Hexanucleotide Element Directs MicroRNA Nuclear Import Hwang, Hun-Way; Wentzel, Erik A; Mendell, Joshua T
Science (American Association for the Advancement of Science),
01/2007, Volume:
315, Issue:
5808
Journal Article
Peer reviewed
MicroRNAs (miRNAs) negatively regulate partially complementary target messenger RNAs. Target selection in animals is dictated primarily by sequences at the miRNA 5' end. We demonstrated that despite ...their small size, specific miRNAs contain additional sequence elements that control their posttranscriptional behavior, including their subcellular localization. We showed that human miR-29b, in contrast to other studied animal miRNAs, is predominantly localized to the nucleus. The distinctive hexanucleotide terminal motif of miR-29b acts as a transferable nuclear localization element that directs nuclear enrichment of miRNAs or small interfering RNAs to which it is attached. Our results indicate that miRNAs sharing common 5' sequences, considered to be largely redundant, might have distinct functions because of the influence of cis-acting regulatory motifs.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Cross-linking and immunoprecipitation (CLIP) ...serves this purpose by exploiting covalent protein-RNA cross-linking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare complementary DNA (cDNA) libraries for sequencing. Here we describe the core steps of CLIP, its primary variations, and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice and discuss the mechanistic and physiologic insights that have already been gained from studies using CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms while providing important biologic and clinically relevant insights.
Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior ...purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.
•cTag-PAPERCLIP generates in vivo profiles of cell-type-specific polyadenylated mRNAs•cTag-PAPERCLIP preserves tissue integrity, capturing mRNAs in the cell’s native state•Distinct APA preference contributes to protein diversity between brain cell types•Activated brain microglia upregulate full-length kinase-active ARAF through APA
Hwang et al. develop cTag-PAPERCLIP to profile mRNA 3′ ends in individual cell types from intact tissues in vivo. They use cTag-PAPERCLIP to show that mRNA alternative polyadenylation contributes to protein diversity between different cell types and cellular states within the brain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Alternative polyadenylation (APA) generates mRNA isoforms and diversifies gene expression. Here we report the discovery that the mTORC1 signaling pathway balances the expression of two
Trim9/TRIM9
...isoforms through APA regulation in human and mouse. We showed that CFIm components, CPSF6 and NUDT21, promote the short
Trim9/TRIM9
isoform (
Trim9-S/TRIM9-S
) expression. In addition, we identified an evolutionarily conserved twin UGUA motif, UGUAYUGUA, in
TRIM9-S
polyadenylation site (PAS) that is critical for its regulation by CPSF6. We found additional CPSF6-regulated PASs with similar twin UGUA motifs in human and experimentally validated the twin UGUA motif functionality in
BMPR1B
,
MOB4
, and
BRD4-L
. Importantly, we showed that inserting a twin UGUA motif into a heterologous PAS was sufficient to confer regulation by CPSF6 and mTORC1. Our study reveals an evolutionarily conserved mechanism to regulate gene isoform expression by mTORC1 and implicates possible gene isoform imbalance in cancer and neurological disorders with mTORC1 pathway dysregulation.
Alternative polyadenylation (APA), an RNA processing mechanism that results in mRNA with distinct 3’ termini, is a rapidly expanding area of research that in recent studies has been linked to the ...mechanistic target of rapamycin (mTOR) signaling pathway, a key regulatory pathway in physiology and metabolism. Despite the recent implications of APA in mTOR signaling, the mechanistic link between mTOR signaling and APA remains poorly understood. We previously leveraged our cTag‐PAPERCLIP technique to generate a dataset of in vivo APA shifts following neuronal mTOR induction and identified TRIM9, an E3 ubiquitin ligase with a role in neurodevelopment, as a gene of interest. In this study, we further characterized the regulation of the mTOR‐induced TRIM9 APA shift observed in mouse neurons in vivo. Further study of the regulation of TRIM9 APA by the core protein complexes of the cleavage and polyadenylation (CP) machinery revealed CSPF6, a component of the CFIm complex, as essential for physiological regulation of TRIM9 isoforms, with loss of CPSF6 leading to an enrichment of the distal TRIM9 isoform. Additional study into the 3’UTR sequence elements of TRIM9 isoforms revealed multiple UGUA sequence motifs, the binding sequence element of the CFIm complex, upstream of the TRIM9 proximal polyA site (PAS). In order to identify the key sequence elements essential for CPSF6‐mediated regulation of the proximal TRIM9 PAS, we developed a RT‐qPCR PAS competition assay to quantify sequence‐mediated usage of PASs. Utilizing this assay, we assessed usage of the TRIM9 proximal PAS in both the absence and the presence of CPSF6. Additionally, we generated constructs containing mutated UGUA sequences in order to ascertain the importance of the UGUA motif to TRIM9 proximal PAS usage. We found that loss of CPSF6 leads to reduced usage of the TRIM9 proximal PAS. Furthermore, mutation of a twin UGUA motif (UGUACUGUA) lead to a reduction in TRIM9 proximal PAS usage. Our results demonstrate a direct role of CPSF6 and identify a key cis‐acting motif in promoting TRIM9 proximal PAS usage. Furthermore, our results also suggest a possible link between neurological disorders with mTOR pathway dysregulation (“mTORopathies”) and neurodevelopment through TRIM9.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Direct control of microRNA (miRNA) expression by oncogenic and tumor suppressor networks results in frequent dysregulation of miRNAs in cancer cells and contributes to tumorigenesis. We previously ...demonstrated that activation of the c-Myc oncogenic transcription factor (Myc) broadly influences miRNA expression and in particular leads to widespread miRNA down-regulation. miRNA transcripts repressed by Myc include several with potent tumor suppressor activity such as miR-15a/16-1, miR-34a, and let-7 family members. In this study, we have investigated mechanisms downstream of Myc that contribute to miRNA repression. Consistent with transcriptional down-regulation, Myc activity results in the decreased abundance of multiple miRNA primary transcripts. Surprisingly, however, primary transcripts encoding several let-7 miRNAs are not reduced in the high Myc state, suggesting a posttranscriptional mechanism of repression. The Lin-28 and Lin-28B RNA binding proteins were recently demonstrated to negatively regulate let-7 biogenesis. We now show that Myc induces Lin-28B expression in multiple human and mouse tumor models. Chromatin immunoprecipitation and reporter assays reveal direct association of Myc with the Lin-28B promoter resulting in transcriptional transactivation. Moreover, we document that activation of Lin-28B is necessary and sufficient for Myc-mediated let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation. These findings highlight an important role for Lin-28B in Myc-driven cellular phenotypes and uncover an orchestration of transcriptional and posttranscriptional mechanisms in Myc-mediated reprogramming of miRNA expression.
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Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we ...investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in ...oncogenesis; however, its effects on cancer progression remain poorly understood. Here, to address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed dedicated regression-based methods to analyse ribosome profiling and alternative polyadenylation data, and identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as a translational controller of a specific mRNA regulon. We found that HNRNPC is downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3' untranslated region lengthening and, subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. In addition, the reduced expression of HNRNPC and its regulon is associated with the worse prognosis in breast cancer patient cohorts.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
MicroRNAs (miRNAs) are 18- to 24-nt RNA molecules that regulate messenger RNAs (mRNAs). Posttranscriptional mechanisms regulate miRNA abundance during development as well as in cancer cells where ...miRNAs frequently exhibit dysregulated expression. The molecular mechanisms that govern the global efficiency of miRNA biogenesis in these settings remain incompletely understood, and experimental systems for the biochemical dissection of these pathways are currently lacking. Here, we demonstrate that miRNAs are subject to dynamic posttranscriptional regulation in widely used cell culture systems. As diverse mammalian and Drosophila cell lines are grown to increasing density, miRNA biogenesis is globally activated, leading to elevated mature miRNA levels and stronger repression of target constructs. This broad increase in miRNA abundance is associated with enhanced processing of miRNAs by Drosha and more efficient formation of RNA-induced silencing complexes. These findings uncover a critical parameter necessary for accurate analysis of miRNAs in cell culture settings, establish a tractable system for the study of regulated miRNA biogenesis, and may provide insight into mechanisms that influence miRNA expression in physiologic and pathophysiologic states.
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Accurate and precise annotation of 3′ UTRs is critical for understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here, we describe a method, poly(A) binding ...protein-mediated mRNA 3′ end retrieval by crosslinking immunoprecipitation (PAPERCLIP), that shows high specificity for mRNA 3′ ends and compares favorably with existing 3′ end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of which contain a downstream GUKKU motif. Furthermore, in the mouse brain, PAPERCLIP discovers extended 3′ UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts, including one in Atp2b2 that is evolutionarily conserved in humans and results in the gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo.
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•PAPERCLIP is a high-performance mRNA 3′ end mapping method based on the CLIP technique•PAPERCLIP provides insights into the regulation of alternative polyadenylation•PAPERCLIP expands knowledge of 3′-UTR-mediated mRNA regulation
Hwang et al. develop PAPERCLIP, a high-performance mRNA 3′ end mapping method based on the CLIP technique. They use PAPERCLIP to show that CstF64/64tau promotes non-canonical poly(A) site usage through a GUKKU motif. Furthermore, they also discover shifts in alternative polyadenylation and identify novel microRNA binding sites during mouse brain development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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