Antagonistic and cooperative AGO2-PUM interactions in regulating mRNAs.

Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3' untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3'UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3'UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent

PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs

A large number of RNA binding proteins (RBPs) and miRNAs bind to the 3′ untranslated regions of mRNA, but methods to dissect their function and interactions are lacking. Here the authors introduce post-transcriptional regulatory element sequencing (PTRE-seq) to dissect sequence preferences, interactions and consequences of RBP and miRNA binding

Genome-Wide Analysis of RNA Secondary Structure in Eukaryotes

The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. Over the past decades, myriad studies have revealed specific examples of structural elements that direct the expression and function of both protein-coding messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In this work, we develop and apply a novel high-throughput, sequencing-based, structure mapping approach to study RNA secondary structure in three eukaryotic organisms. First, we assess global patterns of secondary structure across protein-coding transcripts and identify a conserved mark of strongly reduced base pairing at transcription start and stop sites, which we hypothesize helps with ribosome recruitment and function. We also find empirical evidence for reduced base pairing within microRNA (miRNA) target sites, lending further support to the notion that even mRNAs have additional selective pressures outside of their protein coding sequence. Next, we integrate our structure mapping approaches with transcriptome-wide sequencing of ribosomal RNA-depleted (RNA-seq), small (smRNA-seq), and ribosome-bound (ribo-seq) RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in the model organism Arabidopsis thaliana. We find that secondary structure and mRNA abundance are strongly anti-correlated, which is likely due to the propensity for highly structured transcripts to be degraded and/or processed into smRNAs. Finally, we develop a likelihood model and Bayesian Markov chain Monte Carlo (MCMC) algorithm that utilizes the sequencing data from our structure mapping approaches to generate single-nucleotide resolution predictions of RNA secondary structure. We show that this likelihood framework resolves ambiguities that arise from the sequencing protocol and leads to significantly increased prediction accuracy. In total, our findings provide on a global scale both validation of existing hypotheses regarding RNA biology as well as new insights into the regulatory and functional consequences of RNA secondary structure. Furthermore, the development of a statistical approach to structure prediction from sequencing data offers the promise of true genome-wide determination of RNA secondary structure

Inferring Transcriptional and Post-Transcriptional Network Structure by Exploiting Natural Sequence Variation

Understanding how cellular processes of an organism translate its genome into its phenotype is one of the grand challenges in biology. Linkage studies seek to identify allelic variants that manifest themselves as phenotypic variation between individuals in a population. The advent of high-throughput genotyping and gene expression profiling technologies has made it possible to use messenger RNA levels as quantitative traits in linkage studies. This has created new opportunities to study genetic variation at the level of gene regulatory networks rather than individual genes. This thesis consists of four parts, each of which outlines a different strategy for integrating genome-wide expression data and genotype data in order to identify transcriptional and post-transcriptional regulatory mechanisms. The data for these analyses comes from segregating populations of Saccharomyces cerevisiae (baker’s yeast) as well as Caenorhabditis elegans (roundworm). The first study focused on inferring the in vitro binding specificity of RNA-binding proteins (RBPs). We first analyzed a recent compendium of in vivo mRNA binding data to model the sequence specificity of 45 yeast RBPs in the form of a position- specific affinity matrix (PSAM). We were able to recover known consensus nucleotide sequences for 12 RBPs and discovered novel binding preferences for 3 of the RBPs namely, Scp160p, Sik1p and Tdh3p. The second study aimed to identify transacting chromosomal loci that regulate expression of a large number of genes. Traditionally, such loci are discovered by first mapping expression quantitative loci (eQTLs) for individual genes, and then looking for so-called “eQTLs hotspots”. Our method avoids the first step by integrating information across all genes, leading to a more elegant method that has increased statistical power. For yeast, we recovered 70% of the reported eQTL hotspots from two independent studies, and discovered a new transacting locus on chromosome V. For worm, we detected six transacting loci, only two of which were previously reported as eQTL hotspots. The third study focused on post-transcriptional regulatory networks in yeast, by mapping the regulatory activity level of RNA binding proteins (RBPs) as a quantitative trait in so-called “aQTL” analysis. We used the collection of 15 sequence motifs with the associated mRNA region combinations that we obtained in our first study together with mRNA expression data to estimate RBP activities across yeast segregants. Consistent with a previous study, we recovered the MKT1 locus on chromosome XIV as a genetic modulator of Puf3p activity. We also discovered that Puf3p activity is modulated through distinct loci depending on whether it is binding to 50 or 30 untranslated region (UTR) of its target mRNAs. Furthermore, we identified a locus on chromosome XV that includes the IRA2 gene as a putative aQTL for Puf4p; this prediction was validated using expression data for an IRA2 allele replacement strain. Our fourth study focused on the detection of loci whose allelic variation modulates the in vivo regulatory connectivity between a transcription factor and its target genes. We call these loci connectivity QTLs or “cQTLs”. We mapped the DIG2 locus on chromosome IV as a cQTL for the transcription factor Ste12p. Dig2p is indeed a known inhibitor of yeast mating response activator Ste12p. The coding region of the DIG2 gene contains a single non-synonymous mutation (T83I). We are experimentally testing the functional impact of this mutation in allele replacement strains. We also identified the TAF13 locus as a putative modulator of GCN4p connectivity

Transcriptome-Wide Characterization of APOBEC1-Catalyzed RNA Editing Events in Macrophages

RNA editing refers to the process by which the sequence of RNA is altered through the insertion, deletion or modification of specific nucleotides. Editing of mRNA transcripts can increase the informational complexity encoded by the genome by producing alternative protein isoforms through specific posttranscriptional RNA editing events. Additionally, RNA editing in non-coding regions of mRNA transcripts has been shown to influence gene expression in a tissue-specific manner. In mammals, mRNA editing serves a diverse set of biological roles in neuronal function, host defense and lipid metabolism. The major mRNA editors acting in mammals include the adenosine deaminases acting on RNA (ADARs) and Apolipoprotein B mRNA Editing Catalytic polypeptide-1 (APOBEC1). The ADARs and APOBEC1 were originally characterized as catalysts for previously characterized biologically important RNA-editing events that resulted in specific coding changes; study of additional editing activity was limited by standard sequencing techniques. APOBEC1 in particular was characterized in the small intestine as mediating a specific editing event in the coding region of Apolipoprotein B (Apob). APOBEC1-dependent RNA editing in Apob mediates the tissue-specific differential expression of Apob isoforms, a process important for intestinal lipid metabolism and transport. The development of next-generation sequencing has allowed for transcriptome-wide discovery of RNA editing activity and has resulted in the identification of more than 10,000 RNA editing events, pointing to more biological functions for RNA editing than had been previously appreciated. To search for additional APOBEC1 editing events, our lab developed a comparative RNA-Seq screen for the transcriptome-wide identification of enzyme-specific RNA editing events. Applying this technique to small intestine enterocytes, the site of known APOBEC1 activity, we identified over 30 novel APOBEC1 editing events in transcript 3’UTRs, which represents the first example of physiological APOBEC1 editing outside of the Apob transcript. These newly identified editing events were located in evolutionarily conserved regions of transcript 3’UTRs, suggesting that this editing activity may have functional relevance. The discovery of additional editing activity for APOBEC1, as well as the fact that it is expressed in a number of immune cell types, suggests that APOBEC1, like other members of the AID/APOBEC family, may contribute to cellular immune processes. The focus of the work presented in this thesis is the identification and characterization of physiological APOBEC1 editing activity in bone marrow derived macrophages (BMDMs). Using a comparative RNA-Seq screen, I identified more than 100 novel APOBEC1 editing events in BMDMs. This APOBEC1 activity occurred in two distinct editing patterns and fell within evolutionarily conserved regions of transcript 3’UTRs. Luciferase reporter assays were utilized to assess the consequences of APOBEC1 3’UTR editing on protein expression and identified a number of combinations of editing events that affect translational outcomes. To determine if APOBEC1 editing could modulate protein expression by altering miRNA targeting, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of the Argonaute (Ago) proteins was performed on wild-type and APOBEC1-deficient cells. HITS-CLIP yielded transcriptome-wide maps of Ago binding and potential miRNA seed regions. While there was considerable overlap between loci targeted by both Ago and APOBEC1, little evidence was found for APOBEC1 disruption or creation of miRNA seed targets. Overall, this work characterizes abundant APOBEC1 activity in BMDMs that can modulate protein expression levels by a miRNA-independent mechanism. These results point to broader functions for APOBEC1 in transcript regulation and host defense

Transcriptome-Wide Characterization of APOBEC1-Catalyzed RNA Editing Events in Macrophages

RNA editing refers to the process by which the sequence of RNA is altered through the insertion, deletion or modification of specific nucleotides. Editing of mRNA transcripts can increase the informational complexity encoded by the genome by producing alternative protein isoforms through specific posttranscriptional RNA editing events. Additionally, RNA editing in non-coding regions of mRNA transcripts has been shown to influence gene expression in a tissue-specific manner. In mammals, mRNA editing serves a diverse set of biological roles in neuronal function, host defense and lipid metabolism. The major mRNA editors acting in mammals include the adenosine deaminases acting on RNA (ADARs) and Apolipoprotein B mRNA Editing Catalytic polypeptide-1 (APOBEC1). The ADARs and APOBEC1 were originally characterized as catalysts for previously characterized biologically important RNA-editing events that resulted in specific coding changes; study of additional editing activity was limited by standard sequencing techniques. APOBEC1 in particular was characterized in the small intestine as mediating a specific editing event in the coding region of Apolipoprotein B (Apob). APOBEC1-dependent RNA editing in Apob mediates the tissue-specific differential expression of Apob isoforms, a process important for intestinal lipid metabolism and transport. The development of next-generation sequencing has allowed for transcriptome-wide discovery of RNA editing activity and has resulted in the identification of more than 10,000 RNA editing events, pointing to more biological functions for RNA editing than had been previously appreciated. To search for additional APOBEC1 editing events, our lab developed a comparative RNA-Seq screen for the transcriptome-wide identification of enzyme-specific RNA editing events. Applying this technique to small intestine enterocytes, the site of known APOBEC1 activity, we identified over 30 novel APOBEC1 editing events in transcript 3’UTRs, which represents the first example of physiological APOBEC1 editing outside of the Apob transcript. These newly identified editing events were located in evolutionarily conserved regions of transcript 3’UTRs, suggesting that this editing activity may have functional relevance. The discovery of additional editing activity for APOBEC1, as well as the fact that it is expressed in a number of immune cell types, suggests that APOBEC1, like other members of the AID/APOBEC family, may contribute to cellular immune processes. The focus of the work presented in this thesis is the identification and characterization of physiological APOBEC1 editing activity in bone marrow derived macrophages (BMDMs). Using a comparative RNA-Seq screen, I identified more than 100 novel APOBEC1 editing events in BMDMs. This APOBEC1 activity occurred in two distinct editing patterns and fell within evolutionarily conserved regions of transcript 3’UTRs. Luciferase reporter assays were utilized to assess the consequences of APOBEC1 3’UTR editing on protein expression and identified a number of combinations of editing events that affect translational outcomes. To determine if APOBEC1 editing could modulate protein expression by altering miRNA targeting, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of the Argonaute (Ago) proteins was performed on wild-type and APOBEC1-deficient cells. HITS-CLIP yielded transcriptome-wide maps of Ago binding and potential miRNA seed regions. While there was considerable overlap between loci targeted by both Ago and APOBEC1, little evidence was found for APOBEC1 disruption or creation of miRNA seed targets. Overall, this work characterizes abundant APOBEC1 activity in BMDMs that can modulate protein expression levels by a miRNA-independent mechanism. These results point to broader functions for APOBEC1 in transcript regulation and host defense

Ciphers and Executioners: How 3′-Untranslated Regions Determine the Fate of Messenger RNAs

The sequences and structures of 3′-untranslated regions (3′UTRs) of messenger RNAs govern their stability, localization, and expression. 3′UTR regulatory elements are recognized by a wide variety of trans-acting factors that include microRNAs (miRNAs), their associated machinery, and RNA-binding proteins (RBPs). In turn, these factors instigate common mechanistic strategies to execute the regulatory programs encoded by 3′UTRs. Here, we review classes of factors that recognize 3′UTR regulatory elements and the effector machineries they guide toward mRNAs to dictate their expression and fate. We outline illustrative examples of competitive, cooperative, and coordinated interplay such as mRNA localization and localized translation. We further review the recent advances in the study of mRNP granules and phase transition, and their possible significance for the functions of 3′UTRs. Finally, we highlight some of the most recent strategies aimed at deciphering the complexity of the regulatory codes of 3′UTRs, and identify some of the important remaining challenges

Exploring mRNA 3'-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening

Abstract: Guanine-rich RNA sequences can fold into noncanonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 30-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 30-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo. The 30- UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 30-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression