Hyeshik Chang

Microbiology: RNAs at fever pitch

Nature 502, 7470 (2013). doi:10.1038/nature12563

Authors: Franz Narberhaus

Some normally innocuous bacteria can turn into serious pathogens. It seems that one such species, Neisseria meningitidis, uses three RNA-based thermosensors to escape the immune response of its human host. See Letter p.237

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Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules.

RNA. 2013 Oct 8;

Authors: Cox EM, Sagan SM, Mortimer SA, Doudna JA, Sarnow P

Abstract
The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5' end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3' noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.

PMID: 24106328 [PubMed - as supplied by publisher]

Nature Protocols 8, 2158 (2013). doi:10.1038/nprot.2013.136

Authors: Sandra G Zimmerman, Nathaniel C Peters, Ariel E Altaras & Celeste A Berg

In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)–RNA FISH double

Dhirendra K. Simanshu, Yoshihiro Yamaguchi, Jung-Ho Park, Masayori Inouye, Dinshaw J. Patel. MazF is an mRNA interferase, which, upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the....

Craig R. Stumpf, Melissa V. Moreno, Adam B. Olshen, Barry S. Taylor, Davide Ruggero. Gene regulation during cell-cycle progression is an intricately choreographed process, ensuring accurate DNA replication and division. However, the translational landscape of gene expression under....

Certain mammalian cells can use RNA interference in the innate defense against invading viruses. [Also see Perspective by Sagan and Sarnow] Authors: P. V. Maillard, C. Ciaudo, A. Marchais, Y. Li, F. Jay, S. W. Ding, Olivier Voinnet

Article

The ratio between ATP and ADP within the cell is a key indicator of metabolic status. Tantama et al . describe a ratiometric, genetically encoded fluorescent sensor for ATP:ADP that is now optimized for mammalian cells, and demonstrate that it can detect physiological changes in energy consumption and production.

Nature Communications doi: 10.1038/ncomms3550

Authors: Mathew Tantama, Juan Ramón Martínez-François, Rebecca Mongeon, Gary Yellen

Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system.

pLogo: a probabilistic approach to visualizing sequence motifs.

Nat Methods. 2013 Oct 6;

Authors: O'Shea JP, Chou MF, Quader SA, Ryan JK, Church GM, Schwartz D

Abstract
Methods for visualizing protein or nucleic acid motifs have traditionally relied upon residue frequencies to graphically scale character heights. We describe the pLogo, a motif visualization in which residue heights are scaled relative to their statistical significance. A pLogo generation tool is publicly available at http://plogo.uconn.edu/ and supports real-time conditional probability calculations and visualizations.

PMID: 24097270 [PubMed - as supplied by publisher]

Nature Biotechnology 31, 898 (2013). doi:10.1038/nbt.2682

Authors: Lior Zangi, Kathy O Lui, Alexander von Gise, Qing Ma, Wataru Ebina, Leon M Ptaszek, Daniela Später, Huansheng Xu, Mohammadsharif Tabebordbar, Rostic Gorbatov, Brena Sena, Matthias Nahrendorf, David M Briscoe, Ronald A Li, Amy J Wagers, Derrick J Rossi, William T Pu & Kenneth R Chien

Nature Reviews Genetics. doi:10.1038/nrg3483

Author: David L. Stern

The exoribonuclease Rrp6 forms a complex with Rrp47 that functions in RNA processing and turnover pathways in eukaryotic cells. Using a novel experimental strategy we call DECOID (decreased expression of complexes by overexpression of interacting domains), Rrp47 is resolved from the Rrp6 exoribonuclease, and the two proteins are shown to have separable functions. This study demonstrates a general role for Rrp47 in Rrp6-mediated processing reactions prior to catalysis and reveals a redundant requirement for the two exoribonucleases Rrp6 and Rex1 in the 3' maturation of box C/D snoRNAs.

Nature advance online publication 09 October 2013. doi:10.1038/nature12598

Authors: Annalisa Di Ruscio, Alexander K. Ebralidze, Touati Benoukraf, Giovanni Amabile, Loyal A. Goff, Jolyon Terragni, Maria Eugenia Figueroa, Lorena Lobo De Figueiredo Pontes, Meritxell Alberich-Jorda, Pu Zhang, Mengchu Wu, Francesco D’Alò, Ari Melnick, Giuseppe Leone, Konstantin K. Ebralidze, Sriharsa Pradhan, John L. Rinn & Daniel G. Tenen

Impacts of early instructors can last for decades

Annual contest adds new prize and sponsor

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.

Nature Medicine 19, 1227 (2013). doi:10.1038/nm.3338

Author: Thomas C Südhof

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

Recently, the ability to interact with messenger RNA (mRNA) has been reported for a number of known RNA-binding proteins, but surprisingly also for different proteins without recognizable RNA binding domains including several transcription factors and metabolic enzymes. Moreover, direct binding to cognate mRNAs has been detected for multiple proteins, thus creating a strong impetus to search for functional significance and basic physico-chemical principles behind such interactions. Here, we derive interaction preferences between amino acids and RNA bases by analyzing binding interfaces in the known 3D structures of protein–RNA complexes. By applying this tool to human proteome, we reveal statistically significant matching between the composition of mRNA sequences and base-binding preferences of protein sequences they code for. For example, purine density profiles of mRNA sequences mirror guanine affinity profiles of cognate protein sequences with quantitative accuracy (median Pearson correlation coefficient R = –0.80 across the entire human proteome). Notably, statistically significant anti-matching is seen only in the case of adenine. Our results provide strong evidence for the stereo-chemical foundation of the genetic code and suggest that mRNAs and cognate proteins may in general be directly complementary to each other and associate, especially if unstructured.

The prediction of pairing between microRNAs (miRNAs) and the miRNA recognition elements (MREs) on mRNAs is expected to be an important tool for understanding gene regulation. Here, we show that mRNAs that contain Pumilio recognition elements (PRE) in the proximity of predicted miRNA-binding sites are more likely to form stable secondary structures within their 3'-UTR, and we demonstrated using a PUM1 and PUM2 double knockdown that Pumilio proteins are general regulators of miRNA accessibility. On the basis of these findings, we developed a computational method for predicting miRNA targets that accounts for the presence of PRE in the proximity of seed-match sequences within poorly accessible structures. Moreover, we implement the miRNA-MRE duplex pairing as a two-step model, which better fits the available structural data. This algorithm, called MREdictor, allows for the identification of miRNA targets in poorly accessible regions and is not restricted to a perfect seed-match; these features are not present in other computational prediction methods.

We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3' splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3'ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

Nature Structural & Molecular Biology 20, 1227 (2013). doi:10.1038/nsmb.2665

Authors: Robert L Judson, Tobias S Greve, Ronald J Parchem & Robert Blelloch

Nature Structural & Molecular Biology 20, 1214 (2013). doi:10.1038/nsmb.2664

Authors: Chenguang Gong, Yalan Tang & Lynne E Maquat

Nature Structural & Molecular Biology 20, 1145 (2013). doi:10.1038/nsmb.2695

Author: Arianne Heinrichs

Nature Structural & Molecular Biology 20, 1147 (2013). doi:10.1038/nsmb.2669

Authors: Luciano Di Croce & Kristian Helin

Nature Structural & Molecular Biology 20, 1141 (2013). doi:10.1038/nsmb.2684

Author: Anders Liljas

Translation initiation in eukaryotes is a complex and highly regulated process during which several initiation factors cooperate to recruit an initiator tRNA to the small ribosomal subunit, where the mRNA is scanned for an AUG start codon. Two recent reports provide new structural insights into this process and reveal key functions of initiation factors 1 (eIF1) and 1A (eIF1A) in start-codon selection in atomic detail.

Rongmin Chen, Matthew D’Alessandro, Choogon Lee. BackgroundCircadian clocks coordinate an organism’s activities and regulate metabolic homeostasis in relation to daily environmental changes, most notably light/dark cycles. As in other organisms,....

Cori A. Anderson, Umut Eser, Therese Korndorf, Mark E. Borsuk, Jan M. Skotheim, Amy S. Gladfelter. BackgroundCurrent models of cell-cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling....

Small RNA from a fungal pathogen is transferred to cells of a plant host where it silences the mRNA for defense proteins. [Also see Report by Weiberg et al.] Author: David Baulcombe