Post-transcriptional Regulatory Programs Research Articles

RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex.

In the neocortex, functionally distinct areas process specific types of information. Area identity is established by morphogens and transcriptional master regulators, but downstream mechanisms driving area-specific neuronal specification remain unclear. Here, we reveal a role for RNA-binding proteins in defining area-specific cytoarchitecture. Mice lacking Pum2 or overexpressing human TDP-43 show apparent 'motorization' of layers IV and V of primary somatosensory cortex (S1), characterized by dramatic expansion of cells co-expressing Sox5 and Bcl11b/Ctip2, a hallmark of subcerebral projection neurons, at the expense of cells expressing the layer IV neuronal marker Rorβ. Moreover, retrograde labeling experiments with cholera toxin B in Pum2; Emx1-Cre and TDP43A315T mice revealed a corresponding increase in subcerebral connectivity of these neurons in S1. Intriguingly, other key features of somatosensory area identity are largely preserved, suggesting that Pum2 and TDP-43 may function in a downstream program, rather than controlling area identity per se. Transfection of primary neurons and in utero electroporation (IUE) suggest cell-autonomous and post-mitotic modulation of Sox5, Bcl11b/Ctip2, and Rorβ levels. Mechanistically, we find that Pum2 and TDP-43 directly interact with and affect the translation of mRNAs encoding Sox5, Bcl11b/Ctip2, and Rorβ. In contrast, effects on the levels of these mRNAs were not detectable in qRT-PCR or single-molecule fluorescent in situ hybridization assays, and we also did not detect effects on their splicing or polyadenylation patterns. Our results support the notion that post-transcriptional regulatory programs involving translational regulation and mediated by Pum2 and TDP-43 contribute to elaboration of area-specific neuronal identity and connectivity in the neocortex.

APC regulation of ESRP1 and p120-catenin isoforms in colorectal cancer cells.

The adenomatous polyposis coli (APC) tumor suppressor protein is associated with the regulation of Wnt signaling; however, APC also controls other cellular processes including the regulation of cell adhesion and migration. The expression of full-length APC in SW480 colorectal cancer cells (SW480+APC) not only reduces Wnt signaling, but increases membrane E-cadherin and restores cell–cell adhesion. This report describes the effects of full-length, wild-type APC (fl-APC) on cell–cell adhesion genes and p120-catenin isoform switching in SW480 colon cancer cells: fl-APC increased the expression of genes implicated in cell–cell adhesion, whereas the expression of negative regulators of E-cadherin was decreased. Analysis of cell–cell adhesion-related proteins in SW480+APC cells revealed an increase in p120-catenin isoform 3A; similarly, depletion of APC altered the p120-catenin protein isoform profile. Expression of ESRP1 (epithelial splice regulatory protein 1) is increased in SW480+APC cells, and its depletion results in reversion to the p120-catenin isoform 1A phenotype and reduced cell–cell adhesion. The ESRP1 transcript is reduced in primary colorectal cancer, and its expression correlates with the level of APC. Pyrvinium pamoate, which inhibits Wnt signaling, promotes ESRP1 expression. We conclude that re-expression of APC restores the cell–cell adhesion gene and posttranscriptional regulatory programs leading to p120-catenin isoform switching and associated changes in cell–cell adhesion.

A multi-layer post-transcriptional gene regulatory program fuels cancer angiogenesis and metastasis

Metastasis is the process by which cancer cells spread from the primary tumor to surrounding tissues and to distant organs. It is the primary cause of cancer morbidity and mortality, and represent a major clinical problem, where it is estimated to account for ~90% of cancer deaths (1). It has been suggested that metastasis can be represented as a biphasic process, starting with the physical translocation of cancer cells to a distant organ, followed by their capacity to spread, invade, and thrive in non-native environments (1). Several hypotheses have been proposed to describe the cellular processes involved in cancer metastasis such as epithelial mesenchymal transition, accumulation of mutations, macrophage facilitation process, and macrophage transformation or fusion hybridization with neoplastic cells. However, metastasis process remains one of the most inexplicable aspects of cancer at the molecular level (1). Therefore, finding the right molecular targets for therapeutic intervention is crucial for prevention and treatment success.

RNA editing alterations in a multi-ethnic Alzheimer disease cohort converge on immune and endocytic molecular pathways.

Little is known about the post-transcriptional mechanisms that modulate the genetic effects in the molecular pathways underlying Alzheimer disease (AD), and even less is known about how these changes might differ across diverse populations. RNA editing, the process that alters individual bases of RNA, may contribute to AD pathogenesis due to its roles in neuronal development and immune regulation. Here, we pursued one of the first transcriptome-wide RNA editing studies in AD by examining RNA sequencing data from individuals of both African-American (AA) and non-Hispanic White (NHW) ethnicities. Whole transcriptome RNA sequencing and RNA editing analysis were performed on peripheral blood specimens from 216 AD cases (105 AA, 111 NHW) and 212 gender matched controls (105 AA, 107 NHW). 449 positions in 254 genes and 723 positions in 371 genes were differentially edited in AA and NHW, respectively. While most differentially edited sites localized to different genes in AA and NHW populations, these events converged on the same pathways across both ethnicities, especially endocytic and inflammatory response pathways. Furthermore, these differentially edited sites were preferentially predicted to disrupt miRNA binding and induce nonsynonymous coding changes in genes previously associated with AD in molecular studies, including PAFAH1B2 and HNRNPA1. These findings suggest RNA editing is an important post-transcriptional regulatory program in AD pathogenesis.

Emerging roles of RNA-binding proteins in diabetes and their therapeutic potential in diabetic complications.

Diabetes is a debilitating health care problem affecting 422 million people around the world. Diabetic patients suffer from multisystemic complications that can cause mortality and morbidity. Recent advancements in high-throughput next-generation RNA-sequencing and computational algorithms led to the discovery of aberrant posttranscriptional gene regulatory programs in diabetes. However, very little is known about how these regulatory programs are mis-regulated in diabetes. RNA-binding proteins (RBPs) are important regulators of posttranscriptional RNA networks, which are also dysregulated in diabetes. Human genetic studies provide new evidence that polymorphisms and mutations in RBPs are linked to diabetes. Therefore, we will discuss the emerging roles of RBPs in abnormal posttranscriptional gene expression in diabetes. Questions that will be addressed are: Which posttranscriptional mechanisms are disrupted in diabetes? Which RBPs are responsible for such changes under diabetic conditions? How are RBPs altered in diabetes? How does dysregulation of RBPs contribute to diabetes? Can we target RBPs using RNA-based methods to restore gene expression profiles in diabetic patients? Studying the evolving roles of RBPs in diabetes is critical not only for a comprehensive understanding of diabetes pathogenesis but also to design RNA-based therapeutic approaches for diabetic complications. WIREs RNA 2018, 9:e1459. doi: 10.1002/wrna.1459 This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing Translation > Translation Regulation.

An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression

Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.

Tail characteristics of Trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner

The intricate life cycle of Trypanosoma brucei requires extensive regulation of gene expression levels of the mtRNAs for adaptation. Post-transcriptional gene regulatory programs, including unencoded mtRNA 3′ tail additions, potentially play major roles in this adaptation process. Intriguingly, T. brucei mitochondrial transcripts possess two distinct unencoded 3′ tails, each with a differing functional role; i.e., while one type is implicated in RNA stability (in-tails), the other type appears associated with translation (ex-tails). We examined the degree to which tail characteristics differ among cytochrome c oxidase subunits I and III (CO1 and CO3), and NADH dehydrogenase subunit 1 (ND1) transcripts, and to what extent these characteristics differ developmentally. We found that CO1, CO3 and ND1 transcripts possess longer in-tails in the mammalian life stage. By mathematically modelling states of in-tail and ex-tail addition, we determined that the typical length at which an in-tail is extended to become an ex-tail differs by transcript and, in the case of ND1, by life stage. To the best of our knowledge, we provide the first evidence that developmental differences exist in tail length distributions of mtRNAs, underscoring the potential involvement of in-tail and ex-tail populations in mitochondrial post-transcriptional regulation mechanisms.

Abstract A34: CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells

Abstract The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3' untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3' UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3' UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3' UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF1's regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present an eleven component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Shebna Cheema, Joseph M. Fachini, Natee Kongchan, Guojun Lu, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A34.

Post-transcriptional Regulation of BRCA2 through Interactions with miR-19a and miR-19b

Breast cancer type 2, early onset susceptibility gene (BRCA2) is a major component of the homologous recombination DNA repair pathway. It acts as a tumor suppressor whose function is often lost in cancers. Patients with specific mutations in the BRCA2 gene often display discrete clinical, histopathological, and molecular features. However, a subset of sporadic cancers has wild type BRCA2 and display defects in the homology-directed repair pathway, which is the hallmark of ‘BRCAness.’ The mechanisms by which BRCAness arises are not well understood but post-transcriptional regulation of BRCA2 gene expression by microRNAs (miRNAs) may contribute to this phenotype. Here, we examine the post-transcriptional effects that some members of the six-miRNA cluster known as the miR-17/92 cluster have on the abundance of BRCA2’s messenger RNA (mRNA) and protein. We discuss two interactions involving the miR-19a and miR-19b members of the cluster and the 3′UTR of BRCA2’s mRNA. We investigated these miRNA:mRNA interactions in 15 cell lines derived from pancreatic, breast, colon, and kidney tissue. We show that over-expression of these two miRNAs results in a concomitant decrease of BRCA2’s mRNA and protein expression in a subset of the tested cell lines. Additionally, using luciferase reporter assays we identified direct interactions between miR-19a/miR-19b and a miRNA response element (MRE) in BRCA2’s 3′UTR. Our results suggest that BRCA2 is subject to a complex post-transcriptional regulatory program that has specific dependencies on the genetic and phenotypic background of cell types.

Abstract B58: CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells

Abstract The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3′ untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3′ UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3′ UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3′ UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF1's regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present a distinctive model of gene regulation, invisible to traditional transcriptional profiling, in which the CELF1 protein serves as a central node controlling translational activation of the EMT program and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Natee Kongchan, Joseph M. Fachini, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B58.

Abstract A46: Tissue biology is shaped by noncoding RNAs that depend on gender, population, and race

Abstract Transcriptome profiling using next generation sequencing has revealed the existence of many RNA species that do not code for proteins and are differentially abundant between normal and tumor. Despite great advances in elucidating their roles in the cell, the vast majority of these non-coding RNAs (ncRNAs) remain to be functionalized. Through several recent studies, we found that the populations of transcriptional products from many human ncRNA loci are far more dynamic than currently believed. Specifically, we have discovered that the human genome loci that harbor microRNAs (mIRNAs) and transfer RNAs (tRNAs) respectively produce a multitude of constitutive, previously unsuspected transcripts that differ between normal and tumor and whose composition and abundance depend on several variables that include a patient's gender, population, and race. Such patient attributes will thus need to be considered explicitly in studies of post-transcriptional regulatory programs in cancer. The conventional view holds that miRNA precursors produce at most one consequential mature miRNA from each arm of the precursor hairpin. In the public repositories, each of the known genomic miRNA loci is represented by at most two such sequences, one per arm. We refer to this representative product of a hairpin arm as the arm's “archetype” miRNA. The increasing numbers of transcriptomic datasets have uncovered a different reality wherein each miRNA precursor arm produces multiple isoforms (the “isomiRs”) of the archetype miRNA. These isomiRs enter the RNA interference pathway just like the archetype miRNA sequences and have sequence-dependent downstream regulatory effects. By analyzing transcriptomic data from the 1,000 Genome Project (1KG) and from The Cancer Genome Atlas (TCGA), we found that the production of isomIRs from a miRNA locus is marshaled and depends on a person's gender, population, and race. IsomiR production from a miRNA locus also depends on the tissue under consideration and on disease type/subtype. Moreover, co-expressed isomiRs from the same miRNA locus are generally not well-correlated. Lastly, in a cancer model cell line, we over-expressed isoforms of a miRNA with race-depended abundance in TCGA data: we found that these isoforms had targetomes that had little overlap even though they originated from the same miRNA locus. tRNAs have long been considered to serve as adaptor molecules with well-defined roles in the translation of messenger RNA (mRNA) into amino acid sequences. In recent years, this view is starting to change. Key to the renewed attention to tRNAs has been the discovery of tRNA fragments (tRFs), which are produced in parallel to the mature tRNAs that are used in translation. Increasing numbers of reports have been linking tRFs to cell growth, cell proliferation, response to DNA damage, translation initiation, response to stress, etc. and have shown that some tRFs are loaded on Argonaute and thus enter the RNAi pathway. Our analyses of data from the 1KG and the TCGA projects have revealed that nuclear and mitochondrial tRNAs produce a multitude of tRFs with variable, yet quantized, lengths. We found that, in complete analogy to isomiRs, the tRFs are constitutive in nature, exhibit composition and abundance that differ across tissues and between normal and tumor samples, and depend on a person's gender, population, and race. We also discovered a novel and very rich category of tRFs, the internal-tRFs or i-tRFs: i-tRFs have variable quantized lengths, and variable starting and ending points that are wholly contained within the span of the mature tRNA. Of note, i-tRFs contribute much of the difference that we observe across the gender, population, race, tissue, and disease subtype boundaries. Lastly, using fragment-specific approaches, we were able to validate several i-tRFs in clinical samples and in model cell lines. Citation Format: Isidore Rigoutsos, Aristeidis Telonis, Phillipe Loher, Eric Londin. Tissue biology is shaped by noncoding RNAs that depend on gender, population, and race. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A46.

Distal Alternative Last Exons Localize mRNAs to Neural Projections

Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer.

Systematic Identification of Regulatory Elements in Conserved 3′ UTRs of Human Transcripts

Posttranscriptional regulatory programs governing diverse aspects of RNA biology remain largely uncharacterized. Understanding the functional roles of RNA cis-regulatory elements is essential for decoding complex programs that underlie the dynamic regulation of transcript stability, splicing, localization, and translation. Here, we describe a combined experimental/computational technology to reveal a catalog of functional regulatory elements embedded in 3' UTRs of human transcripts. We used a bidirectional reporter system coupled with flow cytometry and high-throughput sequencing to measure the effect of short, noncoding, vertebrate-conserved RNA sequences on transcript stability and translation. Information-theoretic motif analysis of the resulting sequence-to-gene-expression mapping revealed linear and structural RNA cis-regulatory elements that positively and negatively modulate the posttranscriptional fates of human transcripts. This combined experimental/computational strategy can be used to systematically characterize the vast landscape of posttranscriptional regulatory elements controlling physiological and pathological cellular state transitions.

Abstract 19105: β2 Integrin-Dependent HuR-Mediated mRNA Stabilization in Macrophages is Required for Lymphatic Vessel Formation in Inflammatory Lymphangiogenesis

Macrophages play a central role in the response to acute and chronic tissue injuries including infection, ischemia, and atherosclerosis, where their effector functions in injured tissue include cytokine, growth factor, and matrix remodeling factor production. β2 integrin engagement promotes stabilization of labile mRNAs bearing adenylate-uridylate-rich elements (AREs) by inducing the rapid nuclear-to-cytoplasmic translocation of the RNA binding protein HuR. The secretory role of macrophages in lymphangiogenesis, together with predicted HuR binding sites in the VEGF-C 3’-UTR, led us to hypothesize that HuR regulates VEGF-C production in macrophages in response to integrin engagement. In bone marrow-derived macrophages from mice harboring a myeloid-specific deletion of HuR, we observed that VEGF-C production is impaired due to an increase in the rate of VEGF-C mRNA decay. A conserved ARE was the degradation target and was necessary to mediate HuR-dependent transcript stability that in turn resulted in increased VEGF-C protein. The HuR knockout mice exhibit an abnormal lymphangiogenic response to intraperitoneal LPS injection despite intact macrophage recruitment. Preliminary data indicates a functional interaction between the mRNA stabilizing effect of HuR and the destabilizing effect of microRNA recognition sequences in the 3’-UTR of VEGF-C and that of other integrin-induced lymphangiogenic gene products including cytokines and matrix remodeling proteins. VEGF-C likely acts in concert with these other factors to help guide vessel formation at sites of tissue injury. In conclusion, β2 integrin signaling through HuR promotes VEGF-C production as a central component of a post-transcriptional gene regulatory program essential for macrophage-directed lymphangiogenesis, a finding with implications for processes including tissue ischemia, wound repair, extracellular fluid trafficking, and atherosclerosis.

Lineage- and Developmental Stage-Specific Patterns Of Auf-1 Isoform Expression Contribute To The Regulation Of Erythroid-Specific mRNAs

Post-transcriptional events that regulate the stabilities of one or more specific mRNAs are increasingly recognized for their importance to cell development and differentiation: genome-wide analyses attribute ∼50% of changes in gene expression to alterations in the stabilities of their encoded mRNAs. Processes that differentially regulate the half-lives of mRNAs are particularly important in definitive erythropoiesis, as they control the relative levels of actively translating transcripts in the interval when the nucleus is transcriptionally silenced and is ultimately extruded. We recently identified AUF-1 (AU-binding factor 1; hnRNP D) as a trans-acting factor that stabilizes human β-globin mRNA, and noted previous reports that this RNA-binding protein also regulates the half-lives of other mRNAs encoding factors that are critical to normal erythropoiesis, including p16INK4a (CDKN2A), p21 (CDKN1A), cyclin D1 (CCND1), and thymidylate synthase. Based upon this evidence, we posited that AUF-1 plays a role in post-transcriptional erythropoiesis that is far broader than previously imagined. Our analyses of umbilical cord blood CD34+ cells validated this expectation, demonstrating the presence of two AUF-1 isoforms (p45AUF1 and p40AUF1) that differ by the inclusion/exclusion of exon 7-encoded sequence. Cells that are induced to erythroid differentiation initially express p45AUF1 and p40AUF1 at equal levels, but shift after several days to express only the p40AUF1 isoform. In contrast, cells that are induced to granulocyte-monocyte differentiation initially express only p45AUF1, and continue to express this single isoform through the remainder of development. These observations demonstrate lineage-specific AUF-1 isoform expression that is likely to be important to both erythroid and non-erythroid developmental programs. We subsequently investigated the functional consequences of the erythropoietic AUF-1 isoform switch by analyzing the abilities of p45AUF1 and p40AUF1 to stabilize informative erythroid-specific and -restricted mRNAs. Using an AUF-1 isoform-specific siRNA knockdown strategy, we demonstrated that a reduction in p40AUF-1 (but not p45AUF-1) effected a two-fold decrease in β-globin mRNA in K562 cells that we engineered to express this gene at high levels. p45AUF1 and p40AUF1 knockdown had a different effect on mRNAs encoding cyclin D1 and p21, both of which were unaffected by single-isoform depletion but--in contrast to β-globin mRNA--were increased by coordinate knockdown of both p45AUF1 and p40AUF1. While we expected that the erythropoietic AUF-1 isoform switch was likely due to alternative splicing of exon 7 from the AUF-1 pre-mRNA, we observed instead that the switch results from the transfer of p40AUF1 from the nucleus to the cytoplasm during the developmental interval that immediately precedes nuclear extrusion. This observation implicates developmentally regulated trafficking of p40AUF1 during erythropoiesis that is consistent with its demonstrated post-transcriptional mRNA-stabilizing effects. Finally, we assessed expression of HuR--a competitive antagonist of AUF-1 that has been implicated in stabilizing the mRNAs encoding FasL and cyclin E1 (CCNE1)--in erythroid-induced CD34+ cells, and observed that levels of HuR mRNA increase significantly during the developmental interval that coincides with the erythropoietic p45:p40AUF1 isoform switch. Collectively, the lineage- and developmental-stage specific expression of p45AUF1, p40AUF1, and HuR--combined with their mRNA-specific binding properties--illustrate both the existence and the complexity of post-transcriptional regulatory programs that contribute to the normal development of both erythroid and nonerythroid cells. We are currently conducting RNA-seq analyses in erythroid-differentiated CD34+ cells to identify transcripts that differentially bind p45AUF1, p40AUF1, and HuR; as well as factor-specific shRNA knock-down analyses to characterize corresponding functional effects. In sum, our present work describes a novel mechanism through which erythroid progenitors maintain dynamic regulatory control during an interval when transcriptional processes are silenced. Disclosures:No relevant conflicts of interest to declare.

Global identification of conserved post-transcriptional regulatory programs in trypanosomatids.

While regulatory programs are extensively studied at the level of transcription, elements that are involved in regulation of post-transcriptional processes are largely unknown, and methods for systematic identification of these elements are in early stages. Here, using a novel computational framework, we have integrated sequence information with several functional genomics data sets to characterize conserved regulatory programs of trypanosomatids, a group of eukaryotes that almost entirely rely on post-transcriptional processes for regulation of mRNA abundance. This analysis revealed a complex network of linear and structural RNA elements that potentially govern mRNA abundance across different life stages and environmental conditions. Furthermore, we show that the conserved regulatory network that we have identified is responsive to chemical perturbation of several biological functions in trypanosomatids. We have further characterized one of the most abundant regulatory RNA elements that we discovered, an AU-rich element (ARE) that can be found in 3′ untranslated region of many trypanosomatid genes. Using bioinformatics approaches as well as in vitro and in vivo experiments, we have identified three ELAV-like homologs, including the developmentally critical protein TbRBP6, which regulate abundance of a large number of trypanosomatid ARE-containing transcripts. Together, these studies lay out a roadmap for characterization of mechanisms that modulate development and metabolic pathways in trypanosomatids.

Systematic discovery of structural elements governing stability of mammalian messenger RNAs

Decoding post-transcriptional regulatory programs in RNA is a critical step in the larger goal to develop predictive dynamical models of cellular behavior. Despite recent efforts1–3, the vast landscape of RNA regulatory elements remain largely uncharacterized. A longstanding obstacle is the contribution of local RNA secondary structure in defining interaction partners in a variety of regulatory contexts, including but not limited to transcript stability3, alternative splicing4 and localization3. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (e.g. human cardiac troponin T) or affects other aspects of RNA biology5. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence3,6. We have developed a computational framework based on context-free grammars3,7 and mutual information2 that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behavior. The application of this framework to genome-wide mammalian mRNA stability data revealed eight highly significant elements with substantial structural information, for the strongest of which we showed a major role in global mRNA regulation. Through biochemistry, mass-spectrometry, and in vivo binding studies, we identified HNRPA2B1 as the key regulator that binds this element and stabilizes a large number of its target genes. Ultimately, we created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach can also be employed to reveal the structural elements that modulate other aspects of RNA behavior.

The Ins and Outs of the Epithelial to Mesenchymal Transition in Health and Disease

The epithelial to mesenchymal transition (EMT) converts epithelial cells into migratory and invasive cells and is a fundamental event in morphogenesis. Although its relevance in the progression of cancer and organ fibrosis had been debated until recently, the EMT is now established as an important step in the metastatic cascade of epithelial tumors. The similarities between pathological and developmental EMTs validate the embryo as the best model to understand the molecular and cellular mechanisms involved in this process, identifying those that are hijacked during the progression of cancer and organ degeneration. Our ever-increasing understanding of how transcription factors regulate the EMT has revealed complex regulatory loops coupled to posttranscriptional and epigenetic regulatory programs. The EMT is now integrated into the systemic activities of whole organisms, establishing links with cell survival, stemness, inflammation, and immunity. In addition, the EMT now constitutes a promising target for the treatment of cancer and organ-degenerative diseases.

Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome

Post-transcriptional regulation of gene expression, including mRNA localization, translation and decay, is ubiquitous yet still largely unexplored. How is the post-transcriptional regulatory program of each mRNA encoded in its sequence? Hundreds of specific RNA-binding proteins (RBPs) appear to play roles in mediating the post-transcriptional regulatory program, akin to the roles of specific DNA-binding proteins in transcription. As a step toward decoding the regulatory programs encoded in each mRNA, we focused on specific mRNA–protein interactions. We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements. Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3. Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA–protein interactions are evolutionarily conserved.