Koji Numata ^{1, 2}, Akio Kanai ², Rintaro Saito ^{2, 4}, Shinji Kondo ⁴, Jun Adachi ⁴, Laurens G. Wilming ⁶, David A. Hume ⁷, RIKEN GER Group ⁴, GSL Members ^{5, 8}, Yoshihide Hayashizaki ^{4, 5} and Masaru Tomita ^{2, 3, 9}

¹ Graduate School of Media and Governance, Bioinformatics Program, Keio University, Fujisawa, Kanagawa
252-8520, Japan;
² Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa 252-8520, Japan;
³ Department of Environmental Information, Keio University, Fujisawa, Kanagawa 252-8520, Japan;
⁴ Laboratory for Genome Exploration Research Group, RIKEN Genomic Science Center (GSC), RIKEN Yokohama Institute, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan;
⁵ Genome Science Laboratory, RIKEN, Hirosawa, Wako, Saitama 351-0198, Japan;
⁶ Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK;
⁷ Institute for Molecular Bioscience and School of Molecular and Microbial Sciences, University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia

⁸ Takahiro Arakawa, Piero Carninci, and Jun Kawai.

⁹ Corresponding author.
E-MAIL:   mt@sfc.keio.ac.jp    FAX:  + 81 (466) 47-5099.

( Supplemental material is available online at:  http://www.genome.org )

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)  ( RNA molecules that are not translated to protein products ) has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described non-mouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in
a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals. 

Noncoding RNA (ncRNA) is a global term for transcripts that lack an apparent open reading frame (ORF) and do not encode a protein product. Until recently, the only known functional ncRNAs were ribosomal RNA, transfer RNA, and several small nucleolar RNAs. Other classes of small ncRNAs—such as microRNAs (miRNAs), C/D box snoRNAs, small interfering RNAs(siRNAs), and small temporal RNAs (stRNAs)—have been identified and characterized in all three domains of life (bacteria, archaea, and eukarya) based on experimental expression analysis and computational screening (Sharp 2001; Eddy 2002; Grosshans and Slack 2002; Tang et al. 2002; Wassarman 2002). Lau et al. (2001) had reported that abundantly expressed 21- to 24-nt-long miRNAs are found in Caenorhabditis elegans, and the pattern of their expression varies with developmental stages (Lau et al. 2001). These classes of small ncRNAs are believed to contribute to processes such as transcriptional regulation, translational repression, and mRNA degradation (Storz 2002).

Longer ncRNAs, sometimes referred to as mRNA-like ncRNAs, form a quite distinct class. Unlike the classes noted above, they are processed like mRNA; that is, they are transcribed by RNA polymerase II, spliced, polyadenylated, and conceivably capped (Erdmann et al. 2000, 2001). For example, Xist, which acts as an X-chromosome inactivator to achieve dosage compensation in mammalian females, encodes a 17-kb-long RNA molecule with no significant ORFs, even though it is comprised of seven exons and polyadenylated (Hong et al. 1999; Nesterova et al. 2001). Such mRNA-like processed ncRNAs have been identified in plants and animals and are expressed in a tissue-specific manner (Erdmann et al. 2001). The diversity of such transcripts in the mammalian transcriptome has not been evident from genome sequencing, because exon boundaries are difficult to define, and ncRNAs tend to be less conserved between mammalian species. In this situation, the RIKEN mouse full-length cDNA collection, called FANTOM2 clone set (Okazaki et al. 2002), provides the largest available resource in any mammal for the discovery of candidate functional ncRNAs. The set contains many sequences that do not show apparent protein coding regions according to human-curated annotation. Some of these are likely to be the result of incomplete cDNA synthesis or incompletely processed transcripts (e.g., 3' untranslated regions [UTRs]), or perhaps transcriptional "noise," so the identification of strong candidate functional ncRNAs requires additional annotation criteria.

There are two ways to identify ncRNAs computationally. One is the "genome based" approach, which detects ncRNAs from genomic sequence. More than 200 candidate ncRNA genes are predicted in Escherichia coli by computational comparative genomics using "intergenic" sequence data from four related bacteria (Rivas et al. 2001). Two similar approaches are reported by other groups, and at least 20 ncRNA genes have been experimentally confirmed in E. coli (Argaman et al. 2001; Wassarman et al. 2001). This approach is not feasible in the more complex mammalian genomes.

The other approach is "transcripts-based." MacIntosh et al.(2001) attempted to identify and characterize new ncRNAs by using Arabidopsis thaliana EST sequences. Through systematic computational screening, they extracted dozens of ncRNA candidates and putative RNAs encoding small peptides. The investigators concluded that there are numerous functional ncRNAs in A. thaliana.)

This study describes the initial effort at comprehensive identification of mammalian mRNA-like processed ncRNAs based on the comprehensive mouse transcriptome survey provided by the RIKEN Mouse Gene Encyclopedia project.

Results and Discussion:

Characterization of Previously Reported ncRNAs in FANTOM2 Clone Set
To assess the representation of ncRNAs in the FANTOM2 clone set (see below), we initially identified previously described ncRNAs. The set of previously reported ncRNAs was based on the Noncoding RNAs Database (http://biobases.ibch.poznan.pl/ncRNA/),which was constructed by Erdmann et al (2000, 2001). The query sequence set from the database contains ncRNAs from both mammalian and nonmammalian origins, including plants and flies.

We used a homology search based on BLASTN and Ssearch (Pearson1991), to search for 18 of the murine known ncRNAs, and nine of them were identified in the FANTOM2 clone set. Likewise, putative homologs of several ncRNAs previously described in other mammalian organisms were also found (Table 1). We have newly identified homologs of rat NTAB (French et al. 2001),7H4 (Velleca et al. 1994), human NTT (Liu et al. 1997), NCRMS (Chan et al. 2002), U19 snoRNA host gene (Bortolin and Kiss 1998), and hamster adapt33 (Wang et al. 1996) in the FANTOM2 clone set. These results indicate that ncRNAs are well represented in the FANTOM2 clone set, and sequence conservation across species is a useful criterion in annotation and validation.

Table 1. Summary of Previously Identified ncRNAs or Their Possible Homologs Found in FANTOM2 Clone Set
 

FANTOM2 clone ID	Homologous noncoding RNA	GenBank/EMBL/DDBJ accession no.	Status of identity	Organism	References
C130002M05	KvLQT-as	AF119385	97.8% in 2467 nt	Mm	Smilinich et al. 1999
3830421G02	U17 snoRNA host gene	AJ006836	99.7% in 383 nt	Mm	Pelczar and Filipowicz 1998
A730062M15	U22 snoRNA host gene	U40654	100% in 476 nt	Mm	Tycowski et al. 1996
E130201N16	G90	AJ132433	98.4% in 833 nt	Mm	Krause et al. 1999
A430022B11	XIST	L04961	99.3% in 1497 nt	Mm	Hong et al. 1999
B230105C16	IPW	U69888	97.9% in 617 nt	Mm	Wevrick and Francke 1997
1100001A04	H19	NM_023123	99.2% in 870 nt	Mm	Hurst and Smith 1999
9630004F23	CIOR	AF140607	99.9% in 2118 nt	Mm	Inoue et al. 2002
6430597C21	Rian	AB063319	100% in 1496 nt	Mm	Hatada et al. 2001
2900019G14	NTAB	AY035551	89.7% in 955 nt	Rn	French et al. 2001
5930439P04	Synapse-specific 7H4	L33722	79.1% in 3668 nt	Rn	Velleca et al. 1994
A530032L19	NTT	U54776	61.0% in 2635 nt	Hs	Liu et al. 1997
D630034O16	DGCR5	X91348	73.7% in 243 nt	Hs	Sutherland et al. 1996
D930049J19	NCRMS	XR_000104	72.6% in 446 nt	Hs	Chan et al. 2002
E430001E02	U19 snoRNA host gene	AJ224166	80.7% in 451 nt	Hs	Bortolin and Kiss 1998
5430416N02	adapt33	U29660	63.6% in 676 nt	Cg	Wang et al. 1996

Table 1. Summary of Previously Identified ncRNAs or Their Possible Homologs Found in FANTOM2 Clone Set
Homology search was initially performed by BLASTN. Then the best hit sequence having its e value <1.0e- 10
in same orientation were picked up. For this sequence, Ssearch (Pearson 1991) was carried out for stricter alignment. Hs indicates Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; and Cg, Cricetulus grisseus.

Computational Screening of the Novel ncRNA Candidates
The FANTOM2 clone set contains 60,770 cDNA clones selected from>260 normalized, subtracted, and full-length enriched cDNA libraries of C57BL/6J strain of mouse. The 60,770 clones of FANTOM2-set were clustered into 33,409 transcriptional units (TUs), and approximately half of the TUs contained a deduced  protein sequence (Representative Protein Set [RPS]) based on ORF prediction and/or homology with known proteins. The remaining set of cDNAs (15,815 sequences) that are defined as non–protein-coding TUs (Okazaki et al. 2002) represent the starting set for identification of ncRNAs. To eliminate other possible sources of transcripts that lack an apparent functional ORF, such as UTR-only sequences (incomplete cDNA synthesis), unprocessed mRNAs with retained introns, and chimeric cDNA clones, we applied the following strategy of computational filtering. In addition to removing the RPS, we eliminated sequences that showed any homology with known protein sequences, even if they did not contain any evidence of an ORF. We then mapped the remaining sequences to the mouse genome (MGSCv3). Comparison between the alignments with the genome and exon predictions by GENSCAN (Burge and Karlin 1997) was also considered. Because GENSCAN may fail to identify untranslated regions of protein-coding transcripts, we eliminated any sequences that mapped within 10 kb of any predicted exon on the grounds that they may be part of the same TU, for example, an alternative 3'UTR or splice variant (see Methods).

Consequently, we extracted 4280 transcripts as the candidate set of ncRNAs. The procedures for computational screening and number of remaining sequences in each filtering step are shown in Table 2. The average length of the candidate clones was 1778.9 nt, and that of predicted protein-coding transcriptsin RTS was 2131.8 nt. Likewise, the average length of the longest ORF of resulting candidates was 200.6 nt, whereas that of transcripts in RPS was 1088.7 nt. A full set of accession numbers of the candidate set is provided in the Supplementary tables, and the annotation of each individual candidate can be assessed at the FANTOM2 Web interface as described in the overview of the RIKEN project (Okazaki et al. 2002).

Table 2. Summary of Computational Screening for Novel ncRNA Candidates.
 

Procedures for computational screening	No. of remaining clones
Defined as Non-protein-coding TU	15,815
No homologies to other known protein sequences (BLASTX)	12,382
Could be aligned to mouse genome sequence (identity >90%, length >90%)	11,652
CDS prediction could not be made by GENSCAN (10 kb around mapped region)	4,280

Table 2. Summary of Computational Screening for Novel ncRNA Candidates.

Characterization of the Candidate Set
The strategy used here is conservative, and we know that not all known ncRNAs would meet these criteria. Several previously reported ncRNAs were eliminated at each filtering step of the strategy described above, but as we would hope, the frequency within the remaining set increased (albeit the number of knownncRNAs is too small to assess the statistical validity of this assessment). To characterize the candidate set further, we used several additional criteria. First, we conducted a homology search against publicly available EST sequences of mouse, human,and rat, as well as against the human genome. We additionally searched CpG islands in 5' boundary regions and polyadenylation signals in 3' ends (see more details in Methods). CpG islands and polyadenylation signal are observed not only in a large number of protein-coding genes but also in several known ncRNAs.This can be taken as supporting evidence that they are actually transcribed by RNA polymerase II. We also determined the intron–exon boundaries of the loci encoding the transcripts, and we identified the subset of candidates that is produced by splicing of a primary transcript. These results are summarized in Table 3.

Table 3 Summary of Further Characterization of the ncRNA Candidates.
 

Evidence	Hit no. (%)
Mouse EST hit	1,200 (28.0)
Human EST hit	111 (2.6)
Rat EST hit	252 (5.8)
Human genome homology	454 (10.6)
Potential CpG islands	919 (21.5)
Potential PolyA signal	1,395 (32.6)
Spliced sequences (no. of exons >2)	1,150 (26.9)

Table 3 Summary of Further Characterization of the ncRNA Candidates.

There were 1200 (28.0%) ncRNA candidates, which were homologous with mouse EST sequences (BLASTN with e value lower than 1.0e- 100). This indicates that approximately one fourth of the candidates have independent evidence of reproducible expression.This is not an especially stringent criterion, because the RIKEN Project is itself the largest mouse EST project, and library construction involves strong selection to avoid redundancy. Furthermore, functional ncRNAs may not be abundant transcripts. More interestingly, 111 (2.6%) clones showed strong homology with human ESTs, and 252 (5.8%) clones showed strong homology to rat ESTs. Furthermore, 454 (10.6%) clones could be aligned with the human genome sequence at >50% homology and >70% of length. Sequence conservation is also not a strict criterion for exclusion or functional significance, because several known ncRNAs would fail to meet this criterion.

We identified CpG islands in the 5' boundary genomic region for 919 (21.5%) of the clones. CpG islands are associated with ~40% of promoters for mammalian genes, most commonly those of housekeeping genes that are widely expressed (Takai and Jones 2002). A polyadenylation signal was found at the 3' end of 1395 (32.6%) of the clones. This is a rather more stringent criterion, implying that the transcript is a genuine polyadenylated mRNA-like molecule. However, cDNAs that lack this signal might have arisen by internal oligo-dT priming but might still be bona fide ncRNAs. To clarify the robustness of these criteria, we plotted the average of CpG observed/expected (O/E) ratio and frequency of poly-A–like signals around the 5' boundaries of mapped genomic regions, and the 3' end of cDNA sequences, respectively. As shown in Figures 1 and 2, there is a clear peak of the CpG O/E ratio and poly-A signals that delineates the subsets of transcripts in the candidate set into separate classes.

Figure 1Average of CpG observed/expected (O/E) value around putative transcription start site.

Figure 1 Average of CpG observed/expected (O/E) value around putative transcription start site. The average of CpG O/E ratio for each transcription start site (from 3 kb upstream to 0.5 kb downstream) of the 4280 largest-candidate set was plotted. The set contains 919 sequences, which have potential CpG islands surrounding transcription start site, as referred in Table 3. Putative transcription start sites (TSSs) were defined by 5' boundaries of mapped genomic regions as indicated by an arrow. CpG O/E ratio was calculated every 200-bp window with sliding 20 bp. The formula for producing CpG O/E ratio is described in Methods.

Figure 2Frequencies of polyadenylation signal-like sequences upstream of 3' end.

Figure 2 Frequencies of polyadenylation signal-like sequences upstream of 3' end. Frequencies of polyadenylation signal-like sequences located upstream of 3' end sites were plotted. The sequence pattern AATAAA/ATTAAA was searched for every position from the 3' end of the 4280 largest-candidate set. The set contains 1395 sequences, which contain polyA-signal like sequence in the 3' end, as mentioned in Table 3.

Among the candidate set that could be mapped to the genome,1150 (26.9%) revealed multiple exons. Several known ncRNAs such as Xist, Gas5, and BIC (Smith and Steitz 1998; Hong et al. 1999; Tam 2001) are known to undergo splicing. Again, this criterion provides a strong indication that the transcript is a genuine product of RNAPol II–mediated transcription and is likely to be functional.

In a separate analysis, the FANTOM2 cDNA set was found to contain>2400 sense–antisense pairs (Okazaki et al. 2002;Kiyosawa et al. 2003). Three hundred twenty-three (7.5%) members of the candidate set are also included in the antisense transcript candidates. Antisense transcripts have been implicated in transcription control, especially in genomic imprinting. For example, AIR, which is an antisense transcript from Igf2r locus, silences three kinds of paternal imprinting genes (Sleutels et al. 2002). In another study (Holmes et al. 2003), new and novel antisense transcripts from among the FANTOM2 set were mapped to the imprinted GNAS locus.

Among the candidate set, 68.0% of the clones fitted at least one of the criteria, and 54.8% of these fitted clones satisfied more than two criteria, as shown in Table 3. Therefore, we believe that the set contains many potential ncRNAs and that a substantial subset will be shown to be functional in some aspect of mammalian biology. Twenty-five strong candidates extracted by our filtering are listed as examples for putative ncRNAs in Table 4. The average length of the longest ORFs of them is 195.2 nt. As an additional index that the transcripts are unlikely to code for even a small peptide, in all of the clones except TF14562 and TF9816, the longest ORFs are not started from the first ATG of each clone, which is normally used as the initiation codon in >90% of mammalian protein coding transcripts. It remains possible that a small subset does, indeed, encode small proteins. Detailed annotation of candidate CDS encoding proteins between 50 and 99 amino acids is described elsewhere (Grimmond et al. 2003), and candidates that arise from this analysis will be eliminated from the candidate set.

Table 4: Twenty-Five Examples of ncRNA Candidates
 

				EST hit
ID	Chromosome no.	Length (nt)	Tissues library	Mm	Hs	Rn	Hs genome homolog
TF14562	Chr1 (4)	408	18-day embryo	37	0	0	None
TF15290	Chr10 (5)	1511	Testis	0	0	0	None
TF16305	Chr10 (4)	1742	Testis	0	0	0	1390 (0.625)
TF8960	Chr11 (3)	1658	Stomach/C. striatam/cecum	23	12	3	None
TF8434	Chr11 (2)	891	Cerebellum/embryo10 + embryo11/pancreas	21	0	0	None
TF15141	Chr11 (5)	1878	Embryo/testis/thymus	10	0	0	None
TF23639	Chr12 (1)	1584	Testis (embryo15)	12	0	2	1551 (0.613)
TF19580	Chr13 (2)	2816	Cerebellum	1	0	20	2585 (0.663)
TF9816	Chr13 (2)	1152	C. striatum/embryo 13-head	15	1	0	None
TF9905	Chr17 (2)	350	Testis	1	0	0	273 (0.675)
TF12671	Chr18 (6)	1901	Cecum	0	0	0	None
TF31326	Chr18 (1)	1636	ES cells/E12 upper body	7	0	1	1226 (0.659)
TF28373	Chr19 (4)	2349	N7 Cerebellum/embryo 9/bone	64	1	0	None
TF14099	Chr2 (8)	1174	Testis	0	0	0	None
TE70920	Chr2 (2)	1318	U. bladder/N0 thymus	7	0	2	1160 (0.681)
TE14398	Chr3 (1)	317	Testis	1	0	3	223 (0.562)
TF11833	Chr2 (3)	454	Embryo 8	0	0	0	387 (0.872)
TF27297	Chr3 (2)	1321	Aorta and vein	1	0	0	1247 (0.752)
TF12549	Chr5 (5)	604	Head (neonate 6 day)/E12 upper body	10	0	0	None
TF22931	Chr5 (6)	2408	Forelimb (embryo13)/head	4	0	0	None
TF33090	Chr5 (3)	2703	N0 eyeball	0	0	0	2337 (0.715)
TF13219	Chr6 (3)	479	Testis	0	0	0	343 (0.724)
TF13544	Chr9 (5)	999	Embryo10 + embryo11	25	0	0	None
TF21967	Chr9 (2)	573	Embryo10 + embryo11	3	0	2	571 (0.806)
TF8931	ChrX (7)	3433	Embryo10 + embryo11/tongue/hippocampus/skin	12	0	1	None

Table 4: Twenty-Five Examples of ncRNA Candidates
Examples of ncRNA candidates were listed with their length, mapped chromosome (the number in parenthesis indicates mapped number of exons), tissue library, and number of EST hit (Mm indicates Mus musculus; Hs, Homo sapiens; and Rn, Rattus norvegicus). Status of human genome homology with aligned length and identity (in parenthesis) was also noted. None indicates that no significant homology was observed under the threshold of >70% length, >50% identity. All of these candidates were selected based on visual inspection through FANTOM2 Web interface ( http://fantom2.gsc.riken.go.jp/db/ ), and observed both of CpG islands in their 5' upstream region of transcription start site and polyadenylation signal-like sequence in the 3' end.

The next stage in further validation of these candidate ncRNAs is documentation of their regulation and expression profiles.A number of candidates are isolated from tissue-specific and stage-specific libraries (Table 4). The ncRNAs that show tissue-restricted expression would clearly be candidates for functions in differentiation and development. Examples in this class would include BC1 and NTAB (French et al. 2001; Muddashetty et al. 2002), which are specifically transcribed in the brain. They make a complex with certain ribonucleoproteins (RNPs) and regulate the RNA translation, transport, and turnover. The FANTOM2 Web interface for all candidate transcripts provides some indication of expression pattern based on the profile of libraries from which ESTs have been identified. The RIKEN project includes systematic cDNA microarray analysis of all of the clones in the FANTOM2 set, and some of this information is already in the public domain (Bono et al. 2003).

In conclusion, the FANTOM2 cDNA collection clearly contains thousands of candidate ncRNAs, a significant subset of which has all the characteristics of an mRNA other than protein-coding function. The era of the central dogma, DNA-RNA-protein, as the major conduit for expression of genome-encoded biological information, is clearly at an end.

Methods:

Computational Screening of the Candidates
The Mouse Representative Transcripts Set (RTS) was used as the nonredundant sequence set for this transcriptome screening. First, the RTS sequences that are defined as non–protein coding TU by FANTOM consortium, that is, RTS that cannot produce proteins in the Representative Protein Set (RPS), were extracted (Okazaki et al. 2002). Homology search with known amino-acid sequences (ftp://us.expasy.org/databases/sp_tr_nrdb/) according to BLASTX (http://www.ncbi.nlm.nih.gov/blast) was performed to eliminate any likelihood of homology with a protein-coding transcript. If the result of BLASTX was produced with e value <1.0e - .05, the clone was eliminated. It should be noted that this criterion would eliminate transcripts from expressed pseudogenes. As the next filtering step, cDNA sequences were aligned to genomic sequence by using BLAST and SIM4 (http://globin.cse.psu.edu/dist/sim4/).If they were aligned at >90% identity over >90% of their length, cDNA clones were kept, otherwise they were discarded. In addition, the prediction of protein-coding regions in genomic sequences by GENSCAN (http://genes.mit.edu/GENSCANinfo.html) was also carried out. The remaining sequences were kept as the candidates, if the entire 10-kb region of genome sequence around the mapped region—the size was determined according to previous work (MacIntosh et al. 2001)—did not overlap with protein-coding regions predicted by GENSCAN.

Further Characterization of the Largest Candidate Set
All of the homology searches with publicly available EST sequences were performed by BLASTN. Only EST sequences with e value <1.0e - 100 were regarded as corresponding homologous mouseESTs (ftp://ftp.ncbi.nih.gov/blast/db/est_mouse.Z), and sequences with E-values lower than 1.0e - 50 were regarded as the likely human (ftp://ftp.ncbi.nih.gov/blast/db/est_human.Z) and rat (ftp://ftp.ncbi.nih.gov/genomes/R_norvegicus/rn_est.gz) orthologous ESTs. Reverse hits were not considered.

A homology search with human genomic sequences was performed according to BLAST and SIM4 in the identical way as gene mapping to the mouse genome sequence (see Computational Screening ofthe Candidates). Because there is less selection pressure on contiguous homology of noncoding sequences, hits of 70% of the full-length, and at least 50% of nucleotide identity, were considered significant.

The CpG island analysis was performed according to calculation of CpG O/E ratio and (G+C) content for every 200-bp window with moving 1-bp intervals around 5' boundaries of aligned region of cDNAs. If the region had (G+C) content >50% and CpG O/E ratio > 0.6, it was considered as a CpG island. CpG O/E ratio was calculated by using the Gardiner-Garden and Frommer formula (Gardiner-Garden and Frommer 1987), ([number of CG x N]/[number of C x number of G]), where N denotes the total number of nucleotides in the analyzed sequence. The search of polyadenylation signal is based on statistic pattern search. Two hexamer sequences, AATAAA or ATTAAA, were searched for each 30 nucleotides of the 3' end.

Acknowledgments:

We acknowledge Dr. I. Yamanaka, Dr. H. Bono, and I. Nikaido for technical support; S. Fujimori, A. Sakurai, and H. Kochiwa for helpful discussions; and M. Chishima for surveys of previously identified ncRNAs.

Footnotes:

⁸ Takahiro Arakawa, Piero Carninci, and Jun Kawai.

⁹ Corresponding author.
E-MAIL:    mt@sfc.keio.ac.jp;     FAX: +1- 81 (466) 47-5099.

[Supplemental material is available online at:  http://www.genome.org ]

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Web Site References:

http://biobases.ibch.poznan.pl/ncRNA/; Noncoding RNAs database.

ftp://us.expasy.org/databases/sp_tr_nrdb/; data set for known protein sequences.

ftp://ftp.ncbi.nih.gov/blast/db/; database of mouse EST sequences and human EST sequences.

ftp://ftp.ncbi.nih.gov/genomes/R_norvegicus/; database of rat EST sequences.

http://www.ncbi.nlm.nih.gov/blast; executable files of BLASTN and BLASTX.

http://globin.cse.psu.edu/dist/sim4/; SIM4, a program for gene mapping to the genomic sequences.

http://genes.mit.edu/GENSCANinfo.html; GENSCAN, a program for ORF prediction from genomic sequences.

http://fantom2.gsc.riken.go.jp/db/; Web interface for FANTOM2 database.

Additional References:

1. Saha S, Ansari AZ, Jarell KA, and Ptashne M, "RNA Sequences that Work as Transcriptional Activating Regions".

2. Lee JM, and Sonnhammer ELL, "Genomic Gene Clustering Analysis of Pathways in Eukaryotes".

3. Blumenthal T, Evans D, Link CD, Guffanti A, Lawson D, Thierry-Mieg J, Thierry-Mieg D, Chiu WL, Duke K, Kiraly M, and Kim SK, "A Global Analysis of Caenorhabditis elegans Operons".

4. Storz G, "An Expanding Universe of Noncoding RNAs".

5. Eddy SR, "Non-Coding RNA Genes and the Modern RNA World".

6. Huttenhofer A, Kiefmann M, Meier-Ewert S, O'Brien J, Lehrach H, Bachellerie J-P, and Brosius J, "RNomics: An Experimental Approach that Identifies 201 Candidates for Novel, Small, Non-Messenger RNAs in Mouse".

7. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription".

8. Frenster JH, "Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

9. Special Issue of Genome Research, vol. 13, no. 6b, pp. 1265-1561 (June 2, 2003).
Report of "RIKEN Mouse Genome Encyclopedia" project: the whole system from mouse house to database.

10. Carninci P, et al, "Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia", Genome Research, vol. 13, no. 6b, pp. 1273-1289 (June 2, 2003).

11. Bono H, Yagi K, Kasukawa T, Nikaido I, Tominaga N, Miki R, Mizuno Y, Tomaru Y, Goto H, Nitanda H, Shimizu D, Makino H, Morita T, Fujiyama J, Sakai T, Shimoji T, Hume DA, RIKEN GER Group, Arakawa T, Carninci P, Kawai J, Hayashizaki Y, and Okazaki Y, "Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays", Genome Research, vol. 13, no. 6b, pp. 1318-1323 (June2, 2003).