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Title: Genetics and Regulatory Impact of Alternative Polyadenylation in Human B-Lymphoblastoid Cells}
Article:
Abstract:
Gene expression varies widely between individuals of a population, and regulatory change can underlie phenotypes of evolutionary and biomedical relevance. A key question in the field is how DNA sequence variants impact gene expression, with most mechanistic studies to date focused on the effects of genetic change on regulatory regions upstream of protein-coding sequence. By contrast, the role of RNA 3′-end processing in regulatory variation remains largely unknown, owing in part to the challenge of identifying functional elements in 3′ untranslated regions. In this work, we conducted a genomic survey of transcript ends in lymphoblastoid cells from genetically distinct human individuals. Our analysis mapped the cis-regulatory architecture of 3′ gene ends, finding that transcript end positions did not fall randomly in untranslated regions, but rather preferentially flanked the locations of 3′ regulatory elements, including miRNA sites. The usage of these transcript length forms and motifs varied across human individuals, and polymorphisms in polyadenylation signals and other 3′ motifs were significant predictors of expression levels of the genes in which they lay. Independent single-gene experiments confirmed the effects of polyadenylation variants on steady-state expression of their respective genes, and validated the regulatory function of 3′ cis-regulatory sequence elements that mediated expression of these distinct RNA length forms. Focusing on the immune regulator IRF5, we established the effect of natural variation in RNA 3′-end processing on regulatory response to antigen stimulation. Our results underscore the importance of two mechanisms at play in the genetics of 3′-end variation: the usage of distinct 3′-end processing signals and the effects of 3′ sequence elements that determine transcript fate. Our findings suggest that the strategy of integrating observed 3′-end positions with inferred 3′ regulatory motifs will prove to be a critical tool in continued efforts to interpret human genome variation.
Introduction:
Naturally occurring genetic differences in gene regulation within populations underlie phenotypes of evolutionary and biomedical interest[1]–[3] and can serve as the basis for inference of regulatory networks[4],[5]. A key problem in the field is understanding the molecular mechanisms by which DNA sequence variants give rise to expression change. Recent work has emphasized the importance of sequence differences in regions upstream of gene loci that harbor cis-acting determinants of transcription factor binding[6]–[10] and chromatin architecture[11]–[13]. Much less is known about the role of 3′-end regulation as a determinant of expression variation between individuals. Alternative polyadenylation represents a major regulatory strategy in the human genome, with analysis across tissue types detecting multiple 3′ UTR forms of over half of all human genes[14]. Detailed genetic studies have implicated polymorphisms affecting transcript termination in both Mendelian and complex human disease[15]–[20]. Genomic analyses have hinted at a broader role for genetic differences in RNA 3′-end processing as a driver of expression variation[6],[8],[21],[22], but the prevalence and the mechanisms of these changes are incompletely understood. Progress in dissecting the genetics of 3′-end processing has been limited in part by fundamental questions about the regulatory information encoded in 3′ UTRs. Single-gene studies have made clear that, in addition to its interplay with exonic splicing[23],[24], RNA 3′-end processing can dictate the extent of 3′ UTR sequence incorporated into mature transcripts that governs half-life, translation, and localization[25]–[29]; the efficiency of transcription termination itself can also influence steady-state expression level of a given length form[30]–[34]. In general, however, identifying the regulatory elements that underlie relationships between 3′ UTR sequence and gene expression remains a primary challenge, and for the majority of human genes, the regulatory impact of alternative polyadenylation is unknown. Likewise, the search for molecular players underlying cis-regulation of 3′-end processing at individual gene loci[23] and genome-scale regulation of 3′-end processing in trans[14],[29],[35]–[40] is an area of active research. A complete understanding of the genetics of alternative polyadenylation will require maps of transcript end site usage and 3′ cis-regulatory elements, and analysis strategies to integrate the data. Recently developed short-read sequencing methods for transcript ends[40]–[46] have enabled the possibility of quantitative studies of the regulatory architecture of transcript end forms on a genomic scale. In this work, we set out to investigate mechanisms by which alternative polyadenylation impacts gene expression and its variation across genetically distinct human individuals. We used 3′-end RNA-seq[42] to maximize the genomic coverage and precision of transcript end positions, and to measure quantitative expression levels of transcript forms. The results shed light on the architecture of transcript ends and regulatory elements in human 3′ UTRs and the principles of genetic variation in 3′ length form usage.
Discussion:
Alternative polyadenylation is prevalent in the human transcriptome, and in landmark cases, variation across individuals in the use of 3′ length forms of RNAs has been shown to underlie human disease[15]–[20]. However, for most human genes, the regulatory importance of changes in transcript ends between individuals is incompletely understood, owing to the challenges of measuring 3′-end usage and identifying functional regulatory elements in 3′ UTRs. We have developed a spatially precise, quantitative, high-throughput sequencing approach for 3′ ends, complementing now-classic studies of expressed sequence tags[30],[36],[48],[51],[59]–[61]. We have used the resulting transcript end positions and abundances to pioneer an analysis approach which integrates bioinformatic predictions of 3′ regulatory motifs, genomic analysis, and molecular genetics. With this strategy, we have established a regulatory map of transcript ends and functional elements in the 3′ UTRs of lymphoblastoid cells, and we have abstracted genomic principles of alternative polyadenylation and natural genetic variation in this cell type. Our mapping of 3′-end length forms and sequence motifs in 3′ UTRs revealed an intuitive logic in which the choice between short and long UTR forms governs the incorporation of regulatory elements into mature messages[36],[61]. We note that the 3′ length forms we report in B-lymphoblastoid cells represent a subset of the total complement of 3′ UTR lengths used across tissues. As such, we hypothesize that surveys of tissue types will ultimately reveal transcript forms of many genes, used in particular contexts, that incorporate 3′ regulatory information to different extents. The ability to tune the responsiveness to trans-acting input itself distinguishes alternative polyadenylation from other transcriptional and post-transcriptional regulatory mechanisms, providing a compelling model for the particular advantage of 3′-end processing as a regulatory strategy and a rationale for its prevalence in mammalian genomes. In comparisons across cell lines from genetically distinct individuals, we analyzed the regulatory importance of genetic changes at 3′ transcript ends. We uncovered a key role for polymorphisms in polyadenylation signals as a driver of changes in gene expression, and we detailed the molecular mechanisms at play. The polyadenylation variants we study here dictate the production of transcript forms with different determinants of 3′-end processing, different half-lives, and different recognition sites for trans-acting regulators. These findings establish a connection between observational studies of transcript 3′ length forms across human populations[21],[22] and regulatory effects of this variation. In the case of IRF5, we discovered that a naturally occurring genetic change in usage of 3′ RNA forms can serve to tune the kinetics of recovery of expression after induction by lipopolysaccharide. Thus, against the backdrop of prior studies of this lupus susceptibility gene[15], we have uncovered an additional dimension by which variation in RNA 3′-end processing affects regulatory behavior. Given these case-study results as a validation of our genome-scale analyses, we speculate that many genetic variants with biologically relevant effects mediated by RNA 3′-end processing remain to be discovered in the human population. We have also shown that polymorphisms in 3′ motifs that govern transcript fate can serve as predictors of steady-state levels of the genes in which they lie. In light of the ultimate goal of predicting regulatory and phenotypic effects from human genome sequence, our results indicate that analysis strategies using sequence determinants of transcription initiation and splicing alone are likely to provide an incomplete model of expression variation. Importantly, however, our work makes clear that binding sites for regulators of mRNA localization, half-life, and translation at 3′ ends are themselves only part of the regulatory landscape. Rather, a complete understanding of the genetics of gene expression will integrate the usage of RNA 3′-end processing signals with the effects of 3′ sequence elements that control transcript fate. We anticipate that abundances and positions of transcript ends observed in 3′-end RNA-seq will prove to be a key component in the systems-level modeling of regulatory networks and their variation. In summary, while the genetic study of RNA 3′-end processing is in its infancy, our work and that of others[15]–[22] establishes that variation at 3′ ends can be a critical determinant of regulatory behaviors. However, for the vast majority of human genes, the importance of 3′ regulatory change remains unknown. Our single-gene experiments detail the expression effects of 3′ UTR variation in the disease-associated genes IRF5[15] and DIP2B[55]; the potential for regulatory variants as drivers of human disease will serve as continued motivation for genomic and genetic analyses of expression change.
Messenger RNAs carry the instructions necessary to synthesize proteins that do work for the cell . Extending beyond the protein-coding sequence of a given mRNA is an additional stretch of sequence , harboring signals that govern how much protein is made and how long the mRNA remains in the cell before it is broken down . The incorporation of this end region into mature mRNA is itself subject to change; for the vast majority of human genes , how and why cells use different mRNA ends remains largely unknown . In this work , we surveyed mRNA ends from ∼10 , 000 genes in immune cells from genetically distinct human individuals . We found that mRNA end positions were not randomly distributed , but rather preferentially flanked the locations of regulatory signals that govern mRNA fate . The usage of these mRNA length forms and regulatory elements varied across individuals and could be dissected molecularly . Our results uncover key mechanisms and regulatory effects of transcript end processing , particularly as these are perturbed by genetic differences between humans .