Table of Contents

Volume 9, Issue 1

  January 2012

  Pages 1 - 116

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EDITOR'S CORNER

Open Access Article

Letter from the Editor

Renee Schroeder, Paul P. Gardner and Eva M. Riedmann
Page 1
http://dx.doi.org/10.4161/rna.9.1.18978
Full Text | PDF

Open Access Article

Three years of RNA Families

Paul Gardner
Pages 2 - 3
http://dx.doi.org/10.4161/rna.9.1.18979
Full Text | PDF




LANDES HIGHLIGHTS

Open Access Article

Landes Highlights

Eva M. Riedmann
Pages 4 - 5
http://dx.doi.org/10.4161/rna.9.1.19093
Abstract | Full Text | PDF

Chromatin insulator proteins: Key regulators of transcription
Cancer: Bivalent histone modification patterns in ESCs prepare miRNA loci for hypermethylation 
Novel function for polypyrimidine tract-binding (PTB) protein in cell cycle regulation
Integrating environmental cues in auxin signalling: miR393






RNA FAMILIES

Open Access Article

The HIV-1 Rev response element: An RNA scaffold that directs the cooperative assembly of a homo-oligomeric ribonucleoprotein complex

Jason Fernandes, Bhargavi Jayaraman and Alan Frankel
Pages 6 - 11
http://dx.doi.org/10.4161/rna.9.1.18178
Abstract | Full Text | PDF

The HIV-1 Rev response element (RRE) is a ~350 nucleotide, highly structured, cis-acting RNA element essential for viral replication. It is located in the env coding region of the viral genome and is extremely well conserved across different HIV-1 isolates. It is present on all partially spliced and unspliced viral mRNA transcripts, and serves as an RNA framework onto which multiple molecules of the viral protein Rev assemble. The Rev-RRE oligomeric complex mediates the export of these messages from the nucleus to the cytoplasm, where they are translated to produce essential viral proteins and/or packaged as genomes for new virions.

Open Access Article

Evolutionarily conserved A-to-I editing increases protein stability of the alternative splicing factor Nova1

Manuel Irimia, Amanda Denuc, Jose L. Ferran, Barbara Pernaute, Luis Puelles, Scott W. Roy, Jordi Garcia-Fernàndez and Gemma Marfany
Pages 12 - 21
http://dx.doi.org/10.4161/rna.9.1.18387
Abstract | Full Text | PDF

The structural complexity of the vertebrate brain is mirrored by its unparalleled transcriptome complexity. In particular, two post-transcriptional processes, alternative splicing and RNA editing, greatly diversify brain transcriptomes. Here we report a close connection between these two processes: we show A-to-I RNA editing in Nova1, a key brain-specific regulator of alternative splicing. Nova1 editing levels increase during embryonic development in mouse and chicken brains and show significant variation across postnatal brain regions. Evolutionary conservation of both editing and editing-associated RNA secondary structure of the Nova1 mRNA for 300 million years attests to the functional importance of Nova1 editing. Using a combination of different assays in human HEK293T cell lines, we report a novel post-translational role for this RNA editing. Whereas functional assays showed no effect of RNA editing on the regulatory splicing activity of the encoded proteins, we found evidence that edited forms exhibit reduced proteasome targeting and increased protein half-life. In addition, we found evidence for similar regulation of protein half-life by an evolutionarily conserved alternative splicing event in Nova1. These results open new venues of research on the multi-level integration of gene expression by: (1) revealing the novel role of RNA editing in regulating protein stability, and (2) establishing protein stability as a new target of multifaceted regulation.




POINT-OF-VIEWS

A conserved microRNA/NMD regulatory circuit controls gene expression

Rachid Karam and Miles Wilkinson
Pages 22 - 26
http://dx.doi.org/10.4161/rna.9.1.18010
Abstract | Full Text | PDF

Nonsense-mediated decay (NMD) is a RNA surveillance pathway that degrades subsets of normal and aberrant mRNAs. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Recently, it was shown that NMD is repressed during neural development to allow for the stabilization of NMD mRNA targets. The repression of NMD during development is mediated by a neuron-expressed microRNA, miR-128, which participates in a highly conserved regulatory circuit. miR-128 is induced in differentiating neuronal cells and during brain development, leading to repressed NMD and the consequent upregulation of batteries of mRNAs encoding proteins important for neuron differentiation and function. Together with other results, this suggests the existence of a complex network linking the microRNA and NMD pathways that induce cell-specific transcripts. In this point-of-view article, we will discuss the repercussions of this discovery for neuronal development, brain function and disease.

Pseudogenes are not pseudo any more

Yan-Zi Wen, Ling-Ling Zheng, Liang-Hu Qu, Francisco J Ayala and Zhao-Rong Lun
Pages 27 - 32
http://dx.doi.org/10.4161/rna.9.1.18277
Abstract | Full Text | PDF

Recent significant progress toward understanding the function of pseudogenes in protozoa (Trypanosoma brucei), metazoa (mouse) and plants, make it pertinent to provide a brief overview on what has been learned about this fascinating subject. We discuss the regulatory mechanisms of pseudogenes at the post-transcriptional level and advance new ideas toward understanding the evolution of these, sometimes called “garbage genes” or “junk DNA,” seeking to stimulate the interest of scientists and additional research on the subject. We hope this point-of-view can be helpful to scientists working or seeking to work on these and related issues.




REVIEW

Human mitochondrial tRNA quality control in health and disease: A channelling mechanism?

Ruth Belostotsky, Yaacov Frishberg and Nina Entelis
Pages 33 - 39
http://dx.doi.org/10.4161/rna.9.1.18009
Abstract | Full Text | PDF

Mutations in human mitochondrial tRNA genes are associated with a number of multisystemic disorders. These single nucleotide substitutions in various domains of tRNA molecules may affect different steps of tRNA biogenesis. Often, the prominent decrease of aminoacylation and/or steady-state levels of affected mitochondrial tRNA have been demonstrated in patients' tissues and in cultured cells.

 

Similar effect has been observed for pathogenic mutations in nuclear genes encoding mitochondrial aminoacyl-tRNA-synthetases, while over-expression of mitochondrial aminoacyl-tRNA synthetases or elongation factor EF-Tu rescued mutated tRNAs from degradation.

In this review we summarize experimental data concerning the possible regulatory mechanisms governing mitochondrial tRNA steady-state levels, and propose a hypothesis based on the tRNA channelling principle. According to this hypothesis, interaction of mitochondrial tRNA with proteins ensures not only tRNA synthesis, maturation and function, but also protection from degradation. Mutations perturbing this interaction lead to decreased tRNA stability.




TECHNICAL PAPER

Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1

Hiroaki Mon, Isao Kobayashi, Shinji Ohkubo, Shuichiro Tomita, JaeMan Lee, Hideki Sezutsu, Toshiki Tamura and Takahiro Kusakabe
Pages 40 - 46
http://dx.doi.org/10.4161/rna.9.1.18084
Abstract | Full Text | PDF

RNA interference (RNAi) is a conserved mechanism that catalyzes sequence-specific gene silencing and has been used for loss-of-function genetic screens in many organisms. Here, we demonstrated that the expression of Caenorhabditis elegans SID-1 (CeSID-1) could trigger effective gene silencing in the cultured silkworm cell line, BmN4 (BmN4-SID1). Soaking the BmN4-SID1 in dsRNA corresponding to endogenous target genes induced a significant decrease of the amount of mRNA or protein. A small amount of dsRNA was enough to silence the target gene in a few days. Overexpression of CeSID-1 did not affect the cell viability. Our results suggest that BmN4-SID1 can be used in many applications in silkworm cells and will become a valuable resource for gene analysis.




RESEARCH PAPERS

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis

Ramakanth Madhugiri, Gabriella Pessi, Björn Voss, Julia Hahn, Cynthia M. Sharma, Richard Reinhardt, Jörg Vogel, Wolfgang R. Hess, Hans-Martin Fischer and Elena Evguenieva-Hackenberg
Pages 47 - 58
http://dx.doi.org/10.4161/rna.9.1.18008
Abstract | Full Text | PDF

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Small aminoacyl transfer centers at GU within a larger RNA

Mali Illangasekare and Michael Yarus
Pages 59 - 66
http://dx.doi.org/10.4161/rna.9.1.18039
Abstract | Full Text | PDF

Separate aminoacyl transfer centers related to the small …GUNNN..: NNNU ribozyme seem possible at the frequent GU sequences dispersed throughout an RNA tertiary structure. In fact, such activity is easily detected and varies more than 2 orders in rate, probabably being faster at sites with less structural constraint. Analysis of a particular constrained active site in an rRNA transcript suggests that its difficulty lies not in substrate strand association, but in binding and/or group transfer from the aminoacyl precursor. Efficient aminoacyl transfer requires accurate complementarity between large or small ribozymes and oligoribonucleotide substrates, even when only three or four base pairs link the two. Thus, multi-site active ribozymal superstructures might have coordinated an RNA metabolism, including aiding an early translation apparatus.

Open Access Article

Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells

Cindy Meyer, Katja Eydeler, Eileen Magbanua, Tijana Zivkovic, Nicolas Piganeau, Inken Lorenzen, Joachim Grötzinger, Günter Mayer, Stefan Rose-John and Ulrich Hahn
Pages 67 - 80
http://dx.doi.org/10.4161/rna.9.1.18062
Abstract | Full Text | PDF

Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (K_d = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.

Potential G-quadruplexes in the human long non-coding transcriptome

Gopal Gunanathan Jayaraj, Satyaprakash Pandey, Vinod Scaria and Souvik Maiti
Pages 81 - 86
http://dx.doi.org/10.4161/rna.9.1.18047
Abstract | Full Text | PDF

DNA G-quadruplexes are known as modulators of transcription. More recently G-quadruplexes, located in the untranslated regions of the mRNA of protein coding genes, have been described to negatively regulate gene expression at the post transcriptional/ translational levels. Here we describe the possibility of the existence of G-quadruplexes in non-coding RNA (ncRNA) and discuss their potential biological roles. Using an in house prediction tool (Quadfinder) we observe a significant occurrence and distribution of G-quadruplexes in ncRNA of various sizes. We also observe that most of non-coding RNAs harboring these potential quadruplex motifs peak at the sizes ranging from 200–300 bases. More importantly we report enrichment for single and dinucleotide loops indicating a degree of high stability of these G-quadruplexes and their potential functions in vivo. Subsequent in vitro analyses of a subset of these sequences were performed which support our predictions.

Open Access Article

Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7

Morgane Ravon, Marco Berrera, Martin Ebeling and Ulrich Certa
Pages 87 - 97
http://dx.doi.org/10.4161/rna.9.1.18121
Abstract | Full Text | PDF

We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3′-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3′-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts.

RNase III initiates rapid degradation of proU mRNA upon hypo-osmotic stress in Escherichia coli

Kanstantsin Kavalchuk, Srinivasan Madhusudan and Karin Schnetz
Pages 98 - 109
http://dx.doi.org/10.4161/rna.9.1.18228
Abstract | Full Text | PDF

Hyper-osmotic stress strongly induces expression of the Escherichia coli proU operon encoding a high affinity uptake system for the osmoprotectants glycine betaine and proline betaine. Osmoregulation of proU takes place at the transcriptional level by upregulation of the promoter at high osmolarity and repression of transcription by the nucleoid-associated protein H-NS at low osmolarity. In the present study, we describe an additional level of proU osmoregulation that is independent of transcriptional regulation. We show that osmoregulation occurs at a post-transcriptional level involving RNase III. RNase III specifically processes the proU mRNA within a conserved secondary structure extending from position +203 to +293 of the transcript. Processing is efficient at low osmolarity, but inhibited at high osmolarity. Blocking of RNase III processing by mutation of the processing site eliminates post-transcriptional osmoregulation of proU. Further, the proU mRNA is relatively stable at high osmolarity with a half-life of approximately 65 sec. However, upon osmotic downshift, RNase III immediately processes the proU mRNA which reduces its half-life to less than 4 sec. The data suggest that the primary role of RNase III-mediated processing of proU mRNA is to ensure rapid shutdown of proU upon hypo-osmotic stress.

Characterization of a carcinogenesis-associated long non-coding RNA

Feng Yang, Fan Yi, Zhiguo Zheng, Zhiqiang Ling, Jianv Ding, Jiangfeng Guo, Weimin Mao, Xiongbiao Wang, Xiaoxia Wang, Xianfeng Ding, Zicai Liang and Quan Du
Pages 110 - 116
http://dx.doi.org/10.4161/rna.9.1.18332
Abstract | Full Text | PDF

A negative selection strategy was used in the present study to isolate long polyA-minus RNAs from the total transcriptome and a long non-coding RNA named Yiya was identified. Yiya is a 1.9 kb long intergenic ncRNA gene mapped to chromosome 1q41, a well-established cancer susceptibility locus. Expression profiling revealed a general and regulated expression pattern of Yiya in major tissues, and more interestingly, identified elevated mRNA levels in different cancers. Quantitative analysis further demonstrated a dynamic regulation of Yiya expression in cell cycle progression, suggesting that it was involved in cell cycle regulation. Supporting this, overexpression of Yiya promotes cell cycle progression at the G1/S transition, therefore identifying Yiya as a cell-cycle-associated long non-coding RNA.