UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Robert Lebbink

Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells.
Mayr C, Bartel DP.
Cell
August 21, 2009
1Howard Hughes Medical Institute 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
In cancer cells, genetic alterations can activate proto-oncogenes, thereby contributing to tumorigenesis. However, the protein products of oncogenes are sometimes overexpressed without alteration of the proto-oncogene. Helping to explain this phenomenon, we found that when compared to similarly proliferating nontransformed cell lines, cancer cell lines often expressed substantial amounts of mRNA isoforms with shorter 3' untranslated regions (UTRs). These shorter isoforms usually resulted from alternative cleavage and polyadenylation (APA). The APA had functional consequences, with the shorter mRNA isoforms exhibiting increased stability and typically producing ten-fold more protein, in part through the loss of microRNA-mediated repression. Moreover, expression of the shorter mRNA isoform of the proto-oncogene IGF2BP1/IMP-1 led to far more oncogenic transformation than did expression of the full-length, annotated mRNA. The high incidence of APA in cancer cells, with consequent loss of 3'UTR repressive elements, suggests a pervasive role for APA in oncogene activation without genetic alteration.
Date: 
November 16, 2009
Where: 
HSW 1057

Chong Park

Downregulation of miRNA-200c Links Breast Cancer Stem Cells with Normal Stem Cells
Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula SP, Chiao E, Dirbas FM, Somlo G, Pera RA, Lao K, Clarke MF.
Cell
August 7, 2009
Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 1050 Arastradero Road, Palo Alto, CA 94304, USA.
Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.
Date: 
November 9, 2009
Where: 
HSW 1057

Jason Barbour

microRNAs miR-124, let-7d and miR-181a regulate Cocaine-induced Plasticity
Vijay Chandrasekar, Jean-Luc Dreyer
Molecular and Cellular Neuroscience
August 11, 2009
Division of Biochemistry, Department of Medicine, University of Fribourg, Rue du Musée 5, Chemin du Musée 5, CH-1700 Fribourg, Switzerland
MicroRNAs play key regulatory roles in cellular processes including neurogenesis, synapse development and plasticity in the brain. Psychostimulants induces strong neuroadaptive changes through a surfeit of gene regulatory mechanisms leading to addiction. MicroRNA profiling for identifying miRNAs regulating cocaine induced,plasticity-related genes revealed signi!cant regulation of a set of miRNAs upon cocaine administration, especially let-7d, miR-181a and the brain-speci!c miR-124. These miRNAs target many genes involved in cocaine addiction. Precursor and mature miRNA quanti!cation by qRT-PCR showed that miR-124 and let-7d are significantly downregulated, whereas miR-181a is induced in the mesolimbic dopaminergic system under chronic cocaine administration. Results were confirmed by in situ hybridization, Northern blots, FISH analysis and RNase protection assay. Using lentiviral-mediated miRNA expression, we show a significant downregulation of BDNF and D3R both at mRNA and protein levels by miR-124 and let-7d, respectively. Our data suggest that miR-124, let-7d and miR-181a may be involved in a complex feedback loop with cocaine-responsive plasticity genes, highlighting the possibility that some miRNAs are key regulators of the reward circuit and may be implicated in addiction.
Date: 
November 2, 2009
Where: 
HSW 1057

Matthew Hangauer

Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA.
Chen LL and Carmichael GG.
Molecular Cell
August 28, 2009
University of Connecticut Stem Cell Institute
In many cells, mRNAs containing inverted repeats (Alu repeats in humans) in their 3' untranslated regions (3'UTRs) are inefficiently exported to the cytoplasm. Nuclear retention correlates with adenosine-to-inosine editing and is in paraspeckle-associated complexes containing the proteins p54(nrb), PSF, and PSP1 alpha. We report that robust editing activity in human embryonic stem cells (hESCs) does not lead to nuclear retention. p54(nrb), PSF, and PSP1 alpha are all expressed in hESCs, but paraspeckles are absent and only appear upon differentiation. Paraspeckle assembly and function depend on expression of a long nuclear-retained noncoding RNA, NEAT1. This RNA is not detectable in hESCs but is induced upon differentiation. Knockdown of NEAT1 in HeLa cells results both in loss of paraspeckles and in enhanced nucleocytoplasmic export of mRNAs containing inverted Alu repeats. Taken together, these results assign a biological function to a large noncoding nuclear RNA in the regulation of mRNA export.
Date: 
October 26, 2009
Where: 
HSW 1057

Mikael Langner

Structural insights into mechanisms of the small RNA HEN1
Huang Y, Ji L, Huang Q, Vassylyev DG, Chen X, Ma JB.
Nature
October 8, 2009
1Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. 2Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, California 92521, USA. 3Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of approximately 20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg(2+)-dependent 2'-O-methylation mechanism.
Date: 
October 19, 2009
Where: 
HSW 1057

Yun Choi

An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA
Maida Y, Yasukawa M, Furuuchi M, Lassmann T, Possemato R, Okamoto N, Kasim V, Hayashizaki Y, Hahn WC, Masutomi K.
Nature
September 10, 2009
Cancer Stem Cell Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase reverse transcriptase catalytic subunit (TERT) contributes to cell physiology independently of its ability to elongate telomeres. Here we show that TERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome cartilage-hair hypoplasia. Human TERT and RMRP form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer (also known as DICER1)-dependent manner. These observations identify a mammalian RdRP composed of TERT in complex with RMRP.
Date: 
October 12, 2009
Where: 
HSW 1057

Hwajung Choi

c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism
Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, Zeller KI, De Marzo AM, Van Eyk JE, Mendell JT, Dang CV.
Nature
April 9, 2009
Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. [email protected]
Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis
Date: 
October 5, 2009
Where: 
HSW 1057

Hunter Richards

Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps
Sung Wook Chi1, Julie B. Zang1, Aldo Mele1 & Robert B. Darnell1
Nature
July 23, 2009
Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA
MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein–RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein–RNA complexes in mouse brain. This produced two simultaneous data sets—Ago–miRNA and Ago–mRNA binding sites—that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA–mRNA interactions.
Date: 
September 28, 2009
Where: 
HSW 1057

Yelena Bronevetsky

RNA Polymerase III Detects Cytosolic DNA and Induces Type I Interferons through through the RIG-I Pathway
Chiu YH, Macmillan JB, Chen ZJ.
Cell
August 7, 2009
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
Type I interferons (IFNs) are important for antiviral and autoimmune responses. Retinoic acid-induced gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) proteins mediate IFN production in response to cytosolic double-stranded RNA or single-stranded RNA containing 5'-triphosphate (5'-ppp). Cytosolic B form double-stranded DNA, such as poly(dA-dT)*poly(dA-dT) [poly(dA-dT)], can also induce IFN-beta, but the underlying mechanism is unknown. Here, we show that the cytosolic poly(dA-dT) DNA is converted into 5'-ppp RNA to induce IFN-beta through the RIG-I pathway. Biochemical purification led to the identification of DNA-dependent RNA polymerase III (Pol-III) as the enzyme responsible for synthesizing 5'-ppp RNA from the poly(dA-dT) template. Inhibition of RNA Pol-III prevents IFN-beta induction by transfection of DNA or infection with DNA viruses. Furthermore, Pol-III inhibition abrogates IFN-beta induction by the intracellular bacterium Legionella pneumophila and promotes the bacterial growth. These results suggest that RNA Pol-III is a cytosolic DNA sensor involved in innate immune responses.
Date: 
September 14, 2009
Where: 
HSW 1057

Bryan Burke

Active turnover modulates mature microRNA activity in Caenorhabditis elegans
Chatterjee S, Großhans H.
Nature
September 6, 2009
Friedrich Miescher Institute for Biomedical Research, PO Box 2543, CH-4002 Basel, Switzerland.
MicroRNAs (miRNAs) constitute a large class of regulatory RNAs that repress target messenger RNAs to control various biological processes. Accordingly, miRNA biogenesis is highly regulated, controlled at both transcriptional and post-transcriptional levels, and overexpression and underexpression of miRNAs are linked to various human diseases, particularly cancers. As RNA concentrations are generally a function of biogenesis and turnover, active miRNA degradation might also modulate miRNA accumulation, and the plant 3'-->5' exonuclease SDN1 has been implicated in miRNA turnover. Here we report that degradation of mature miRNAs in the nematode Caenorhabditis elegans, mediated by the 5'-->3' exoribonuclease XRN-2, affects functional miRNA homeostasis in vivo. We recapitulate XRN-2-dependent miRNA turnover in larval lysates, where processing of precursor-miRNA (pre-miRNA) by Dicer, unannealing of the miRNA duplex and loading of the mature miRNA into the Argonaute protein of the miRNA-induced silencing complex (miRISC) are coupled processes that precede degradation of the mature miRNA. Although Argonaute:miRNA complexes are highly resistant to salt, larval lysate promotes efficient release of the miRNA, exposing it to degradation by XRN-2. Release and degradation can both be blocked by the addition of miRNA target RNA. Our results therefore suggest the presence of an additional layer of regulation of animal miRNA activity that might be important for rapid changes of miRNA expression profiles during developmental transitions and for the maintenance of steady-state concentrations of miRNAs. This pathway might represent a potential target for therapeutic intervention on miRNA expression.
Date: 
September 21, 2009
Where: 
HSW 1057