and heterologous HIV-1 viral isolates; 3) to characterize the sensitivity of HIV-1 subtype G viral isolates against broadly neutralizing antibodies, to gain insight into the neutralization vulnerabilities of the subtype G viruses