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

Cottrell, Kyle A.; Chaudhari, Hemangi G.; Cohen, Barak A.; Djuranović, Sergej

doi:10.1038/s41467-017-02745-0

Cited by 38 publications

(53 citation statements)

References 71 publications

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“…It is important to note that several of our RIP-Seq identified targets overlapped with mRNAs previously identified in HeLa (Galgano et al, 2008) and HEK293 (Bohn et al, 2018, Hafner et al, 2010 cells, validating our results. However, it is also important to bear in mind that PUM-mediated activation or repression, or lack of PUM regulation may be cell-typespecific (Cottrell, Chaudhari et al, 2018). Therefore, we can expect only a partial target overlap when PUM targets from different types of cells are compared.…”

Section: Discussionmentioning

confidence: 99%

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

Ilaslan

et al. 2019

Preprint

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Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins with wideranging roles, including germ cell development that has functional implications in fertility.Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity, particularly related to their roles in fertility. Here, by RNA sequencing (RNA-Seq) approaches, we identified separate mRNA pools regulated by PUM1 and PUM2 proteins in human male germ cells.Using global mass spectrometry-based profiling, we identified distinct PUM1-and PUM2bound putative protein cofactors, most of them involved in RNA processing. Combinatorial analysis of RNA-Seq and mass spectrometry findings revealed that PUM1 and PUM2 may form distinct RNA-regulatory networks, with different roles in human reproduction and testicular tumorigenesis. Our findings highlight the functional divergence and versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

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Section: Discussionmentioning

confidence: 99%

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

Ilaslan

et al. 2019

Preprint

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“…MPRAs quantify activity of putative regulatory elements by coupling them to a reporter gene and counting transcribed, element-specific tags ("barcodes"), using sequencing to determine the ratio of (expressed RNA barcode)/(delivered DNA barcode). This experimental framework has been applied to human splicing (21)(22)(23), RNA editing (24), protein translation (25), UTRs (26)(27)(28)(29)(30), and, most broadly, transcriptional (i.e., cis-) regulators. MPRAs thus offer a flexible framework to study regulatory phenomenon, including transcription factor (TF) and RNA binding protein (RBP) actions, transcript stability, and ribosome occupancy.…”

Section: Part 1: Mpras For Identification Of Sequence Variants With Fmentioning

confidence: 99%

“…As shown in Figure 1C and 1D, the same architecture and RNA/DNA expression metric can be used to assess UTR effects on transcript stability. UTR MPRAs have yet to be implemented to study regulatory variants directly, but have been used to study the regulatory grammar of the ASD/ID-implicated CELF proteins and related RBPs (26,50,51), features conferring transcript stability via 3'UTRs (27,28,30), and 5'UTR influences on translation (52) and prediction of functional 5'UTR variants (29). Across enhancer and UTR MPRAs, several key forms of disease-associated noncoding variation can be assessed for functional consequences using a variety of model systems and delivery approaches (Figure 1E-F).…”

Section: Part 1: Mpras For Identification Of Sequence Variants With Fmentioning

confidence: 99%

“…Complimentarily, MPRAs focused on neuropsychiatric disorder associated variation stand to benefit from high-information datasets by aiding variant prioritization for assay inclusion. Several datasets on synthetic UTRs (27,29), RNA binding proteins (59,60), and postmortem human brain multi-omics (61-70) have become available in recent years. Integrative computational analyses have brought these datasets together predict functional variation in SCZ, bipolar disorder, and ASDs (71,72); however, these predictions have not yet been systematically tested.…”

Section: Mpras Enable Identification Of Functional Regulators and Varmentioning

confidence: 99%

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The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

Mulvey¹,

Lagunas²,

Dougherty³

2020

Preprint

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Neuropsychiatric phenotypes have been long known to be influenced by heritable risk factors. The past decade of genetic studies have confirmed this directly, revealing specific common and rare genetic variants enriched in disease cohorts. However, the early hope for these studies-that only a small set of genes would be responsible for a given disorder-proved false. The picture that has emerged is far more complex: a given disorder may be influenced by myriad coding and noncoding variants of small effect size, and/or by rare but severe variants of large effect size, many de novo.Noncoding genomic sequences harbor a large portion of these variants, the molecular functions of which cannot usually be inferred from sequence alone. This creates a substantial barrier to understanding the higher-order molecular and biological systems underlying disease risk. Fortunately, a proliferation of genetic technologies-namely, scalable oligonucleotide synthesis, high-throughput RNA sequencing, CRISPR, and CRISPR derivatives-have opened novel avenues to experimentally identify biologically significant variants en masse. These advances have yielded an especially versatile technique adaptable to large-scale functional assays of variation in both untranscribed and untranslated regulatory features: Massively Parallel Reporter Assays (MPRAs). MPRAs are powerful molecular genetic tools that can be used to screen tens of thousands of predefined sequences for functional effects in a single experiment. This approach has several ideal features for psychiatric genetics, but remains underutilized in the field to date. To emphasize the opportunities MPRA holds for dissecting psychiatric polygenicity, we review here its applications in the literature, discuss its ability to test several biological variables implicated in psychiatric disorders, illustrate this flexibility with a proof-of-principle, in vivo cell-type specific implementation of the assay, and envision future outcomes of applying MPRA to both computational and experimental neurogenetics.

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“…Recent studies by multiple groups, using a variety of approaches, indicate that regulation by miRNAs is only a modest fraction of the regulatory information found in mammalian 3′UTRs [75][76][77][78][79]. While our method has been applied primarily to the characterization of selective pressures acting on miRNAs, we also analyzed selective pressures operating on all possible 8mer motifs, and successfully recovered motifs corresponding to RNA binding proteins with regulatory functions.…”

Section: Identifying and Characterizing Selective Pressures Acting Onmentioning

confidence: 99%

Evolutionary Dynamics of microRNA target sites across vertebrate evolution

Simkin

Geißler

McIntyre

et al. 2019

Preprint

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MicroRNAs (miRNAs) control the abundance of the majority of the vertebrate transcriptome. The recognition sequences, or target sites, for bilaterian miRNAs are found predominantly in the 3′ untranslated regions (3′UTRs) of mRNAs, and are amongst the most highly conserved motifs within 3′UTRs. However, little is known regarding the evolutionary pressures that lead to loss and gain of such target sites. Here, we quantify the selective pressures that act upon miRNA target sites.Notably, selective pressure extends beyond deeply conserved binding sites to those that have undergone recent substitutions. Our approach reveals that even amongst ancient animal miRNAs, which exert the strongest selective pressures on 3′UTR sequences, there are striking differences in patterns of target site evolution between miRNAs. Considering only ancient animal miRNAs, we find three distinct miRNA groups, each exhibiting characteristic rates of target site gain and loss during mammalian evolution. The first group both loses and gains sites rarely. The second group shows selection only against site loss, with site gains occurring at a neutral rate, whereas the third loses and gains sites at neutral or above expected rates. Furthermore, mutations that alter strength of existing target sites are disfavored. Applying our approach to individual transcripts reveals variation in the distribution of selective pressure across the transcriptome and between miRNAs, ranging from strong selection acting on a small subset of targets of some miRNAs, to weak selection on many targets for other miRNAs. miR-20 and miR-30, and many other miRNAs, exhibit broad, deeply conserved targeting, while several other comparably ancient miRNAs show a lack of selective constraint, and a small number, including mir-146, exhibit striking evidence of rapidly evolving target sites. Our approach adds valuable perspective on the evolution of miRNAs and their targets, and can also be applied to characterize other 3′UTR regulatory motifs. AUTHOR SUMMARYGene regulation typically involves two components; the first is a regulator, and the second is a target that the regulator acts on. Examining the evolution of both components allows us to make inferences regarding how gene regulation originated and how it is changing over time. One gene regulatory system, which is widespread in mammals and other animals, relies upon regulators known as microRNAs (miRNAs), which recognize target sites within mRNAs, leading to posttranscriptional repression of the targeted mRNAs. This system has been studied extensively from the perspective of the evolution of the miRNA regulators. Moreover, a subset of the target sites themselves are known to be deeply conserved. Here, we have systematically examined the rate at which target sites for individual miRNAs are created and destroyed across mammalian evolution.We find that mutations that strengthen or weaken existing target sites are strongly disfavored. For ancient microRNAs, even recently evolved target sites are under strong selective constraint, a...

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

Cited by 38 publications

References 71 publications

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

Evolutionary Dynamics of microRNA target sites across vertebrate evolution

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