Nature Structural & Molecular Biology 20, 543 (2013). doi:10.1038/nsmb.2582
Author: Elisa Izaurralde
A recent study on the mechanism of microRNA–mediated gene silencing suggests that microRNA–induced silencing complexes inhibit ribosome scanning by recruiting the DEAD-box RNA helicase eIF4AII through an interaction with the NOT1 subunit of the CCR4–NOT deadenylase complex.
Nature advance online publication 05 May 2013. doi:10.1038/nature12108
Authors: Heidi Dvinge, Anna Git, Stefan Gräf, Mali Salmon-Divon, Christina Curtis, Andrea Sottoriva, Yongjun Zhao, Martin Hirst, Javier Armisen, Eric A. Miska, Suet-Feung Chin, Elena Provenzano, Gulisa Turashvili, Andrew Green, Ian Ellis, Sam Aparicio & Carlos Caldas
MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA–mRNA interactions rather than as on–off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.
The EMBO Journal. doi:10.1038/emboj.2013.99
Authors: Ken-ichi Takayama, Kuniko Horie-Inoue, Shintaro Katayama, Takashi Suzuki, Shuichi Tsutsumi, Kazuhiro Ikeda, Tomohiko Urano, Tetsuya Fujimura, Kiyoshi Takagi, Satoru Takahashi, Yukio Homma, Yasuyoshi Ouchi, Hiroyuki Aburatani, Yoshihide Hayashizaki & Satoshi Inoue
Expression levels of microRNA machinery components Drosha, Dicer and DGCR8 in human (AGS, HepG2, and KEYSE-30) cancer cell lines.
Int J Clin Exp Med. 2013;6(4):269-74
Authors: Jafari N, Dogaheh HP, Bohlooli S, Oyong GG, Shirzad Z, Alibeiki F, Asl SH, Zargar SJ
Abstract
UNLABELLED: MicroRNAs (miRNAs) have recently been shown to play fundamental roles in diverse cellular processes and linked to variety of cancers. Dicer and Drosha are two major enzymes in the miRNA maturation process. DGCR8 is the assistant of Drosha in the microprocessor complex. In this study, we evaluated the mRNA expression profiles of major miRNA processing machinery Drosha, Dicer, and DGCR8 in human gastrointestinal (AGS, KYSE30 and HepG2) cancer cell lines.
MATERIALS AND METHODS: The cells were cultured and harvested, and total cellular RNA was isolated from cells. Then, first-strand cDNA was synthesized from the RNA of cells. Afterward, Quantitative analysis was performed by real-time RT-PCR using the PowerSYBR Green PCR Master Mix.
RESULTS: Expression levels of Drosha in AGS and HepG2 cells were higher than the controls, whereas, Drosha's expression level in KYSE-30 cell line was lower. The Dicer expression levels in AGS and HepG2 cells were higher, while, its expression level in KYSE-30 cell was lower. The DGCR8 expression levels in all three cell lines were significantly higher than the control samples.
CONCLUSION: Expression levels of the two most important enzymes of the miRNA machinery, Drosha and Dicer, and microprocessor complex component, DGCR8 were noticeably dysregulated when compared to healthy controls.
PMID: 23641303 [PubMed - in process]
Nature advance online publication 24 April 2013. doi:10.1038/nature12121
Authors: Vicent Pelechano, Wu Wei & Lars M. Steinmetz
Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation, or produce truncated proteins that differ in localization or function. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.
Nature advance online publication 01 May 2013. doi:10.1038/nature12080
Authors: Jia Shen, Weiya Xia, Yekaterina B. Khotskaya, Longfei Huo, Kotaro Nakanishi, Seung-Oe Lim, Yi Du, Yan Wang, Wei-Chao Chang, Chung-Hsuan Chen, Jennifer L. Hsu, Yun Wu, Yung Carmen Lam, Brian P. James, Xiuping Liu, Chang-Gong Liu, Dinshaw J. Patel & Mien-Chie Hung
MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.
One of the most important functional features of nuclear factor TDP-43 is its ability to bind UG-repeats with high efficiency. Several cross-linking and immunoprecipitation (CLIP) and RNA immunoprecipitation-sequencing (RIP-seq) analyses have indicated that TDP-43 in vivo can also specifically bind loosely conserved UG/GU-rich repeats interspersed by other nucleotides. These sequences are predominantly localized within long introns and in the 3'UTR of various genes. Most importantly, some of these sequences have been found to exist in the 3'UTR region of TDP-43 itself. In the TDP-43 3'UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop. In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches. We show that the binding affinity to the UG-like sequences has a dissociation constant (Kd) of ~110 nM compared with a Kd of 8 nM for straight UGs, and have mapped the region of contact between protein and RNA. In addition, our results indicate that the local concentration of UG dinucleotides in the CLIP sequences is one of the major factors influencing the interaction of these RNA sequences with TDP-43.
Enhancer elements are essential for tissue-specific gene regulation during mammalian development. Although these regulatory elements are often distant from their target genes, they affect gene expression by recruiting transcription factors to specific promoter regions. Because of this long-range action, the annotation of enhancer element–target promoter pairs remains elusive. Here, we developed a novel analysis methodology that takes advantage of Hi-C data to comprehensively identify these interactions throughout the human genome. To do this, we used a geometric distribution-based model to identify DNA–DNA interaction hotspots that contact gene promoters with high confidence. We observed that these promoter-interacting hotspots significantly overlap with known enhancer-associated histone modifications and DNase I hypersensitive sites. Thus, we defined thousands of candidate enhancer elements by incorporating these features, and found that they have a significant propensity to be bound by p300, an enhancer binding transcription factor. Furthermore, we revealed that their target genes are significantly bound by RNA Polymerase II and demonstrate tissue-specific expression. Finally, we uncovered that these elements are generally found within 1 Mb of their targets, and often regulate multiple genes. In total, our study presents a novel high-throughput workflow for confident, genome-wide discovery of enhancer–target promoter pairs, which will significantly improve our understanding of these regulatory interactions.
Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo. Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.
A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions. Drugs that trap these complexes have proven to be potent therapeutics in both cancer and infectious disease. Nonetheless, current assays for DNA–protein adducts are cumbersome, limiting both mechanistic studies and translational applications. We have developed a rapid and sensitive assay that enables quantitative immunodetection of protein–DNA adducts. This new ‘RADAR’ (rapid approach to DNA adduct recovery) assay accelerates processing time 4-fold, increases sample throughput 20-fold and requires 50-fold less starting material than the current standard. It can be used to detect topoisomerase 1-DNA adducts in as little as 60 ng of DNA, corresponding to 10 000 human cells. We apply the RADAR assay to demonstrate that expression of SLFN11 does not increase camptothecin sensitivity by promoting accumulation of topoisomerase 1-DNA adducts. The RADAR assay will be useful for analysis of the mechanisms of formation and resolution of DNA–protein adducts in living cells, and identification and characterization of reactions in which covalent DNA adducts are transient intermediates. The assay also has potential application to drug discovery and individualized medicine.
Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.
Nature advance online publication 01 May 2013. doi:10.1038/nature12178
Authors: Julie A. Law, Jiamu Du, Christopher J. Hale, Suhua Feng, Krzysztof Krajewski, Ana Marie S. Palanca, Brian D. Strahl, Dinshaw J. Patel & Steven E. Jacobsen
DNA methylation is an epigenetic modification that has critical roles in gene silencing, development and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24-nucleotide small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis, and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a Pol-IV-interacting protein. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets, and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin-binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals, a further understanding of this early targeting step may aid our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.
Ultraviolet-B-mediated induction of protein–protein interactions in mammalian cells
Nature Communications 4, 1779 (2013). doi:10.1038/ncomms2800
Authors: Remco P. Crefcoeur, Ruohe Yin, Roman Ulm & Thanos D. Halazonetis
FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA
Nature Communications 4, 1798 (2013). doi:10.1038/ncomms2822
Authors: Ye Fu, Guifang Jia, Xueqin Pang, Richard N. Wang, Xiao Wang, Charles J. Li, Scott Smemo, Qing Dai, Kathleen A. Bailey, Marcelo A. Nobrega, Ke-Li Han, Qiang Cui & Chuan He
Structures of the human and Drosophila 80S ribosome
Nature 497, 7447 (2013). doi:10.1038/nature12104
Authors: Andreas M. Anger, Jean-Paul Armache, Otto Berninghausen, Michael Habeck, Marion Subklewe, Daniel N. Wilson & Roland Beckmann
Protein synthesis in all cells is carried out by macromolecular machines called ribosomes. Although the structures of prokaryotic, yeast and protist ribosomes have been determined, the more complex molecular architecture of metazoan 80S ribosomes has so far remained elusive. Here we present structures of Drosophila
High-throughput sequencing (HTS) methods can provide short sequence reads for many millions of individual molecules in a sample, allowing the use of sequencing to measure the abundance of RNA molecules. To quantify the amount of a particular sequence in a sample of large RNAs (e.g., mRNAs), it is important to fragment the RNA into short pieces that can be ligated to oligonucleotides that allow polymerase chain reaction (PCR) amplification and sequencing. The most desired end structure of RNA for such ligation steps is a 5' phosphate and a 3' OH. Thus, enzymes that leave these groups after cleavage are of particular utility, avoiding the need to dephosphorylate the 3' end with phosphatases or phosphorylate the 5' end with kinase before proceeding. One such enzyme, RNase III, is widely available. Although it primarily cuts duplex RNA, this specificity is salt- and concentration-dependent, and many RNAs that lack strong extended duplexes are nonetheless susceptible to cleavage at many spots. RNA fragmentation by RNase III does not seem to grossly affect the distribution of RNA sequencing reads. Thus, it has become a standard method for creating nominally representative pools of transcriptome sequences with 5' phosphates and 3' OH for library construction. Three steps in preparing fragmented transcriptome RNA for sequencing library construction are described here: (1) fragmenting the RNA with RNase III to the extent that ~60–100-nucleotide fragments are created, (2) purifying the RNA from the RNase III reaction, and (3) analyzing the digestion products for their suitability in library production.
Off-Target Effect of Endogenous siRNA Derived from RMRP in Human Cells.
Int J Mol Sci. 2013;14(5):9305-18
Authors: Maida Y, Kyo S, Lassmann T, Hayashizaki Y, Masutomi K
Abstract
Endogenous siRNAs (endo-siRNAs) are key regulators of RNA silencing in plants and worms; however, the biogenesis and function of endogenous siRNAs in mammals remain largely unknown. We previously demonstrated that human telomerase reverse transcriptase produces a self-targeting endogenous siRNA from non-coding RMRP RNA via RNA-dependent RNA polymerase (RdRP) activity. Here, we investigated whether the endo-siRNA derived from RMRP targets other genes in addition to RMRP. Four algorithms for microRNA target prediction were used to identify possible targets of the endo-siRNA, and the phytanoyl-CoA hydroxylase-interacting protein-like gene (PHYHIPL) was identified as the most promising candidate. The 3' UTR of PHYHIPL was found to contain three possible target sites with perfect seed pairing; deletion of each of these sites resulted in recovery of upstream luciferase expression. In addition, sequence-specific inhibition of the RMRP-derived endo-siRNA increased expression of PHYHIPL mRNA. The results described here suggest that the endo-siRNA uses silencing mechanisms that are similar to those used by microRNAs for gene silencing. To our knowledge, this study is the first confirmation of the off-target effect of human endogenous siRNA produced by RdRP activity.
PMID: 23629666 [PubMed - in process]
Non-polyadenylated transcription in embryonic stem cells reveals novel non-coding RNA related to pluripotency and differentiation.
Nucleic Acids Res. 2013 Apr 29;
Authors: Livyatan I, Harikumar A, Nissim-Rafinia M, Duttagupta R, Gingeras TR, Meshorer E
Abstract
The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.
PMID: 23630323 [PubMed - as supplied by publisher]
Defining miRNA Targets: Balancing Simplicity with Complexity.
Circulation. 2013 Apr 26;
Authors: Freedman JE, Tanriverdi K
Abstract
Micro RNAs (miRNAs) are small RNAs that play an important role in the negative regulation of gene expression by suppressing protein translation. Animal genomes contain an abundance of small genes that produce regulatory RNAs of about 22 nucleotides in length. The Ambros lab identified the first miRNAs in 1993 while characterizing a genetic locus involved in the control of developmental timing in C. elegans.(1) It has since been shown that these miRNAs are diverse in sequence and expression patterns and are evolutionarily widespread, suggesting that they may participate in a wide range of genetic and regulatory pathways. Since their initial discovery, thousands of papers have been published characterizing miRNA properties, defining their expression, and demonstrating function. MiRNAs are initially transcribed as long primary miRNAs (pri-miRNAs) that are processed by the RNase III enzyme Drosha to generate stem-loop precursor miRNAs (pre-miRNAs) approximately 70 nucleotides in length.(2) These precursors are exported into the cytoplasm and, subsequently, the cytoplasmic enzyme Dicer cleaves the pre-miRNA to release the mature miRNA.(3) Binding of miRNA to a messenger RNA (mRNA) with Ago proteins inhibits protein translation. It is estimated that the human genome encodes about 1500 miRNAs that are thought to regulate more than 30% of protein-coding genes.(4) As interindividual variation of miRNA expression levels influences the expression of a myriad of miRNA target genes; these processes likely contribute to phenotypic differences and susceptibility to common and complex disorders.
PMID: 23625958 [PubMed - as supplied by publisher]
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miRNA_Targets: a database for miRNA target predictions in coding and non-coding regions of mRNAs.
Genomics. 2012 Dec;100(6):352-6
Authors: Kumar A, Wong AK, Tizard ML, Moore RJ, Lefèvre C
Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that play a role in post-transcriptional regulation of gene expression in most eukaryotes. They help in fine-tuning gene expression by targeting messenger RNAs (mRNA). The interactions of miRNAs and mRNAs are sequence specific and computational tools have been developed to predict miRNA target sites on mRNAs, but miRNA research has been mainly focused on target sites within 3' untranslated regions (UTRs) of genes. There is a need for an easily accessible repository of genome wide full length mRNA - miRNA target predictions with versatile search capabilities and visualization tools. We have created a web accessible database of miRNA target predictions for human, mouse, cow, chicken, Zebra fish, fruit fly and Caenorhabditis elegans using two different target prediction algorithms, The database has target predictions for miRNA's on 5' UTRs, coding region and 3' UTRs of all mRNAs. This database can be freely accessed at http://mamsap.it.deakin.edu.au/mirna_targets/.
PMID: 22940442 [PubMed - indexed for MEDLINE]
Substrate-specific structural rearrangements of human Dicer.
Nat Struct Mol Biol. 2013 Apr 28;
Authors: Taylor DW, Ma E, Shigematsu H, Cianfrocco MA, Noland CL, Nagayama K, Nogales E, Doudna JA, Wang HW
Abstract
Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer-RNA complexes to gain insight into the structural basis for human Dicer's substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer.
PMID: 23624860 [PubMed - as supplied by publisher]
by James A. Gagnon, Jill A. Kreiling, Erin A. Powrie, Timothy R. Wood, Kimberly L. Mowry
Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.Nature Methods 10, 384 (2013). doi:10.1038/nmeth.2460
Nature Methods 10, (2013). doi:10.1038/nmeth.f.363
Authors: Lindsay Freeberg, Scott Kuersten & Fraz Syed
Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins.
by Holly M. Bik, Miriam C. Goldstein
by Kohila Mahadevan, Hui Zhang, Abdalla Akef, Xianying A. Cui, Serge Gueroussov, Can Cenik, Frederick P. Roth, Alexander F. Palazzo
In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX) pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER) and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins.