Table of Contents

Volume 9, Issue 6

  June 2012

Special Issue: RNA and Disease

Guest Editor: Sven Diederichs

REVIEWS

Macro lncRNAs: A new layer of cis-regulatory information in the mammalian genome

Philipp Guenzl and Denise Barlow
Abstract | Full Text | PDF

Long non-protein-coding RNAs (lncRNAs) have been shown in the past ten years to comprise a major part of the mammalian transcriptome and are predicted from their highly specific expression patterns, to play a role in regulating protein-coding gene expression in development and disease. Many lncRNAs particularly those lying in imprinted clusters, are predominantly unspliced 'macro' transcripts that can also show a low level of splicing, and both the unspliced and spliced transcript have the potential to be functional. Three known imprinted macro lncRNAs have been shown to silence from three to ten genes in cis in imprinted gene clusters. We review here the potential for functional macro lncRNAs, defined here as 'inefficiently-spliced lncRNAs' to play a wider cis-regulatory role in the mammalian genome. This potential has been underestimated by the inability of current RNA-seq technology to annotate unspliced macro lncRNAs.

Battle of the midgets: Innate microRNA Networking

Shlomo Elias and Ofer Mandelboim
Abstract | Full Text

Natural killer (NK) cells play an important role in the direct killing of cancerous and virus-infected cells. One of the important activating receptors which mediates this killing is NKG2D. This receptor recognizes various stress-induced ligands including the major histocompatibility complex class I–related chain A and B (MICA and MICB respectively). The mechanisms controlling the expression of the NKG2D ligands are not completely understood, yet various studies have demonstrated that the expression of the NKG2D ligands is manipulated by viruses and by tumor cells in order to escape the NKG2D detection. Cumulative data have emphasized that various microRNAs (miRNAs) of both human and viral origin control the expression of NKG2D ligands, particularly MICB. Herein we review recent findings regarding the miRNA regulation of the NKG2D ligands. We propose that these miRNAs generate a complex network of interactions that control the expression of the NKG2D ligands under normal conditions and during disease development.

RNA-binding protein nucleolin in disease

Kotb Abdelmohsen and Myriam Gorospe
Abstract | Full Text | PDF

Nucleolin is a multifunctional protein localized primarily in the nucleolus, but also found in the nucleoplasm, cytoplasm and cell membrane. It is involved in several aspects of DNA metabolism, and participates extensively in RNA regulatory mechanisms, including transcription, ribosome assembly, mRNA stability and translation, and microRNA processing. Nucleolin’s implication in disease is linked to its ability to associate with target RNAs via its four RNA-binding domains and its arginine/glycin-rich domain. By modulating the post-transcriptional fate of target mRNAs, which typically bear AU-rich and/or G-rich elements, nucleolin has been linked to cellular events that influence disease, notably cell proliferation and protection against apoptotic death. Through its diverse RNA functions, nucleolin is increasingly implicated in pathological processes, particularly cancer and viral infection. Here, we review the RNA-binding activities of nucleolin, its influence on gene expression patterns, and its impact upon diseases. We also discuss the rising interest in targeting nucleolin therapeutically.

Viroid-specific small RNA in plant disease

Christian Hammann and Gerhard Steger
Abstract | Full Text | PDF

Viroids are the smallest autonomous infectious nucleic acids known today. They are non-coding, unencapsidated, circular RNAs with sizes ranging from 250 to 400 nucleotides and infect certain plants. These RNAs are transcribed by rolling-circle mechanisms in the plant host’s nuclei (Pospiviroidae) or chloroplasts (Avsunviroidae). Since viroids lack any open reading frame, their pathogenicity has for a long time been a conundrum. Recent findings, however, show that viroid infection is associated with the appearance of viroid-specific small RNA (vsRNA). These have sizes similar to endogenous small interfering RNA and microRNA and thus might alter the normal gene expression in the host plant. In this review we will summarize the current knowledge on vsRNA and discuss the current hypotheses how they connect to the induced symptoms, which vary dramatically, depending on both the plant cultivar and the viroid strain.

RNA mediated Toll-like receptor stimulation in health and disease

Alexander Dalpke and Mark Helm
Abstract | Full Text | PDF

Besides their well known functions in storage and translation of information nucleic acids have emerged as a target of pattern recognition receptors that drive activation of innate immunity. Due to the paucity of building block monomers used in nucleic acids, discrimination of host and microbial nucleic acids as a means of self/foreign discrimination is a complicated task. Pattern recognition receptors rely on discrimination by sequence, structural features and spatial compartmentalization to differentiate microbial derived nucleic acids from host ones. Microbial nucleic acid detection is important for the sensing of infectious danger and initiating an immune response to microbial attack. Failures in the underlying recognitions systems can have severe consequences: thus, inefficient recognition of microbial nucleic acids may increase susceptibility to infectious diseases. On the other hand, excessive immune responses as a result of failed self/foreign discrimination are associated with autoimmune diseases. This review gives a general overview over the underlying concepts of nucleic acid sensing by Toll-like receptors. Within this general framework, we focus on bacterial RNA and synthetic RNA oligomers.

Telomeres and disease: Enter TERRA

André Maicher, Lisa Kastner and Brian Luke
Abstract | PDF

Telomere function is tightly regulated in order to maintain chromosomal stability. When telomeres become dysfunctional, the replicative capacity of cells diminishes and cellular senescence ensues. This can lead to impaired tissue replenishment and eventually degenerative disorders, referred to as telomere syndromes. Cancer can also develop as a result of the genomic instability associated with telomere dysfunction. TERRA (TElomeric Repeat containing RNA) is a long non-coding transcript that stems from sub-telomeric regions and continues into the telomeric tract and is therefore a hybrid of both sub-telomeric and telomeric sequence. In general, increased TERRA transcription is associated with telomere shortening and compromised telomere function. Here we will briefly outline the general principles behind telomere dysfunction-associated diseases. Furthermore, we will discuss the few known links that exist between telomere transcription (TERRA) and disease. Finally, we will speculate on how the understanding, and eventual manipulation, of TERRA transcription could potentially be used in terms of therapeutic strategies.




RESEARCH PAPERS

Open Access Article

CpG island hypermethylation-associated silencing of small nucleolar RNAs in human cancer

Humberto J. Ferreira, Holger Heyn, Catia Moutinho and Manel Esteller
Abstract | Full Text | PDF

Much effort in cancer research has focused on the tiny part of our genome that codes for mRNA. However, it has recently been recognized that microRNAs also contribute decisively to tumorigenesis. Studies have also shown that epigenetic silencing by CpG island hypermethylation of microRNAs with tumor suppressor activities is a common feature of human cancer. The importance of other classes of non-coding RNAs, such as long intergenic ncRNAs (lincRNAs) and transcribed ultraconserved regions (T-UCRs) as altered elements in neoplasia, is also gaining recognition. Thus, we wondered whether there were other ncRNAs undergoing CpG island hypermethylation-associated inactivation in cancer cells. We focused on the small nucleolar RNAs (snoRNAs), a subset of ncRNA with a wide variety of cellular functions, such as chemical modification of RNA, pre-RNA processing and control of alternative splicing. By data mining snoRNA databases and the scientific literature, we selected 49 snoRNAs that had a CpG island within ≤ 2 Kb or that were processed from a host gene with a 5′-CpG island. Bisulfite genomic sequencing of multiple clones in normal colon mucosa and the colorectal cancer cell line HCT-116 showed that 46 snoRNAs were equally methylated in the two samples: completely unmethylated (n = 26) or fully methylated (n = 20). Most interestingly, the host gene-associated 5′-CpG islands of the snoRNAs SNORD123, U70C and ACA59B were hypermethylated in the cancer cells but not in the corresponding normal tissue. CpG island hypermethylation was associated with the transcriptional silencing of the respective snoRNAs. Results of a DNA methylation microarray platform in a comprehensive collection of normal tissues, cancer cell lines and primary malignancies demonstrated that the observed hypermethylation of snoRNAs was a common feature of various tumor types, particularly in leukemias. Overall, our findings indicate the existence of a new subclass of ncRNAs, snoRNAs, that are targeted by epigenetic inactivation in human cancer.

Complexities of 5'splice site definition: Implications in clinical analyses

Laura De Conti, Natasa Skoko, Emanuele Buratti and Marco Baralle
Abstract | PDF

In higher eukaryotes, the 5' splice site (5'ss) is initially recognized through an RNA-RNA interaction by U1 small nuclear ribonucleoprotein (U1 snRNP). This event represents one of the key steps in initial spliceosomal assembly and many disease-associated mutations in humans often disrupt this process. Beside base pair complementarity, 5'ss recognition can also be modified by additional factors such as RNA secondary structures or the specific binding of other nuclear proteins. In this work, we have focused on investigating a few examples of changes detected within the 5'ss in patients, that would not be immediately considered "disease causing mutations". We show that the splicing outcome of very similar mutations can be very different due to variations in trans-acting factor(s) interactions and specific context influences. Using several NF1 donor sites and SELEX approaches as experimental models, we have examined the binding properties of particular sequence motifs such as GGGU found in donor sites, and how the sequence context can change their interaction with hnRNPs such as H/F and A1/A2. Our results clearly show that even minor differences in local nucleotide context can differentially affect the binding ability of these factors to the GGGU core. Finally, using a previously identified mutation in KCNH2 that resulted in intron retention we show how very similar 5'ss mutations found in patients can have a very different splicing outcome due to the neighbouring sequence context, thus highlighting the general need to approach splicing problems with suitable experimental approaches.





---

RECENTLY ACCEPTED AND COMING SOON

REVIEWS

The Hallmarks of Cancer: A long non-coding RNA point of view

Tony Gutschner and Sven Diederichs
Abstract

With the advent of next generation sequencing methods and progress in transcriptome analysis, it became obvious that the human genome contains much more than just protein-coding genes. In fact, up to 70% of our genome is transcribed into RNA that does not serve as templates for proteins. In this review, we focus on the emerging roles of these long non-coding RNAs (lncRNAs) in the field of tumor biology. Long ncRNAs were found to be deregulated in several human cancers and show tissue-specific expression. Functional studies revealed a broad spectrum of mechanisms applied by lncRNAs such as HOTAIR, MALAT1, ANRIL or lincRNA-p21 to fulfill their functions. Here, we link the cellular processes influenced by long ncRNAs to the hallmarks of cancer and therefore provide an ncRNA point-of-view on tumor biology. This should stimulate new research directions and therapeutic options considering long ncRNAs as novel prognostic markers and therapeutic targets. 


miRNAs and neural stem cells: A team to treat Parkinson's disease?

Thomas Palm, Lamia'a Bahnassawy and Jens Schwamborn
Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder with no proven neuroprotective or neurorestorative therapies. During disease progression, degeneration of dopaminergic neurons of the central nervous system occurs. Therefore, therapies that either aim on the inhibition of this degeneration or on the replacement of the degenerated neurons are needed. On the one hand, arrest of degeneration might be achievable through specific inhibition of disease associated genes like α-Synuclein or Leucine rich repeat kinase 2 (LRRK2). On the other hand, based on neural stem cells that bear the ability to generate new dopaminergic neurons, replacement of degenerated cells could be accomplished. Since both approaches can be regulated by micro-RNAs, these molecules have an enormous therapeutic potential. In this review, we will focus on the neurobiological and neurodegenerative implications of miRNAs and highlight their role in stem cell fate decisions. Finally, we will discuss their potential as therapeutic agents and targets for Parkinson's disease.

The mammalian microRNA response to bacterial infections

Ana Eulalio, Leon Schulte and Jörg Vogel
Abstract

MicroRNAs are small RNAs that post-transcriptionally regulate eukaryotic gene expression. In addition to their involvement in a wide range of physiological and pathological processes, including viral infections, microRNAs are increasingly implicated in the eukaryotic response to bacterial pathogens. Recent studies have characterized changes in host microRNA expression following infection with exclusively extracellular (Helicobacter pylori) or intracellular (Salmonella enterica) Gram-negative bacteria, as well as in the response to Gram-positive (Listeria monocytogenes) and other pathogens (Mycobacterium and Francisella species). In this review, we discuss the emerging roles of microRNAs in mammalian host signaling and defense against bacterial pathogens.

RNA helicases in infection and disease

Lenz Steimer and Dagmar Klostermeier
Abstract

RNA helicases unwind their RNA substrates in an ATP-dependent reaction, and are central to all cellular processes involving RNA. They have important roles in viral life cycles, where RNA helicases are either virus-encoded or recruited from the host. Vertebrate RNA helicases sense viral infections, and trigger the innate antiviral immune response. RNA helicases have been implicated in protozoic, bacterial and fungal infections. They are also linked to neurological disorders, cancer, and ageing processes. Genome-wide studies continue to identify helicase genes that change their expression patterns after infection or disease outbreak, but the mechanism of RNA helicase action has been defined for only a few diseases. RNA helicases are prognostic and diagnostic markers and suitable drug targets, predominantly for antiviral and anti-cancer therapies. This review summarizes the current knowledge on RNA helicases in infection and disease, and their growing potential as drug targets.

Posttranscriptional regulation in metabolic diseases

Wook Kim and Eun Kyung Lee
Abstract

Post-transcriptional gene regulation by microRNAs (miRNAs) and RNA-binding proteins (RBPs) is central to many biological functions.  Aberrant gene expression patterns underlie many metabolic diseases that represent major public health concerns and formidable therapeutic challenges.  Several studies have established a number of post-transcriptional regulators implicated in metabolic diseases such as diabetes and obesity.  In addition, emerging knowledge of metabolically active and insulin-sensitive organs, such as the pancreas, liver, muscle and adipose compartment, is rapidly expanding the panel of potential therapeutic targets for the treatment of metabolic diseases.  Here, we review our current understanding of miRNAs and RBPs that affect glucose and lipid homeostasis, and their roles in normal physiology and metabolic disorders, especially type 2 diabetes and obesity.

MicroRNAs, wild-type and mutant p53: more questions than answers

Matthew Jones and Ashish Lal
Abstract

The tumor suppressor p53 is a sequence-specific transcription factor that activates the expression of genes involved in apoptosis, cell cycle arrest and senescence. p53 can also inhibit gene expression and this effect is partly mediated by inducing several microRNAs (miRNAs). MiRNAs have emerged as a new class of regulators of the expression and function of eukaryotic genomes. Tumor suppressive or oncogenic functions have been attributed to some miRNAs. Recent studies have shown that p53 can alter the transcription of several miRNAs, and in some cases, it can also influence miRNA maturation. Conversely, miRNAs can also modulate the abundance and activity of p53 by direct or indirect mechanisms. Moreover, mutant p53 can actively repress the expression of some miRNAs that are activated by wild-type p53. In this review, we discuss recent evidences of this crosstalk between miRNAs and the p53 network and also highlight its implications in cancer.

MicroRNAs in diabetes and diabetes-associated complications

Johan Lorenzen, Regalla Kumarswamy, Seema Dangwal and Thomas Thum
Abstract

Diabetes mellitus due to its high prevalence and associated complications is a major socioeconomic health problem. Diabetes is characterized by multiple macro- and microvascular complications (e.g. diabetic nephropathy, cardiomyopathy, neuropathy, retinopathy). Research efforts aim to elucidate pathophysiological mechanisms contributing to the disease process. MicroRNAs are endogenous small single stranded molecules regulating targets through mRNA cleavage or translational inhibition. MicroRNAs regulate many biological cellular functions and are often deregulated during diseases. The aim of the present article is to summarize the current knowledge of the impact of microRNAs on the development of diabetes and its associated complications including endothelial and vascular smooth muscle cell dysfunction, diabetic cardiomyopathy, diabetic nephropathy, regulation of pancreatic beta cell function as well as skeletal muscle and hepatic involvement.

Small RNAs have a large impact: Circulating microRNAs as biomarkers for human diseases

Matthew Weiland, Xinghua Gao, Li Zhou and Qing-Sheng Mi
Abstract

The highly conserved RNAs, microRNAs (miRNAs), are a class of small single stranded noncoding RNAs that function through translational repression of specific target mRNAs. miRNAs exhibit a wide range of involvement regulating gene expressions. miRNA expression dysregulated in cancer cells and damaged tissues from different diseases implicates a functional role of miRNAs in the disease development. More recently miRNAs have been detected in cell-free serum, and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA collection and their sensitivity and specificity in diseases has encouraged a pursuit of miRNA biomarker research. As a result, approximately 100 circulating miRNAs have been identified as biomarkers for different diseases, and the number is growing. Here we review recently reported circulating miRNA biomarkers and discuss their values and challenges for the disease biomarkers.

Involvement of long noncoding RNAs in diseases affecting the central nervous system

Chiara Pastori and Claes Wahlestedt
Abstract

DNA sequences associated with protein-coding genes have been the primary focus of most genetic analyses of complex human diseases. Although we are rapidly gaining a comprehensive view of the etiology of certain central nervous system disorders, major gaps in our understanding persist. Recent studies have uncovered that many human genomic sequences are transcribed but not translated, generating an astounding diversity of noncoding RNAs (ncRNAs). This awareness should be taken into account when studying human diseases and may have profound implications on the development of novel biomarkers as well as therapies.





RESEARCH PAPERS

Regulation of BACE1 by miR-29a/b in a cellular model of Spinocerebellar ataxia 17

Reema Roshan, Tanay Ghosh, Mugdha Gadgil and Beena Pillai
Abstract

Polyglutamine diseases are a class of neurodegenerative disorders characterized by expansion of polyglutamine repeats, protein aggregation and neuronal cell death in specific regions of the brain. The expansion of a polyglutamine repeat in the TATA binding protein (TBP) causes a neurodegenerative disease, Spinocerebellar Ataxia 17 (SCA17). This disease is characterized by intranuclear protein aggregates and selective loss of cerebellar neurons, including Purkinje cells. MicroRNAs are small, endogenous, regulatory non-coding RNA molecules that bind to messenger RNAs with partial complementarity and interfere in their expression. Here, we used a cellular model of SCA17 where we expressed TBP with 16 (normal) or 59 (pathogenic) polyglutamines and found differential expression of several microRNAs. Specifically, we found two microRNAs, miR-29a/b, were down-regulated. With miR-29a/b down regulation, we found an increased expression of targets of miR-29a/b -beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), p53 upregulated modulator of apoptosis (PUMA) and BAK, increased cytochrome c release and apoptosis. Restoration of miR-29a/b in the pathogenic polyglutamine background reduced the BACE1expression. While, antagomiRs against miR-29a/b resulted in an increase in BACE1 levels and neuronal apoptosis. In spite of the elevation of BACE1 in Alzhemiers disease, its role in neuronal cell death has not been established. Here, we show that increased BACE1 expression is not sufficient to cause apoptosis. However restoring level of BACE1 to normal in polyglutamine cells partially reduced neuronal apoptosis. We show a role for the miR-29a/b-BACE1 regulatory interaction in SCA17, suggesting that this microRNA could be part of a common molecular mechanism leading to neuronal cell death in multiple neurodegenerative disorders. The identification of a common mechanism of microRNA mediated neurodegeneration not only improves our understanding of the process, but also provides promising and novel therapeutic targets.

MicroRNA expression changes after lung cancer resection: A follow-up study

Petra Leidinger
Abstract

MiRNAs are powerful biomarkers for detecting various diseases from tissue and body fluids. The potential of these molecules to monitor patients over time has, however, been less explored. We followed the fate of the plasma miRNome of lung cancer patients starting prior to surgery and ending 18 months after surgery, with blood taken at three-month intervals. Principal component and clustering analysis showed that the differences of the overall miRNA patterns between the different time points were significantly smaller than between patients. For each patient we found a rather specific fluctuating miRNA pattern. We identified miRNAs that showed a significant correlation between expression level and time distance from surgery. A network analysis revealed twelve correlated miRNAs regulating 48 genes that were deregulated in lung cancer tissue. Our data underline the importance of studies that follow the fate of miRNAs over time, both to further our understanding of the biology of miRNA signatures and to establish these signatures as biomarkers.

MicroRNA-mediated regulation of gene expression is affected by disease-associated SNPs within the 3′-UTR via altered RNA structure

Ulrike Haas, Georg Sczakiel and Sandra Laufer
Abstract

Single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) or their target sites (miR-SNPs) within the 3′-UTR of mRNAs are increasingly thought to play a major role in pathological dysregulation of gene expression. Here, we studied the functional role of miR-SNPs on miRNA-mediated post-transcriptional regulation of gene expression. First, analyses were performed on a SNP located in the miR-155 target site within the 3′-UTR of the Angiotensin II type 1 receptor (AGTR1; rs5186, A>C) mRNA. Second, a SNP in the 3′-UTR of the muscle RAS oncogene homolog (MRAS; rs9818870, C>T) mRNA was studied which is located outside of binding sites of miR-195 and miR-135. Using these SNPs we investigated their effects on local RNA structure, on local structural accessibility, and on functional miRNA binding, respectively. Systematic computational RNA folding analyses of the allelic mRNAs in either case predicted significant changes of local RNA structure in the vicinity of the cognate miRNA binding sites. Consistently, experimental in vitro probing of RNA showing differential cleavage patterns and reporter gene-based assays indicated functional differences of miRNA-mediated regulation of the two AGTR1 and MRAS alleles. In conclusion, we describe a novel model explaining the functional influence of 3′-UTR-located SNPs on miRNA-mediated control of gene expression via SNP-related changes of local RNA structure in non-coding regions of mRNA. This concept substantially extends the meaning of disease-related SNPs identified in non protein-coding transcribed sequences within or close to miRNA binding sites.




REVIEW

MicroRNA-mediated post-transcriptional mechanisms of gene expression in proliferating

and Quiescent Cancer Cells

Olivier LeTonqueze, Ju Lee and Shobha Vasudevan
Abstract

MicroRNAs are small non-coding RNA regulators of gene expression that play important roles in critical biological processes, including cell division, self-renewal and cell state maintenance. Their deregulation leads to extensive clinical consequences in tumorigenesis. Cancers demonstrate heterogeneity in their cell states implicated in their resistance and resurgence. Apart from proliferating cells, cancers harbor a small proportion of assorted quiescent cells that resist conventional therapeutics and contribute to cancer recurrence. MicroRNA expression, targets, microRNPs (microRNA-protein complexes) and their functions have been demonstrated to be regulated in distinct tumor cell states and as an adaptive response to stress signals in tumor-unfavorable environments. In turn, altered microRNPs and their modified post-transcriptional mechanisms of gene expression may contribute to tumor resistance and influence tumor progression. An understanding of distinct microRNA mechanisms in cancer cells would provide extensive insights into the versatile role of microRNAs in the perpetuation of tumors and indicate potential therapeutic avenues.