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Science in China: Series C Life Sciences 2006 Vol.49 No.

3 201ü217

201

DOI: 10.1007/s11427-006-0201-8

Genetic regulation by non-coding RNAs
QI Liwang1, LI Xinmin2, ZHANG Shougong1 & AN Daochang3
1. Laboratory of Cell Biology, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China; 2. Functional Genomics Facility, Division of Biological Science, University of Chicago, Chicago 60637, USA; 3. China National Center for Biotechnology and Development, Ministry of Science and Technology, Beijing 100081, China Correspondence should be addressed to Qi Liwang (email: lwqi@caf.ac.cn)

Received January 27, 2006; accepted Feburuary 27, 2006

Abstract Large scale cDNA sequencing and genome tiling array studies have shown that around 50% of genomic DNA in humans is transcribed, of which 2% is translated into proteins and the remaining 98% is non-coding RNAs (ncRNAs). There is mounting evidence that these ncRNAs play critical roles in regulating DNA structure, RNA expression, protein translation and protein functions through multiple genetic mechanisms, and thus affect normal development of organisms at all levels. Today, we know very little about the regulatory mechanisms and functions of these ncRNAs, which is clearly essential knowledge for understanding the secret of life. To promote this emerging research subject of critical importance, in this paper we review (1) ncRNAs’ past and present, (2) regulatory mechanisms and their functions, (3) experimental strategies for identifying novel ncRNAs, (4) experimental strategies for investigating their functions, and (5) methodologies and examples of the application of ncRNAs.
Keywords: non-coding RNA, genetic regulation, cDNA library, gene.

RNAs can be divided into two distinct classes: protein-coding RNAs (mRNAs) and nonprotein-coding RNAs (ncRNAs). The protein-coding RNAs have classical characteristics of “genes” including open reading frames (ORFs), polyadenylation signals, conserved promoter regions and splice sites. It was traditionally believed that protein-coding RNAs controlled cellular functions, and the repertoire of protein-coding genes determined the complexity of an organism[1]. Consequently, for much of the last twenty years up to recently completed genome projects, interest in the transcriptional activity of the genome has been focused almost exclusively on the discovery of protein-coding genes[2]. For ncRNAs, it was previously believed that there were only a few species, such as tRNAs, rRNAs and spliceosomal RNAs, and they
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were considered accessory molecules involved in mediating transcription and translation[3]. These traditional points of view have recently been challenged. First of all, the large scale cDNA sequencing and comparative analyses showed that there is only a weak correlation between biological complexity and the number of protein-coding RNAs. If one uses the numbers of protein-coding RNAs as a basis for evaluating biological complexity, one would reach the conclusions that insects are less complex than nematodes, and rice is more complex than humans. It is clear that protein-coding RNAs are not the only source for determining biological functions. Secondly, the large scale gene expression profiling, molecular cloning and tiling array analyses[4 6] showed that around 50% of human genomic DNA is transcribed while only 2% of

ki. constitutively expressed and necessary for cell viability (Table 2). spliceosome components rRNA modifications trans-translation telomeric DNA synthesis.edu/biochem/rna-sequence/ http://www.mbio. miRNAs inhibit translation by binding with imperfect homology to the untranslated region of mRNA. Now. A group of 21 23 bp RNAs with regulatory functions when associated with a protein complex.edu/tRNAdb/ http://psyche.ag.poznan. in contrast.ncsu. small nucleolar RNAs (snoRNA) functioning in RNA modifications. A group of 21 23 bp RNAs with regulatory functions when associated with a protein complex. they constitute the vast majority of the genomic output of complex organisms[9.edu/smallRNA/Database/Telomere-R NA http://www. Acronym ncRNA miRNA siRNA tncRNA snRNA snoRNA Table 1 Definition of some recently described ncRNAs Definition A group of RNAs that are transcribed but are not translated into proteins. Apparently.org. Crick[13] predicted the existence of a functional RNA adaptor that mediates between the triplet genetic code and the encoded amino acid.cn translation & ribosome components translation pre-mRNA splicing.auburn.fr/databases/hoppsigen. Similar percentages were also observed in mouse[5.202 Science in China: Series C Life Sciences the transcripts is translated into proteins and the remaining 98% is ncRNAs[7].narna.edu/mirror/tmRDB/ http://mbcr.ugent. a breakthrough that is essential for revealing the genetics regulation of the cells.ibch.bio.cgb.wustl. some have regulatory funcnoncoding RNA tions. see Table 1). Name Table 2 Functional classification of major non-coding RNAs Type Early discovred structural ncRNAs rRNA tRNA snRNA snoRNA tmRNA Telomerase RNA Function Database or research tool http://intra.psb.uk/sequences/ http://siRNA. Because both tRNAs and rRNAs assist in translation they are called housekeeping RNAs.ncl. In vitro synthesized double-stranded RNA in 21 23 bp introduced into the cell can ‘silence’ a gene of interest. 1 ncRNAs: Past and present As early as 1958.ac.umass. Reside within the nucleole of eukaryotic cells and participate in methylation or ribosomal RNA modifismall nucleolar RNA cation or processing.sanger.12]. we call these adaptors transfer RNAs (tRNAs). It was subsequently found that some other RNA species are also involved in housekeeping roles. ncRNAs are not limited to a few species in helping RNA transcription and protein translation. such as small nuclear RNAs (snRNA) functioning in pre-RNA splicing. siRNAs small interfering RNA bind to mRNA with perfect bp homology causing mRNA degradation.se http://pbil.17] (for ncRNAs abbreviation.be:8080/rRNA/ http://rna. component of telomerase Ribonuclease P maturation of 5 ends of pre-tRNA Recently discovered regulatory ncRNAs miRNA various regulatory functions siRNA regulation of gene expression Expressed pseudogenes regulation of gene expression Antisense RNAs regulation of gene expression Riboregulators regulation of gene expression Others various regulatory functions .ac. how many have functions? How do they function and what are their functions? Answering these questions represents a major breakthrough in the life sciences[3.bioinfo. Similar in length to miRNAs but are apparently not processed from a miRNA-like hairpin precursor and tiny noncoding RNA are not phylogenetically conserved Reside within the nucleus of eukaryotic cells and involved in RNA splicing or histone mRNA terminasmall nuclear RNA tion. and RNAs important for the transport and insertion of proteins into membranes and telomeric sequence addition[16.html http://www.html http://www.uk/ http://biobases.6] and Drosophila[8]. This intriguing observation raised several fundamental questions: do they all have biological functions? If not.tmc. tRNAs represent the second class of ncRNAs after ribosome RNAs (rRNAs)[15].11. In micro-RNA animals.uthct.edu/RNaseP/ http://microrna.pl/ncRNA http://noncode.edu/dbs/uRNADB/uRNADB. These early discovered housekeeping RNAs are usually small.univ-lyon1. Crick’s adaptor hypothesis was later biochemically demonstrated by Hoagland and his colleagues[14].bcm.10].

both are excised from longer. Antisense RNAs are another group of ncRNAs with regulatory functions. double-stranded RNA precursors. Vol 309).27]. Dicer. stability and translation[33. The definition of regulatory ncRNAs can be traced back to 1969 when Britte and Davidson first explicitly pointed out that ncRNAs can regulate gene expression through complementary base-paring[18].34]. or an epigenetic control of gene expression[37]. Because miRNA can function with a partial complementarity to target mRNAs. (2) siRNAs arise from a long (hundreds to thousands of base pairs). but miRNAs are initially transcribed (from DNA) to a single strand of RNA. mobile genetic elements. Previous experiments showed that in many cases the sense and antisense transcripts are produced at the same time and there is no correlation between the amount of the latter and the expression of a protein from the former[35]. A miRNA triggers RNA degradation when it is highly complementary to its target. and inhibits RNA translation when it is poorly complementary to its target. These similarities suggest that siRNAs and miRNAs have a common evolutionary origin.29]. siRNAs and miRNAs are the two best studied families in the ncRNA world[21 24]. people started to realize that the ncRNAs not only passively assist in protein translation but also actively regulate gene transcription and translation. siRNAs function through a RNAi pathway[25] and require full complementarity to target mRNAs. this apparently important hypothesis did not receive deserved attention when it was initially published in Science until the discovery of the catalytic properties of the RNA subunit of ribonuclease P[19] and the self-splicing activity of group I introns[20]. Based on the computer predictions. These two classes of ncRNAs have many common characteristics: they have a similar size (21 23 nucleotides) and a similar mechanism of generation. it has recently been found that ncRNAs represent the main genomic output of transcriptional activities and commonly exist in eukaryotes (Table 2). another large group of ncRNAs have regulatory roles. one siRNA normally has one unique target. one miRNA may have up to 100 target RNAs[28]. It should be pointed out that not all antisense RNAs act as negative regulators of their corresponding protein-coding RNAs. and therefore. miRNAs can guide target degradation by entering the RNAi pathway like siRNAs but can also inhibit the translation of target mRNAs through partial complementarity to their 3′-untranslated region (UTR) by a currently unknown mechanism[26. and other foreign DNA invasions. suggesting that antisense RNAs can function independent of the overlapping protein-coding gene and may participate in some general regulations[36]. they all target protein coding RNAs. It was estimated that about 20% of human genome transcribes antisense RNAs[31]. Through the high-throughput genomic approaches. miRNAs’ targets can potentially account for more than one third of protein-coding genes in human genome[30]. i. but at least some of them can act on their target RNAs by complementary base-pairing and then affect their splicing. recent large scale EST sequencing and analyses indicate that actual percentage of the transcribed antisense RNAs could be even higher[32]. However. double stranded-RNA-precursor molecules by an RNase III-like enzyme. these two groups of the ncRNAs have their own functional characteristics: siRNAs represent a self-defense system protecting the genome from viruses. they all need to be assembled into the RNA Induced Silencing Complex (RISC) and in both cases only one strand functions. the human genome includes approximately 250 1000 miRNAs[21. . Whether a miRNA degrades the target mRNA or inhibits its translation will depend on the degree of complementarity to target mRNA sequence. in contrast. which then folds over forming a relative short (~70 bp) double-stranded ‘hairpin’ precursors. Unfortunately. and are thus called regulatory ncRNAs.e. In addition. After these unexpected findings. see September 2 Issue of Science. they have developed their own personalities including that (1) siRNAs can derive from coding or non-coding RNAs while miRNAs derive exclusively from non-coding RNAs.Genetic regulation by non-coding RNAs 203 Relative to the small group of housekeeping ncRNAs. Therefore. miRNA is an endogenous regulatory system guiding development in an organism. Because of the differences between siRNAs and miRNAs. We do not know to what extent these antisense transcripts are functional. during evolution. These ncRNAs are involved in various genetic regulations through multiple mechanisms (for the recent development in the RNA world.

~3% of which is transcribed[38]. coli E. to humans. In the past 30 years. It seems probable that many transcribed pseudogenes are not transcriptional noise and that they have important functions. However. to mRNA and to protein. recent experiments showed that the sense strand of the expressed pseudogenes can regulate the mRNA stability of their homologous coding genes[42] while their antisense strands can regulate the translation of their homologous coding genes[43]. Table 3 Representative regulatory mechanisms of ncRNAs Mechanism Transcriptional repression Post-transcriptional regulation Translational repression Translational activation DNA methylation DNA demethylation Modification of the histone proteins Regulation of chromatin structure Regulation of mRNA stability Dosage compensation Genomic imprinting X chromosome inactivation X chromosome activation Orgnism Several orgnisms Mouse E. the high organisms require coordinated actions of hundreds of genes to synthesize a specific cell type or tissue at a given time and place[54]. It was previously believed that pseudogenes have no biological functions because they are normally lacking functional promoters and corresponding regulatory elements[41].47]. [55] [56] [57] [58] [59] [37. Similar to the regulatory mechanism of the antisense RNAs. the functions of proteins as regulatory factors are well known.13]. Riboswitches are located in the 5′-untranslated regions (5′-UTRs) of mRNA with structural-recognition elements for specific metabolites. three new regulatory pathways are included: from ncRNA to DNA. It is theoretically possible for scientists to design artificial riboregulator systems with any small molecule to regulate any target genes[45]. In the mouse. It is the central nerve of the regulation and critically important for determining the complexity of organisms. With a few exceptions[46. Due to the prevalence of riboswitches across diverse organisms from prokaryotes. which recently draws scientists’ attention. and easy degradation compared to proteins offers higher regulatory sensitivity[11. Fig. from an evolutionary point of view. ncRNAs as regulators have at least equally. important roles compared to proteins. functions of the recently discovered ncRNAs remain unknown[48 53]. there are many other ncRNAs that do not belong to any above mentioned groups with the size ranging from 100 10000 bp. In fact. Binding of these small molecules leads to alternative structures that can modulate translation initiation or transcription termination[44].40]. Riboswitches (or riboregulators) represent the final group of ncRNAs with significant theoretical and practical potentials. if not more.60] [60. a novel class of riboswitches was recently identified that functions independent of the downstream mRNA sequences to which it is attached[45]. scientists have started to engineer artificial riboregulator systems to target a specific small molecule.204 Science in China: Series C Life Sciences A new family of ncRNAs is the expressed pseudogenes. RNA and protein through multiple genetic mechanisms (Table 3). but the regulatory roles of RNAs are poorly documented. the lack of ORFs makes ncRNAs less sensitive to point mutation. coli Arabidopsis Human Arabidopsis Yeast Mouse Drosophila Human Human Human Example Riboswitches miR-196 DicF RprA miRNA KHPS1a ncRNA ncRNA Makorin1-p1 roX1/roX2 AIR XIST TSIX Ref. eukaryotes. In the direction of genetic regulation. 2 Regulatory mechanisms and functions of ncRNAs During development. In the direction of information flow. ~5000 processed pseudogenes have been identified and. The human genome is estimated to contain up to 20000 pseudogenes. 1 shows revised central dogma that combines the genetic information flow with the genetic regulation. two new information flow pathways are added: from DNA to ncRNA and from mRNA to ncRNA. In addition. These extraordinary discoveries have made it necessary to revise the central dogma. They can regulate DNA. Recent experiments show that during the cellular development. These RNA-mediated genetic network appears equally or more important than protein-mediated regulatory network. to date.61] [62] [38] [63] [64] [65] [66] . 48 have been detected in expressed cDNA libraries[39. RNAs as regulatory molecules have several advantages over proteins: expression of RNA without subsequent translation consumes less energy. respectively.

In Drosophila. Abnormal expression of imprinted loci has been found to underlie several human genetic disorders. chromosome inactivation that is transcribed from the antisense of Xist locus is Tsix. The precise role in X-chromosome silencing is not clear. which triggers doubling of the expression of the genes located on the male X chromosome[68 70]. Angelman syndrome and Prader-Willi syndrome. males and females differ in the number of X chromosomes. protein translation and their functions (dotted grey arrows). mRNA expression and protein translation through the interaction with DNA and mRNA (dotted black arrows). Developed Central Dogma.1 Transcriptional regulation sation Dosage compen- In most animals. Many ncRNAs are known to be derived from the imprinted loci. Maintaining the expression status of the imprinted genes is critical for normal development in mammals. IGF2R/M6PR. including IGF/H19. The MSL complexes then modify histone H4. poly-adenylated ncRNA ever identified. IGF/H19 was not only the first imprinted locus to be identified. It has been proposed that Xist can selectively methylate or demethylate histone isoforms H3 and H4. mRNA expression. One of the best studied ncRNAs is the DsrA RNA in E. The figure was revised on the basis of “Steering wheel of life”[11]. which is involved in two ncRNAs: roX1 and roX2. Xist is transcribed from the inactivated X chromosome and remains associated with the chromosome after transcription. dosage compensation is accomplished by two-fold enhancement of gene expression from the male X chromosome. Tsix can inhibit the function of Xist by base-pairing between Xist and Tsix which might interfere with the binding of proteins to Xist RNA[67].Genetic regulation by non-coding RNAs 205 Fig. Xist (X inactive specific transcript) represents an excellent example of dosage compensation. DLK1-GTL2 and PWS/AS[71]. It is known that ncRNAs play key roles in dosage compensation. The differential methylation represses the paternally derived H19 allele and the maternally derived allele of the adjacent insulin-like growth factor 2 (Igf2). This locus contains a key imprinting control element that is a differentially methylated domain (DMD) 2 4 kb upstream of the H19 transcription start. 2. including DiGeorge syndrome. The different species equalize the expression levels of X-chromosome genes either by X-chromosome inactivation in XX cells or by upregulation of the single X chromosome in XY cells. and that protein regulates DNA structure. The classic central dogma believes that genetic information flows from DNA to protein through mRNA (solid black arrows). Developed central dogma shows that genetic information can also flow from DNA and mRNA to ncRNA (solid red arrows). 2. which lead to the modification of chromatin structure. This modification is important for the deposition of silencing factors. This leads to the . Over-expression of this 87 nucleotide RNA suppresses translation of the global repressor H-NS and stimulates translation of the stress-response σ factor (RpoS) of RNA polymerase. The product of Xist transcription is a 17 kb ncRNA which is able to inactivate one of the X chromosomes in females. coli. another ncRNA associated with X- Genetic imprinting is a process by which differential DNA methylation of one of the two parental alleles of a gene results in preferential silencing of the allele from one parent. KCNQ1. Interestingly. and that ncRNA regulates DNA structure.3 Translational regulation Many ncRNAs are involved in the modulation of translational efficiency at the post-transcriptional level.2 Transcriptional regulation Genetic imprinting 2. These two RNAs are responsible for the assembly of the MSL protein complexes and their targeting to specific sites on the male X chromosome. Of those. This process of equalization is referred to as dosage compensation. but also provided the first example of a spliced. 1.

glucocorticoids and progestins[76]. transposons jump.59]. ncRNAs affect normal development of organisms at various levels (Table 4). such as triggering mRNA degradation. interfering RNA splicing. The existence of 6S RNA has been known for over 3 decades. an antisense non-coding transcript. leading to unspecific alteration of the transcriptional activities within the affected chromosomal region (activation or suppression)[65. in Drosophila. For example. flowers take on shapes unlikely to please a bee. Interaction with the 5′ portion of DsrA competes with the formation of the secondary structure. has been found to be associated in a ribonucleoprotein complex with the steroid receptor coactivator 1 (SRC-1). polyadenylation. The interaction with σ 70 may be responsible for the general reduction in the transcription of σ 70-dependent genes. cell differentiation[83]. including receptors for androgens. lipid metabolism[86]. 2. ncRNAs act on proteins to modulate protein structure. and protein localization[90]. Recently. DsrA inhibits the translation of hns RNA through a similar mechanism of RNARNA interaction. Without these ncRNAs. Numerous experimental data show that ncRNAs regulate various cellular. estrogens. elegans is a classical example in the control of development[107]. Wassarman and Storz[75] used UV crosslinking experiments to demonstrate that 6S RNA regulates the activities of RNA polymerase through directly contacting the σ 70 and beta/beta' subunits of the enzyme. a novel non- coding RNA. and consequently influence their functions.66]. activities and functions[61. including cell proliferation[84]. respectively[84]. DsrA interacts with both 5′ and 3′ portions of the ORF of the hns mRNA[73]. In this case. Interested readers can find many more similar examples of antisense regulation from recent literatures[74]. There is a secondary structure within the 5′UTR of rpoS mRNA that serves as a cis-acting inhibitor of translation. and consequently promotes translation of rpoS[72]. The complementary base-pairing represses translation of the HFE mRNA. and to function as a specific co-activator of several steroid receptors. Through the regulatory mechanisms mentioned above. and cells fail to divide due to lack of functional centromeres[21]. . RNA transportation and formation of translation machinery. originating from the antisense strand of the human HFE gene. organ development[81]. the brain and muscles fail to develop.4 Regulation of protein functions ncRNAs not only regulate transcription and translation through multiple mechanisms.109].77 79]. elegans. bantam miRNA in cells of the fly larva represses Hid protein through a similar regulatory mechanism of Lin-4 to control the selection of programmed cell death and cell proliferation. miRNA lin-4 in C. to affect RNA stability. RNA splicing. ncRNAs can also modify DNA by methylation or demethylation to regulate transcription of the corresponding genes[37. For example. The experiment showed that 6S RNA accumulates as cells reach the stationary phase of growth. Deregulation of ncRNAs can lead to abnormal development of organisms and causes many human genetic diseases. apoptosis[85]. The antisense base-pairing can also suppress protein translation through other mechanisms. plants succumb to viral infection. In humans and mice. editing. and protein translation. two key regulators of early larval developmental transitions in C. ncRNAs act on the promoter of a gene to specifically stimulate or repress transcription of the gene. Translational activation of RpoS by DsrA depends on direct RNARNA interactions between the 5′ untranslated region (UTR) of the rpoS mRNA and the 5′ portion of DsrA. includes a portion complementary to exon 1 of the sense transcript.206 Science in China: Series C Life Sciences induction of two groups of genes: those repressed by H-NS and those activated by RpoS. In summary. but also affect the structure and activities of a protein by directly binding with the protein. Similar roles for lin-4 as temporal regulators of development are also found in other animals[108. ncRNAs act on DNA to modulate chromatin structure. insulin secretion is dysregulated. but its function remains a mystery. Up-regulation of lin-4 RNA in the second larval stage represses the expression of LIN-14 and LIN-28. Another class of ncRNAs that can regulate translation (including siRNAs and miRNAs) depends on antisense interactions with target mRNAs. the steroid receptor activator (SRA) RNA. ncRNAs act on RNAs to induce RNA degradation. physiological and biological events.

start and stop codons.g. yeasts and worms using a similar comparative sequence analysis strategy[115.114]. Numerous novel ncRNAs have also been identified in other E. Rivas et al. [80. mouse and human genomes shows that there is significant conservation outside protein coding sequences. miR-145 PCGEM1 Let-7 H19 HIS-1 miR-15a. miRNAs NRSE. which reveals commonly conserved sequence segments as well as secondary structure.112]. open reading frames. The biological functions of many of these have been experimentally confirmed[113. Cell differentiation Cell proliferation Regulation of apoptosis Fat metabolism Modulation of behaviour Formation of photoreceptors Regulation of insulin secretion Regulation of protein localization Disease events Breast cancer Colon cancer Prostate cancer Lung cancer Liver cancer Myeloid leukemia B-CLL B-cell neoplasia Angelman syndrome Beckwith-Wiedemann Syndrome Schizophrenia and bipolar Spinocerebellar ataxia Prader–Willi syndrome Alzheimer’s disease Psoriasis Russel-Silver syndrome Organism human human Drosophila human Drosophila mouse rat mouse Drosophila human human human human rat mouse human human human human human human human human human human Example Let-7. The computational detection of ncRNAs is far more difficult than the detection of protein-coding genes.81] [82. Based on the comparative sequence analysis. The degree of the conservation is equivalent to or greater than that of protein-coding sequences[110]. The increasing number of sequenced model organisms makes this strategy practically feasible. and therefore are most likely an evolutionary consequence under a specific selection pressure[36]. Comparison of the dog. This finding provides a theoretic foundation for using the comparative sequence analysis strategy to predict ncRNAs. An efficient means of finding ncRNAs is comparative sequence analysis across multiple species. coli strains.83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] 207 3 Strategies for the identification of ncRNAs 3. QRNA is one of the widely utilized statistical package. Although ncRNAs do not have commonly conserved characteristic features like mRNA. polyadenylation sites and so on). miR-143 Bantam ADAPT33 Mir-14 Bc1 TUG1 miR-375 hsr BC200 miR-143.1 Computational predictions of ncRNAs ncRNAs are normally short and often lack the conserved characteristic features of the protein-coding genes (e. applied QRNA to the "intergenic" spacers of Escherichia coli using comparative sequence data from four related bacteria. resulting in insufficient statistical signals by their primary sequence. The size and distribution of the conservation are not consistent with neutral drift from a common ancestral sequence.116]. They predicted 275 candidate ncRNA loci[111.Genetic regulation by non-coding RNAs Table 4 ncRNAs regulate various physiological and pathological events Event Normal events Embryo development. miR-16a BCMS UBE3A/SNURF-SNRPN LIT1 DISC2 SCA8 ZNF127AS BC200 PRINS MESTIT1 Ref. scientists have developed several software products to predict ncRNAs. some signatures are likely to exist within a single class of .

edu/mirscan/ http://www. 3. Table 5 summarizes recent software products that are commonly used for the predicSoftware/database name Software tRNAScan-SE snoscan snoGPS mirscan RNAmotif Erpin PatSearch QRNA DDBRNA MSARI RNAz Database RNAdb NONCODE Rfan Erdmann ASRP miRBase SiRNAdb HuSiDa tRNAdb Plant ncRNA tmRDB RDP-II Predicted/involved molecule tRNAs C/D Box snoRNAs H/ACA snoRNAs miRNAs ncRNAs ncRNAs ncRNAs ncRNAs ncRNAs ncRNAs ncRNAs ncRNA database ncRNA database ncRNA database ncRNA database small RNAs miRNAs siRNAs human siRNA tRNAs ncRNAs tmRNAs rRNAs tion of ncRNAs and related ncRNA databases.edu/snoscan/ http://lowelab.org. ncRNA species usually exhibit sizes between 50 and 500 nt[138].imb.ucsc.cgb. Consequently.it/research/dibernardo/default.ac.mit.edu/ Ref.edu/tRNAscan-SE/ http://lowelab.it/BIG/PatSearch/ http://www. The construction of a ncRNA-cDNA library differs from that of the traditional cDNA library in two aspects.poznan.scripps.cgrb.ucsc. For example. but also significantly faster and more suitable for large-scale genomic screens.mit.edu/PLANTncRNAs/ http://www. Ambion and Invitrogen have recently released reagent kits that are specifically Web address/database http://lowelab. rat. followed by sequence analysis and genomic localization of the genes.prl.html http://tagc.pl/ncRNA/ http://asrp.hu-berlin. It combines comparative sequence analysis.de/~nebulus/sirna/v2/ http://www.univ-mrs.se http://itb.ucsc.edu/mb/case/casegr-sh-3.msu.ag.csail.cnr. ncRNAs can also be identified by experimental screens.125].208 Science in China: Series C Life Sciences ncRNAs.uni-bayreuth.[126].ki.2 Experimental identification of ncRNAs In addition to the above described computational approach.edu/eddy/software http://www.ac.genetics.uq.edu/snoGPS/ http://genes. Fugu. Current experimental approach for the identification of ncRNAs is to construct specialized cDNA libraries. Recently.tbi.edu/ http://biobases. and zebrafish.at/~wash/RNAz http://research.47] [130] [131] [132] [133] [134] [135] [136] [137] Table 5 A summary of software and databases for ncRNAs . This integrated approach is not only much more accurate than previous methods[112. This method does not require pre-knowledge on DNA sequence and possible secondary structure and is thus suitable for the prediction of different classes of ncRNAs.edu.tRNA. First of all.sanger.wustl.ibch.oregonstate.itb.univie. [117] [118] [119] [120] [121] [122] [123] [112] [124] [125] [126] [127] [128] [129] [46.cme. including tRNAScan-SE for tRNAs. The authors applied this method to screening conserved noncoding elements in upstream regions of orthologous genes from human.cn http://rfam.auburn. RNAs that range from 17 to 25 nt should be selected specifically by gel electrophoresis. This new area of research that seeks to identify ncRNA genes by an experimental approach is called “RNomics”.fr/erpin/ http://www. snoscan for snoRNAs and mirscan for microRNAs.tigem.htm http://theory.biologie.uk/sequences/ http://siRNA.ba.de http://www. reported software which is able to quickly predict ncRNAs.wustl.msu.bioinfo. mouse. some custom-designed software have been developed to search for a specific class of ncRNAs (Table 5). They found many conserved RNA secondary structures not described so far to our knowledge and recovered all of the known ncRNAs. it is necessary to perform a size selection by gel electrophoresis before the construction of a library.edu http://microrna.au/rnadb/ http://noncode. These small RNA molecules can be isolated and purified based on the molecular markers on the gel. for the construction of a library aimed at identification of miRNAs and siRNAs. structure prediction and measurement of thermodynamic stability together.5.edu/MSARi/ www.edu/mirror/tmRDB/ http://rdp. Washietl et al.

small-scale experiments of this nature are possible. whether all ncRNAs have biological functions. It should be pointed out that current RNAi methods generally function in the cytoplasm while many ncRNAs are located in the nucleus or nucleolus of the cell. coli[146]. Through cell-based assays. the in vivo functions of ncRNAs can only be confirmed by gene knockout experiments. Drosophila[140].[147] have recently reported a deletion of 2. To avoid redundant expression of already known ncRNA species. In the next few years. followed by phenol extraction (Fig. what percentage of these ncRNAs has function.g. they selected 512 ncRNA sequences from the RIKEN Fantom2 mouse cDNA collection that showed significant conservation with human genomic sequences. Microarray technologies offer a great potential in finding ncRNAs. which includes sequence encoding four novel ncRNA transcripts. a technology that has rapidly evolved in the last few years. and mouse[141]. However. revealing the biological functions of ncRNAs will be a central subject of investigations in the life sciences. ribonucleo-protein particles (RNPs) can be immuno-precipitated with specific antibodies directed against known RNA-binding proteins. first strand reverse transcription cannot be initiated by dT primers. secondary structure analysis and chromosomal localization. and then designed two DNA vector-encoded short hairpin RNAs (shRNAs) for each of the mouse-human conserved ncRNAs.149].3 Mb of mouse genome. 2). of which 6 are essential for cell viability. the method for delivering siRNA needs to be improved. This physiology-based screen identified 8 functional ncRNAs. An alternative to ncRNA-transcript knockouts might be systematic inactivation of ncRNA transcripts by RNAi ‘knock-down’. This shRNA collection was arrayed in 384-well tissue culture plates. 4 Experimental strategies for revealing functions of ncRNAs Early studies show that ncRNAs are involved in various genetic regulations at multiple levels. elegans[139]. this experiment demonstrates the feasibility of using the gene knockout approach to study the functions of multiple genes at the same time. during a specific time and at a specific location. BlastN). novel ncRNAs can be identified by database searches (e. Ultimately. Clues to the functions of the novel ncRNA transcripts might emerge from their expression patterns in a specific tissue. After sequencing. a number of ncRNAs have been isolated from E. The functions of the novel ncRNAs can be potentially predicted if they follow into the same group as those of known functions. 2). Clearly. To identify a specific class of ncRNAs that associates with specific RNA-binding protein(s). Such RNAi knock-down has been applied to the analysis of all of the protein-coding genes in the cells[148. because many ncRNAs lack a polyA tail.Genetic regulation by non-coding RNAs 209 designed for the rapid isolation of a large quantity of small RNAs from the cells or tissues.145] and E. Secondly. testing thousands of transcript knockouts is practically not feasible. which can be used to systematically identify all transcripts in the genome[143]. in contrast. Through the construction of specialized cDNA libraries. followed by RT-PCR amplification. Affymetrix has recently released the genomic tiling arrays. methods to examples Experimental data show that abnormal expression of . Although they did not observe a visible phenotype. The resulting cDNAs are cloned into a vector and sequenced (Fig. Use of cluster analysis approach can classify genes based on their expression patterns. a specific class of ncRNAs that associates with specific RNA-binding protein(s) can be identified[142]. 5 Applications of ncRNAs: From mechanisms. cDNA clones can be spotted on filters in high-density arrays and screened with oligonucleotide probes directed against the most abundant known ncRNA species.[150]. C. Experimental strategies for functional studies of ncRNAs are similar to those for protein coding genes. coli[114]. In this experiment. An excellent example is the experiment recently conducted by Willingham et al. This kind of arrays has been successfully utilized in finding new ncRNAs in humans[144. they can determine the roles of these ncRNAs by monitoring key cellular processes and signaling pathways. Using a similar strategy described above. it requires the addition of linkers of known sequence to both ends of ncRNA by RNA T4 ligase. Nobrega et al. To study the functions of ncRNAs using siRNA technology effectively. However. and has what function remain to be answered.

ncRNAs affects various important traits during development and causes multiple human diseases[151].210 Science in China: Series C Life Sciences Fig. ncRNAs. scientists have successfully induced directional expression of the . As described above. Upper right for a general purpose cDNA library and upper left for a specialized cDNA library encoding a specific ncRNA subclass. current applications of ncRNAs are primarily restricted to siRNAs and miRNAs which we know relatively more regarding their functions compared to other groups of ncRNAs. as a genetic engineering tool. these two groups of ncRNAs can trigger degradation of their target mRNAs or inhibit their translation by complementary base-paring. Strategies for the construction of cDNA libraries of ncRNAs. 2. Therefore. Based on these mechanisms. have a bright future in improving important economic traits and preventing/treating human diseases. Due to the limited functional knowledge on the majority of ncRNAs.

which is able to efficiently protect and deliver siRNAs in vitro and in vivo. The vector-based systems offer the advantage of high delivery efficiency and high level of expression. A set of guidelines for the selection of siRNAs appropriate for a given sequence are available in the literature. However. the vector-based systems need to deliver vector DNA. Bitko et al. The ability to temporally and spatially regulate the expression of siRNA has great values in practice. in collaboration with Novartis and Merck. caffeine content in coffee plants has been markedly reduced by siRNA-mediated suppression of the caffeine synthase gene[159]. a novel class of chemically engineered and cholesterol-conjugated oligonucleotides. the growth of different types of tumors can be inhibited by the direct application of siRNAs targeting key oncogenes[156. topological application. However. Alnylan Pharmaceuticals (http://www.164]. In addition. 6 Conclusions Recent experiments show that more than 50% of genome in higher organisms is transcribed. In contrast. There are two main methods for delivering siRNA: direct delivery and vector-assisted delivery. The efficiency for in vivo siRNA delivery can be improved by forming siRNA complex with some small molecules that have a high affinity with cell membranes. By directly instilling siRNA into the nose of the mouse. At present. Urban-Kleil et al.com). soaking and gene gun shooting. which targets viral amplification gene. The vector-based delivery is first to clone a DNA segment encoding siRNA into a plasmid or viral vector and then deliver the construct to the cells or tissues by the traditional methods. has been developed specifically for targeting miRNAs[157]. tissues or organisms so that the traits that are under the direct control of these genes can be manipulated favorably[151]. their feasibility has been tested and proved in many in vivo experiments[151]. the vast majority of which are ncRNAs.com/ techlib/tb/tb-506. siRNA mediated by a hairpin RNA has been used in cotton to downregulate two key fatty acid desaturase genes resulting in improved fatty acid composition of cotton-seed oil[160]. In addition.[162] showed that infections by respiratory syncytial virus can be specifically prevented and inhibited by the siRNA.html) for details. The genome-wide . Here. Interested readers can refer to relevant papers[152 154] and websites (http://www. into the cells or tissues all together.163. The advantages of direct delivery are ease of use and suitability for high-throughput screening. termed ‘antagomirs’. particularly viral vector DNA. we will focus on the key methodologies for introducing siRNAs into the cells. In another study. which raises safety concerns. It appears that the vector-based delivery has great potential in improving agriculturally important traits while the direct deliver has more practical values in the treatment of human diseases.Genetic regulation by non-coding RNAs 211 target genes by delivering siRNAs into the cells. In several other mouse models. the instability of siRNA molecules in vivo and their often poor uptake into target tissues remain two major practical issues. The design of siRNA has been a subject of intensive studies. siRNA has been used to generate a dominant high-lysine maize variant by knocking out the expression of the 22-kD maize zein storage protein[161].[155] demonstrated that siRNA modified with 2′-fluoro (2′-F) pyrimidines is nuclease-resistant and shows a greater stability in vivo. scientists have tried to increase their stability by chemically modifying siRNA. which compensates for the disadvantages of direct delivery approach. Cystic Fibrosis and so on. specifically develop direct siRNA therapeutics in an attempt to treat Respiratory Syncytial Virus infection. the direct delivery technology has not been formally approved in any case in the treatment of human diseases except for a few clinical trails in the treatment of the skin and eye diseases. Parkinson’s Disease.[156] recently described a polyethylenimine complexation method. The vector-based delivery technology has been successfully used in improving the nutritional value of plants[158]. alnylam. Layzer et al. For example. Pandemic Flu. The safety concerns become a major obstacle in the clinical applications of vector-based siRNA. Direct delivery is to introduce synthesized doublestranded RNA in the length of 21 bp into the cells or tissues by needle injection. However. Spinal Cord Injury.ambion. the vector-based systems allow the incorporation of regulatory elements to the promoter region of the expression plasmid resulting in tissue-specific silencing at a given time. They have recently made some progresses in those therapeutic development programs.

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