A New Frontier for Molecular Medicine: Noncoding Rnas

Biochimica et Biophysica Acta 1756 (2005) 65 – 75 http://www.elsevier.com/locate/bba Review A new frontier for molecular medicine: Noncoding RNAs

Maciej Szymanskia, Miroslawa Z. Barciszewskaa, Volker A. Erdmannb, Jan Barciszewskia,*

aInstitute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12, 61704 Poznan, Poland bInstitut fur Chemie/Biochemie, Freie Universitat Berlin, Thielallee 63, 14195 Berlin, Germany

Received 30 May 2005; received in revised form 27 July 2005; accepted 28 July 2005 Available online 15 August 2005

Abstract

It is now becoming evident that the variety of noncoding RNA (ncRNA) molecules play important roles in many cellular processes and they are not just mere intermediates in transfer of genetic information from DNA to proteins. Recent data, from the analyses of transcriptional activity of human genome, suggest that it may contain roughly equal numbers of protein- and RNA-encoding transcription units. Many of the ncRNAs described in humans as well as in other mammals have been linked, through specific chromosomal localization or expression patterns, with certain diseases including complex congenital syndromes, neurobehavioral and developmental disorders and cancer. These findings clearly indicate that an expression of genes of which end-products are RNA molecules is crucial for development, differentiation and normal functioning of the cells. The ncRNAs expression patterns can therefore be used as molecular markers for specific diagnostic methods. D 2005 Elsevier B.V. All rights reserved.

Keywords: Molecular medicine; RNA; DNA

Contents

1. Introduction ...... 65 2. Noncoding RNAs...... 66 3. Noncoding RNAs in the human genome ...... 67 4. ncRNAs and human diseases ...... 68 5. ncRNAs and human cancers ...... 69 6. Noncoding transcripts from imprinted genes ...... 71 7. Perspectives...... 72 Acknowledgements ...... 72 References ...... 72

1. Introduction genomic DNA itself does not answer all questions concern- ing transmission of genetic information or the molecular Although, there is no doubt that a completion of human basis of human diseases. Immediately, it has been realized genome project marks a beginning of a new era in molecular that not only genetic coding (DNA sequence), but also epi- biology, it is now evident that the sequence of human genetic endowment (beyond DNA sequence) is an important factor in the architecture of the genome and the decoding * Corresponding author. Tel.: +48 61 8528503x132; fax: +48 61 process. The understanding of the genome requires also a 8520532. detailed knowledge on the regulatory mechanisms control- E-mail address: [email protected] (J. Barciszewski). ling the spatial and temporal expression of particular genes.

The most surprising discovery revealed by the analysis of the human genome was the number of protein genes of around 30,000. Their open reading frames (ORFs), comprise less than 2% of the 3.2 billion bases [1–3]. Despite a controversy as to a real number of protein genes, with the latest estimates as low as ¨25,000 [4], these figures are at least 2–3 times lower than it was previously believed. Thus, human and other mammalian genomes contain large portions which do not code proteins. In the humans a significant fraction of genomic DNA is composed of repetitive sequences which account for 46% of the genome [1,3]. The noncoding parts of protein-coding genes (introns, 5V- and 3V-UTRs) comprise ca. 25–27% of the genome (Fig. 1) [2]. Although the functions of the remaining quarter of genomic DNA is largely unknown, it is clear that at least some of its portions are responsible for spatial and temporal coordination of gene expression. One should keep in mind that the above considerations are based on the assumption Fig. 2. Noncoding DNA in selected eukaryotic genomes. An increase in a that any given fragment of genomic DNA is transcribed complexity of organisms is accompanied by the accumulation of DNA only in one direction and is part of one transcription unit. sequences which are not translated, but most likely play a regulatory role This simplified view is, however, not entirely accurate. In either directly or through noncoding RNAs. both human and mouse genomes, there is significant number of loci in which a bi-directional transcription occurs genomes, earlier regarded as a ‘‘junk DNA’’, may play a giving rise to sense–antisense pairs of transcripts [5]. crucial role in regulation of complex mechanisms which The importance of DNA regions which do not code for underlie development and differentiation by means of proteins is evident when one compares their content in the controlling the expression of proteins which can be viewed sequenced genomes. In fact, there is a reverse correlation as a cell’s hardware [8]. between the contribution of protein-coding regions and the complexity of organisms (Fig. 2) [6,7]. In prokaryotes, where intergenic and untranslated regions are short and 2. Noncoding RNAs splicing is an exception rather than the rule, ORFs account for over 90% of genomic DNA. Among eukaryotes, Recent results obtained from the analyses of single noncoding DNA regions constitute 10–40% in simple human chromosomes suggest that approximately half of the unicellular organisms, 70–90% in invertebrates and as human genomic DNA is transcribed [9]. This contrasts with much as ca. 98% in mammals. Thus, noncoding parts of the the assessments of the number of genes which code for proteins. It is now widely accepted that a significant fraction of the transcriptional output from the genome consists of untranslated or noncoding RNAs (ncRNA). A broad definition of these RNAs includes any transcript or its fragment which is not used as a template in ribosomal protein synthesis [8]. The functions of transfer and ribosomal RNAs (tRNA, rRNA) in translation, and small nuclear RNAs (snRNA) in pre-mRNA splicing have been recognized many years ago. It was followed by discoveries of RNA species involved in other housekeeping roles like DNA replication (telomerase RNA) or RNA modifications (snoRNAs). A new class of ncRNAs that has emerged in recent years consists of regulatory RNA molecules (riboregulators) which are key players in many mechanisms controlling expression of genetic information (Fig. 3). Fig. 1. Coding and noncoding DNA in human genome. Based on a protein- Unlike the housekeeping RNAs, the riboregulators are coding capacity, the genomic DNA can be divided into two parts. The usually not constitutively expressed. Their synthesis is often coding part (¨2%) consists of the open reading frames (ORFs). The tissue- or developmental stage-specific, In some instances, noncoding part (¨98%) comprises untranslated parts of protein coding genes-introns, UTRs-(27%) and repetitive sequences (46%). The remaining they are induced as a response to biotic and abiotic changes 25% of the genome is a repository of regulatory elements including in the environment [10]. The majority of eukaryotic noncoding RNA genes. ncRNAs resemble transcripts from protein-coding genes. M. Szymanski et al. / Biochimica et Biophysica Acta 1756 (2005) 65–75 67

mutations, unless they occur within regions crucial for interactions with proteins or other RNAs.

3. Noncoding RNAs in the human genome

Initially, most of known ncRNAs have been identified somehow accidentally as novel transcripts, differentially expressed in various cells or tissue types or in response to changing environmental conditions. More systematic approaches, including large scale full-length cDNA sequencing, revealed that ncRNAs constitute a significant Fig. 3. The ‘‘steering wheel of life’’ shows a flow of genetic information fraction of transcripts from mammalian genomes [21–23]. (solid lines) and regulatory interactions between the proteins and nucleic About 70 human ncRNAs have been at least partial acids (dashed lines). Noncoding RNAs have been shown to be involved in characterized in terms of function or expression profiles. control of molecular processes at all stages of expression of genetic In contrast to proteins, whose activities can be more or less information (white arrows). reliably predicted from the amino acid sequences, it is very difficult to anticipate function of a given RNA from its With a few exceptions, they are products of RNA polymer- nucleotide sequence alone. Even for relatively well-studied ase II (PolII) and their primary transcripts are often a subject ncRNAs (e.g. XIST or H19), the amount of data is rather to alternative splicing. The 5V- and 3V-ends of these mRNA- limited and there are very little clues which could suggest like ncRNA are capped and polyadenylated, respectively possible mechanisms through which these transcripts [10]. The parallels with protein-coding genes are not only actually work. There is also a possibility that, as in case restricted to structural features. Recent analyses of human of human globins or yeast SER3 protein, the control of chromosomes 21 and 22 revealed that the expression of a expression of protein-coding genes may depend on tran- majority of ncRNAs is regulated by the same transcription scriptional activity of intergenic regions which encode factors that control the expression of protein coding genes ncRNAs [24,25]. which implies that these transcripts are in fact functional A significant fraction of human ncRNAs originate as [11]. antisense transcripts from protein-coding genes. A bi-direc- The ncRNAs have been implicated in mechanisms which tional expression occurs frequently in both human and affect all levels of transmission of genetic information from mouse genomes [5]. The role of these antisense transcripts is DNA to proteins (Fig. 3). The RNAs interacting with DNA not clear, but it seems that they do not always serve as mere are involved in modification of chromatin structure which negative regulators of corresponding protein-coding genes alters its transcriptional activity. RNA molecules can fold preventing translation, inducing RNAi pathway or affecting into complex structures providing binding sites for proteins mRNA transport, splicing or stability [26,27]. In many or a variety of small molecules [12]. Binding of an RNA to a cases, the sense and antisense transcripts are produced at the protein can change the properties of the latter and the RNA same time and there is no correlation between the amount of molecules have been shown to act as modulators of the latter and the expression of a protein from the former transcription factors [13–15] and RNA polymerase [11]. Therefore, it is reasonable to speculate that, at least in [16,17]. RNAs can also serve as factors recruiting proteins some cases, functions of those ncRNAs are independent of to particular locations within the cell [18,19]. The RNA– the overlapping protein-coding gene. In some cases, RNA interactions may regulate splicing, editing, mRNA antisense transcripts may be involved in an epigenetic stability and translation. Some unidentified cellular RNAs control of gene expression. Such a mechanism has been may be also responsible for conformational change of demonstrated for the sphingosine kinase-1 (Sphk1) gene. normal prion protein PrPC to the infectious PrPSc form [20]. Tissue-dependent, differential methylation of the Sphk1 The ncRNAs as factors controlling the development and CpG island is responsible for generation of various subtypes differentiation offer an unrivalled plasticity that can be a of Sphk1 protein. An antisense transcript, Khps1, induces driving force of evolution [4,8]. It is now clear that without demethylation of CG sites and methylation of non-CG sites a detailed knowledge about this group of RNAs, the within the tissue-dependent, differentially methylated region understanding of genome will not be possible. (T-DMR), thereby changing the Sphk1 expression patterns It is noteworthy that RNAs as signalling or regulatory [28]. molecules also have an advantage over proteins when one Antisense ncRNAs are often produced from imprinted considers the issue of cellular economy. Expression of genes and their expression is usually tissue-specific or RNA, without subsequent translation, consumes less energy limited to certain stages in the development. The protein- and it can be more easily degraded than proteins. The lack coding (sense) and RNA-coding (antisense) transcripts are of ORFs also makes ncRNAs less sensitive to point reciprocally imprinted and it seems that the ncRNAs may be 68 M. Szymanski et al. / Biochimica et Biophysica Acta 1756 (2005) 65–75 required for the silencing of the protein-coding genes DISC1 encodes a large protein related to other proteins [29,30]. This may be accomplished by RNA-induced expressed in the nervous system, DISC2 produces a number chromatin remodelling results in transcriptional inactivation of transcripts 2.5–9.5 kb long without protein coding of imprinted chromosomal region in a manner similar to X- potential. DISC2 gene is transcribed from the opposite chromosome inactivation mediated by XIST RNA [31]. strand and overlaps the 3V-region of DISC1. It has been proposed that DISC2 RNA may be involved in the regulation of DISC1 expression [32]. The t(1:11)(q43,q14) 4. ncRNAs and human diseases also disrupts another ncRNA gene–PSZA11q14 (putative schizophrenia associated gene from 11q14)–which shows The most interesting aspect of ncRNAs is their implica- significantly reduced expression in patients with schizo- tion in a number of human diseases (Table 1). They include phrenia. A homologous gene was identified in mouse but neurobehavioral and developmental disorders as well as the putative ORF is not conserved which suggests that both certain forms of cancer. The changes of expression levels or transcripts are not translated [33]. Because PSZA11q14 is genetic and epigenetic alterations affecting the ncRNAs transcribed from the antisense strand of the first intron of the accompanying malignant processes strongly support the DLG2 gene it has been proposed that its product may functional role of RNA especially in cases when ncRNA function as its cis-antisense regulator. genes are disrupted by chromosomal aberrations. Non- Another translocation, t(7;13)(q31.2;q21), which was coding transcripts, or at least a majority of them, cannot then identified in an autistic boy confirmed earlier reports be considered as only non-functional products of spurious implicating abnormalities affecting the long arm of chro- transcription. mosome 7 in the origin of autism [34]. This translocation Certain ncRNAs have been mapped to chromosomal disrupts the complex RAY1/ST7 locus which produces at regions associated with neurobehavioral disorders, includ- least 18 transcripts originating from both strands [35].In ing autism, bipolar affective disorder and schizophrenia. A addition to the two major transcripts ST7 and RAY1, two number of schizophrenia patients carry a balanced trans- ncRNAs, ST7OT4 and ST7OT3, are produced from the location t(1:11)(q43,q14). Within the breakpoint region of sense strand. Their transcription initiates within the first and chromosome 1q43, two genes called DISC1 and DISC2 tenth introns, respectively. The two antisense RNAs, (disrupted in schizophrenia 1 and 2) were found. While ST7OT1 and ST7OT2, initiate from the first and ninth

Table 1 A list of noncoding RNAs associated with human disorders ncRNA Chromosome Disorder Ref. BCMS 13q14.3 B-cell neoplasia [59] OCC1 12q24.1 Overexpressed in colon carcinoma (2 variants) [48] MALAT-1 11q13 Metastasis associated in lung adenocarcinoma [60] TRNG10 10q21 Various cancer cells [89] CMPD 17q24–25 Campomyelic displasia [90] HOST2 10 Expressed in ovarian cancer cells [49] NSCLC 6p25–6p24 Expressed in non-small cell lung carcinoma [60] NCRMS 12q21 Increased expression in alveolar rhabdomyosarcoma [61] DD3 9q21–q22 Overexpressed in prostate cancer [49] PCGEM1 2q32 Overexpressed in prostate cancer [51] RAY1/ST7 7q31 Autistic disorder [34] DGCR5 22q11 Disrupted in DiGeorge syndrome [91] 22k48 22q11 HIRA intronic transcript deleted in DiGeorge syndrome [92] PSZA11q14 11q14 Reduced expression in brains of patients with schizophrenia [33] DISC2 1q42.1 Disrupted in schizophrenia [32] MEN1 11q13 Multiple endocrine neoplasia type 1 locus transcripts [93] HANC 1q42–43 Expressed in CD4+T lymphocytes infected with HTLV-1 [96] SRA-Del 5q31.3–32 Steroid receptor activator RNA isoform expressed in breast cancer [54] C6orf37OS 6p21 Antisense transcript from C6orf37 locus within diffuse [94] panbronchiolitis critical region PEG8/IGF2AS 11p15.5 Fetal tumours [47] SCA8 (KLHL1 antisense) 13q21 Spinocerebellar ataxia type 8 [95] MESTIT1 7q32 Russel–Silver syndrome [84] COPG2IT1 7q32 Russel–Silver syndrome [86] IPW 15q12 Prader–Willi syndrome [77] LIT1 11p15 Romano–Ward, Jervell and Lange–Nielsen syndromes [29] UBE3A-AS 15q12 Angelman syndrome [82] H19 11p15.5 Tumours [38] PRINS – Psoriasis [37] M. Szymanski et al. / Biochimica et Biophysica Acta 1756 (2005) 65–75 69 introns, respectively. Although, the apparently noncoding progression [42]. These contradicting results can be transcripts harbour short ORFs and the possibility that they explained assuming that the effects of H19 expression encode small peptides cannot be excluded, yet the putative depend on the type or the developmental stage of cells. It translation products do not show any similarity to known may be also due to expression of cell or tissue-specific proteins and are not conserved in other mammals. The role splice forms [45] or the expression of specific H19 RNA- of noncoding transcripts from RAY1/ST7 locusisnot binding proteins [46]. known, but the antisense transcripts may play a role of Another imprinted gene implicated in cancer is a negative regulators of translation of the protein coding paternally expressed antisense RNA PEG8/IGF2AS. This mRNAs [35]. Interestingly, the RAY1/ST7 gene was also RNA is transcribed from the IGF2 (insulin-like growth described as a tumour suppressor based on the coincidence factor 2) locus. IGF2AS shows significantly elevated of several mutations with certain cases of breast and colon expression levels in Wilms’ tumours and several other cancers [36]. Recently, it has been demonstrated that foetal tumours. The overexpression is restricted to cancer overexpression of ncRNA, PRINS (psoriasis susceptibil- cells and it is not observed in the surrounding normal kidney ity-related RNA gene induced by stress), is associated with tissue. Human IGF2AS contains an open reading frame psoriasis susceptibility. PRINS expression is elevated in encoding a putative 273 aa long protein. This ORF is not cells exposed to certain stress conditions including infection conserved in mouse, and no corresponding protein product by HSV and irradiation with UV-B. It has been suggested has been identified which suggests that it is not translated that PRINS is an element of mechanisms involved in [47]. protection of cells against stress [37]. Specific overexpression of ncRNAs was also found to be a good marker for several other tumours in adults. Colon carcinoma cells show significantly higher levels of the 5. ncRNAs and human cancers OCC-1 (overexpressed in colon carcinoma 1) gene transcripts. OCC-1 is located on human chromosome 12q24.1 The changes in expression of certain ncRNAs has been and produces two, 1.2 and 1.3 kb long, transcripts which associated with some forms of cancer. The first and best- differ at their 5V- and 3V-ends. OCC-1 RNAs show tissue- studied cancer-related RNA is H19 RNA. It was also the specific expression patterns and are absent or expressed at first noncoding transcript identified as a product of very low levels in normal mucosa [48]. In prostatic tumours, imprinted gene. Human H19 gene is located at chromosome there are two ncRNAs which are significantly overexpressed 11p15.5 adjacent to the IGF2 (insulin like growth factor 2) when compared with healthy tissue. The DD3 RNA is a gene. These two genes are reciprocally imprinted with IGF2 product of the DD3/PCA3 (prostate cancer antigen 3) gene and H19 showing paternal and maternal expression, mapped to chromosome 9q21–22, and its transcription respectively. During development, H19 is transcribed at seems to be restricted to prostate. Elevated levels of its very high levels in placenta, embryo and in most of foetal transcript were observed in over 90% of prostate tumour tissues, but after birth its expression is significantly reduced cases [49], and it was shown to be a specific marker which [38]. Although mammalian H19 RNAs show a significant can be used for diagnosis of prostate cancer [50]. Another sequence similarity there are no conserved ORFs, but they prostate specific gene expressing a ncRNA which is up- share, a common RNA secondary structure [39] which is a regulated in tumour tissue is androgens-responsive strong argument for its functionality. It has been proposed PCGEM1 [51]. Overexpression of PCGEM1 correlates that the reciprocal expression patterns of H19 and IGF2 in with increase in proliferation and colony formation which the course of development reflect different roles played by suggested its involvement in regulation of cell growth [52]. the products of these genes. IGF2 is a growth factor which One of the transcripts specific for human ovarian cancer is stimulates cell proliferation while H19 RNA may be HOST-2 RNA (human ovarian cancer specific transcripts). responsible for differentiation [38]. Changes in the methyl- This 2.8 kb long RNA is primarily made of Harlequin LTR ation status of differentially methylated regions (DMR) type elements, does not contain any significant ORF and its upstream of the H19 gene resulting in the loss of imprinting role in the biology and origins of ovarian cancer is not of H19 and IGF2 and biallelic expression of either gene can known [53]. cause malignant cell growth [40]. One of the most interesting ncRNAs is a steroid receptor There are contradicting reports on the role of H19 RNA activator RNA (SRA RNA) involved in the regulation of in cancers. H19 can act as a tumour suppressor gene, gene expression mediated by steroid receptors. SRA RNA expression of which reduces tumourigenicity and growth of in a complex with the SRC-1 protein (steroid receptor co- certain malignant cell lines [41,42]. On the other hand, since activator 1) acts as a strong co-activator of receptors for its expression is elevated in some cancer types including progestins, estrogens, androgens and glucocorticoids [54]. lung, breast and bladder, H19 seems to have oncogenic Its activity is sensitive to inhibition by antisense oligodeoxy- properties [43,44]. Epithelial cells accumulate H19 RNA in ribonucleotides and mutations affecting predicted secon- about 10% of breast cancer cases. In mice, cancer cells dary structure, but insensitive to nonsense mutations within transfected with human H19 were shown to promote tumour a putative ORF [54,55]. The interaction between SRC-1 70 M. Szymanski et al. / Biochimica et Biophysica Acta 1756 (2005) 65–75 and SRA RNA is probably mediated by a subfamily of large chromosomal region including several genes associ- DEAD-box RNA-binding proteins, p72/p68 [56]. SRA ated with muscle development, myogenic regulators Myf5 RNA can also interact with a specific RNA-binding and Myf6 and a growth factor Igf2. This in turn may be a domain of a hormone induced transcriptional repressor, causative agent for cancer development. Similar patterns of SHARP. This suggested that a modulation of expression of NCRMS expression were observed in neuroblastoma and respective genes results from competition between SHARP synovial sarcoma which suggests that these tumours may and steroid receptors for SRA RNA [57]. SRA RNA have common etiology [61]. shows increased expression in breast, uterus, ovarian A class of ncRNAs which is particularly interesting in the cancers which suggested that it may play a role in their context of cancer research is micro RNAs (miRNAs). These pathogenesis [54]. In mouse, an overexpression of SRA small (¨20 nt long) RNA molecules play a pivotal role in RNA led to proliferation, inflammation and apoptosis in the regulation of certain processed related to development in steroid hormone responsive tissues. There was, however, all eukaryotes. Their potential for a specific posttranscrip- no evidence of tumour development and the lifespan of the tional regulation of gene expression combined with their transgenic animals was comparable to that of the wild small size and evolutionary conservation makes them ideal type. These observations suggested that the up-regulation candidates for agents controlling complex gene networks of SRA RNA is not a causative agent in tumourigenesis, governing cell growth and differentiation [62]. Altered but can be in fact a measure to oppose excessive patterns of miRNAs may be therefore responsible for proliferation associated with tumour growth [15]. Recently, changes in the cells’ genetic program which in turn results new variants of the SRA transcripts with an extension at in malignant growth [63]. It has been demonstrated that the 5V-end were identified. These new isoforms possess various cancer cell lines display different miRNA expres- protein-coding potential and could be translated in an in sion patterns. The variations are both qualitative as well as vitro system and were shown to encode proteins in vivo. quantitative as shown in the case of colorectal cancers where This was the first evidence for a gene producing transcripts the levels of precursors of miR-24-2 showed a 50-fold that can act both as mRNA and ncRNA [58]. difference between samples [64]. A detailed analysis of the One of the largest human ncRNA genes is the BCMS (B- distribution of miRNAs on human chromosomes demon- cell neoplasia-associated gene with multiple splicing) strated that a majority of them are located within minimal located within the chromosomal region 13q14.3. Deletions deletion, minimal amplification or breakpoint regions linked affecting BCMS are found in over half cases of B-cell to the origin of certain forms of cancer and that they can act chronic lymphocytic leukaemia (B-CLL) and over 60% of both as tumour suppressors or as oncogenes [65]. mantle cell lymphoma. BCMS spans ¨560 kb and is In a systematic study aimed at determination of ex- composed of at least 50 exons. Alternative splicing of the pression patterns of microRNAs in a large number of primary transcript generates a large number of mature normal and malignant human tissues it has been found that products which show tissue-specific distribution. Interest- the vast majority of known microRNAs showed differential ingly, none of the so far identified alternatively spliced expression in different cancer types. The microRNA variants have a significant protein coding potential and they expression profiles discriminate between tumours which are most probably functional end products [59]. The role of originate from different tissue types as well as between these transcripts and their possible involvement in tumour normal and malignant cells. Interestingly, all of the cancer suppression is not known. types show reduced overall expression of microRNAs. It The expression of ncRNAs can differ between different has been, therefore, suggested that higher levels of micro- subtypes of cancer cells. In non-small cell lung cancer RNAs are associated with differentiated state [66]. Thus, it (NSCLC), metastasis was found to be associated with seems evident that miRNAs may constitute key elements expression of a MALAT-1 gene (metastasis associated in responsible for the maintenance of cell homeostasis by lung adenocarcinoma transcript 1), encoding an 8-kb-long inhibiting expression of genes involved in carcinogenesis. ncRNA. Sequences similar to human MALAT-1 were The miRNA expression also changes upon induction of identified in several species which suggests it has some differentiation as demonstrated in human leukemia cells function. The specific association of MALAT-1 with [67]. One of the regions of human chromosome 13q14 metastasising forms of lung cancer makes it a good frequently deleted in B-cell chronic lymphocytic leukaemia candidate for the diagnosis of lung cancer patients [60].In (B-CLL), in addition to the BCMS gene also harbours two rhabdomyosarcoma (RMS), the two histological subtypes miRNA genes miR-15 and miR-16. It is unclear whether any can be also distinguished based on the expression of the of these miRNAs play a role in B cell differentiation. NCRMS gene (noncoding RNA in RMS). The NCRMS gene However, a putative target for miR16 is arginyl-tRNA consists of at least 11 alternatively spliced exons mapped to synthetase (ArgRS) mRNA and its expression was found to human chromosome 12q21. That NCRMS RNA shows correlate with levels of miR-16, suggesting a posttranscrip- elevated expression in the alveolar RMS but not in the tional regulatory mechanism [68]. embryonal subtype of RMS. The expression of NCRMS Several types of human lymphoma are characterized by may indicate a deregulation of gene expression within the amplification of a 13q31 locus and overexpression of M. Szymanski et al. / Biochimica et Biophysica Acta 1756 (2005) 65–75 71 therein contained c13orf25 gene [69]. The short putative maternal uniparental disomy for chromosome 15, deletion ORFs of c13orf25 are not evolutionarily conserved and this of paternally inherited 15q11–q13 region, paternally region serves a as a host gene for a cluster of seven inherited balanced translocations or imprinting mutations. microRNAs (mir-17-92) which shows a high degree of Such inhibition affects the paternally expressed IPW gene sequence conservation with the mouse orthologue. It has (imprinted in Prader–Willi syndrome), located about 180 kb been found that there were markedly elevated levels of pri- from the imprinting control element. IPW encodes a 2.3-kb mir-17-92 in 65% out of 46 different lymphoma samples. It polyadenylated RNA which probably functions as an RNA has been demonstrated that mir-17-92 promotes tumour [77]. In humans, the IPW is expressed as several alter- development in mice [70]. The expression of mir-17-92 natively spliced ncRNAs, at the same levels in all tissues. cluster is regulated by c-Myc, a transcription factor Interestingly, the mouse homolog which it shares a nearly responsible for regulation of cell growth, division and 80% conserved 319 nt long region shows tissue-specific apoptosis. c-Myc also induces expression of another tran- variations with very high expression in brain [78]. It has scription factor, E2F1 which controls genes responsible for been found that noncoding transcripts between SNRPN and the transition from G1 to S phase. Interestingly, two UBE3A genes, including IPW, function as hosts for intron- microRNAs encoded within the mir-17-92 cluster, miR- encoded snoRNAs. Two snoRNAs, HBII-52 and HBII-85, 17-5p and miR-20, apparently target E2F1 mRNA and which are encoded by long arrays of tandemly repeated negatively regulate its translation. The same may be also units, were found not to be expressed in brains of patients true for several other gene activated by c-Myc and targeted with PWS [79]. This suggested that the two snoRNAs may by other microRNAs from the mir-17-92 cluster [71]. It has be involved in the etiology of this syndrome. HBII-52 been proposed that the increased levels of microRNAs snoRNA shows complementarity to the mRNA encoding reduce pro-apoptotic response to myc overexpression and serotonin receptor 2C at the A-to-I editing site [79]. The promote stem cell properties [70]. mouse homologue of HBII-52 snoRNA promotes 2V-O- Changes in expression of specific microRNAs were also methylation which in turn decreases the efficiency of editing demonstrated in other types of cancer. Glioblastoma cells [80]. A characterization of novel PWS-associated trans- overexpress miR-21, which was suggested to act as an location t(4;15)(q27;q11.2) suggested that PWS arises, at antiapoptotic factor. Inhibition of expression of miR-21 in least in part, due to the absence of HBII-85 snoRNA, but the the glioblastoma tissue culture results in activation of targets of this RNA are not known [81]. caspases and apoptosis [72]. In Burkitt’s lymphoma, there The second disorder associated with defects within is 100-fold increase in expression of miR-155 [73]. Reduced 15q11–q13 region is the Angelman syndrome resulting expression of miR-143 and miR-145 is associated with from disruption of maternal expression of a single gene, colorectal neoplasia [74]. The let-7 microRNA, expression UBE3A. An unusual feature of UBE3A is its biparental of which is greatly reduced in lung cancer, was shown to be expression in most tissues with imprinted, maternally- a negative regulator RAS oncogene. Its overexpression in specific pattern of transcription restricted to certain brain tissue culture was shown to reduce RAS protein expression cells. In both human and mouse there is a paternally-specific and inhibit lung cancer cells growth [75]. antisense transcripts form the UBE3A/Ube3a genes was observed. The antisense transcript, spanning over 460 kb and overlapping the IPW gene, is repressed by the deletion 6. Noncoding transcripts from imprinted genes of PWS-IC which leads to biparental expression of UBE3A and repression of the paternal antisense transcription. This A number of ncRNAs identified in humans are the suggests a direct role of the antisense UBE3A-AS transcript products of imprinted genes. Abnormal pattern of expres- in regulation of the maternally expressed UBE3A. [82].It sion from imprinted gene clusters can result in severe has been proposed that the functional products whose congenital disorders like Prader–Willi, Beckwith–Wiede- aberrant expression may be a causative agent of the PWS mann or Angelman syndromes. The genetic aberrations are the snoRNAs encoded within the UBE3A-AS introns responsible for these disorders affect complex imprinted loci [83]. and cause a number of developmental defects often The Russel–Silver syndrome, is linked to the chromo- associated with mental retardation and other neurobehavio- some 7q32 region which contains two imprinted ncRNA ral phenotypes [76]. genes. MESTIT1 (MEST intronic transcript 1), is a The Angelman (AS) and Prader–Willi syndromes (PWS) paternally expressed antisense RNA initiated from the arise from defects in imprinted genes located within the intron of the MEST/PEG1 (mesoderm specific transcript, chromosome 15q11–q13 region. There are 11 paternally paternally expressed gene 1) gene. The longer 4.2 kb variant and one maternally expressed genes in this region. The is expressed in fibroblasts and foetus tissues while the imprinted expression is regulated by a bipartite imprinting shorter 2.4 kb version (PEG1-AS) was found in testis and center (PWS-IC and AS-IC) upstream of the SNRPN gene. mature spermatozoa [84,85]. Another antisense transcript The PWS is brought about by genomic alterations causing form the 7q32 region, COPG2IT1 (COPG2 intronic tran- inactivation of paternally expressed genes as a result of script 1) origins from an intron 20 of the COPG2 (coatomer 72 M. Szymanski et al. / Biochimica et Biophysica Acta 1756 (2005) 65–75 protein complex gamma 2 subunit) gene. It is expressed controlling cells’ growth and differentiation as well as the form the paternal allele in all foetal tissues [86]. responses to changes in their environment. The unravelling A Beckwith–Wiedeman syndrome (BWS) and several of the involvement of ncRNAs in human diseases can also human cancers have been linked to genetic and epigenetic contribute to design of novel diagnostic and therapeutical abnormalities within the cluster of 13 maternally and 4 methods. paternally expressed genes on the human chromosome 11p15. In the majority of cases the Beckwith–Wiedemann syndrome results from the loss of imprinting at the IGF2 Acknowledgements gene which are not related to methylation and expression of H19 observed in Willms’ tumours [87]. The second, H19- This work was supported by grants from the Polish State independent, imprinting control region was localized within Committee for Scientific Research to JB, and from the the KvLQT1 gene linked to Romano–Ward, Jervell and Fonds der Chemischen Industrie e.V., the Bundesministe- Lange–Nielsen syndromes. 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