Michael Lovci

L1 Retrotransposition in Human Neural Progenitor Cells

Long interspersed element 1 (LINE-1 or L1) retrotransposons have markedly affected the human genome. L1s must retrotranspose in the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells in vitro and in the mouse brain in vivo. Here we demonstrate that neural progenitor cells isolated from human fetal brain and derived from human embryonic stem cells support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus, and in several regions of adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.

Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans

MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is challenging because of uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG-1 in C. elegans. Using cross-linking and ALG-1 immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), we identified extensive ALG-1 interactions with specific 3′ untranslated region (UTR) and coding exon sequences and discovered features that distinguish miRNA complex binding sites in 3′ UTRs from those in other genic regions. Furthermore, our analyses revealed a striking enrichment of Argonaute binding sites in genes important for miRNA function, suggesting an autoregulatory role that may confer robustness to the miRNA pathway.

LIN28 Binds Messenger RNAs at GGAGA Motifs and Regulates Splicing Factor Abundance

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.

BioFeatureFinder: Flexible, unbiased analysis of biological characteristics associated with genomic regions

BioFeatureFinder is a novel algorithm which allows analyses of many biological genomic landmarks (including alternatively spliced exons, DNA/RNA-binding protein binding sites, and gene/transcript functional elements, nucleotide content, conservation, k-mers, secondary structure) to identify distinguishing features. BFF uses a flexible underlying model that combines classical statistical tests with Big Data machine-learning strategies. The model is created using thousands of biological characteristics (features) that are used to build a feature map and interpret category labels in genomic ranges. Our results show that BFF is a reliable platform for analyzing large-scale datasets. We evaluated the RNA binding feature map of 110 eCLIP-seq datasets and were able to recover several well-known features from the literature for RNA-binding proteins; we were also able to uncover novel associations. BioFeatureFinder is available at https://github.com/kbmlab/BioFeatureFinder/.

Prediction of biologically important features related to intron retention events based on machine learning algorithms

The retention of introns represents a class of alternative splicing (AS) events frequently associated with diseases. Despite the recent development of many AS identification tools, most of the tools do not consider their relationship to relevant biological features. We developed a package capable of accessing many features to AS events and evaluated association between events. We identified that retained introns and nearby exons have lower GC content then their nonretained counterparts.