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Pipelines for cross-species and genome-wide prediction of long noncoding RNA binding


Abundant long, noncoding RNAs (lncRNAs) in mammals can bind to DNA sequences and recruit histone- and DNA-modifying enzymes to binding sites to epigenetically regulate target genes. However, most lncRNAs’ binding motifs and target sites are unknown. The large numbers of lncRNAs and target sites in the whole genome make it infeasible to examine lncRNA binding to DNA purely experimentally. Here, we report a protocol for lncRNA/DNA-binding analysis that is built upon a database containing the GENCODE-annotated human and mouse lncRNAs, the orthologs of these lncRNAs in 17 mammals, and the genome sequences of the 17 mammals. Cross-species and genome-wide lncRNA/DNA-binding analysis begins with and is driven by database search. The predicted DNA-binding motifs and binding sites answer the general question of which lncRNAs may epigenetically regulate which genes, and can be used to identify potential sites for genome and epigenome editing. To use the protocol, preliminary knowledge of the base-pairing rules that guide the binding of noncoding RNAs to DNA to form triplexes, as well as the skills required to use the UCSC Genome Browser, are needed. A genome-wide prediction takes from 2 to 10 d, and the results are sent to users automatically by e-mail. The platform is updated continuously, making it possible to study more lncRNAs and larger genomic regions in less computational time.

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Fig. 1: The platform that integrates multiple data sources and programs.
Fig. 2: The number of orthologs of the 13,562 human lncRNAs in the 16 other mammals.
Fig. 3: The workflows in this protocol.
Fig. 4: The web page of the database search.
Fig. 5: The web page showing search results.
Fig. 6: The web page showing database functions.
Fig. 7: The web page for batch TFO/TTS prediction after a database search.
Fig. 8: The web page showing the graphic display of an lncRNA and its orthologs.
Fig. 9: The web page showing the text display of the lncRNA RP11-375H19.2 and its orthologs.
Fig. 10: The TTS distributions of human PTENP1-asRNA and PTENP1 in the human PTEN locus.
Fig. 11: The TFO1 of human PTENP1-asRNA is in exon 1.
Fig. 12: The TTSs of CDKN2B-AS1 and other lncRNAs in the human CDKN2A/2B locus.
Fig. 13: The 83 lncRNAs on human Y chromosome have no TTS in the human CDKN2A/2B locus.

Code availability

The source code is available at our website ( and the GitHub website ( under a GNU Affero General Public License v.3.0.

Data availability

Example datasets that include TFOclass1 and TFOsorted files of all examples are available at our website ( and the GitHub website (


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This work received financial support (to H. Zhu) from the NSFC (31571348 and 31771456), the Special Program for Applied Research on SuperComputation of the NSFC-Guangdong Joint Fund, and the Guangzhou Science and Technology Innovation Committee (201607010067).

Author information




S.H. and X.Y. performed the genome searches; H. Zhang and J.L. built the database; H. Zhang, Y.W., and H. Zhu revised the LongTarget code; H. Zhu designed the study, analyzed the data, and drafted the manuscript.

Corresponding authors

Correspondence to Hai Zhang or Hao Zhu.

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Competing interests

The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

He, S., Zhang, H., Liu, H. & Zhu, H. Bioinformatics 31, 178–186 (2015):

Liu, H., Shang, X. & Zhu, H. Bioinformatics 33, 1431–1436 (2017):

Wang, S. et al. Cell Death Dis. 9, 805 (2018):

Integrated supplementary information

Supplementary Figure 1 The coordinates of some TTSs of RP11-375H19.2 collected in the database.

To open this window that shows TTSs of TFO1, click the blue TFO1 button in the webpage shown in Fig. 9.

Supplementary Figure 2 The initial records in the Excel file that reports the TTS distribution of H19 at all transcripts in the human genome hg38.

“bs” means binding site. bs_chr, bs_start, and bs_end indicate the chromosome number, start coordinate, and end coordinate of a TTS. TTS_area is the area of the peak of a TTS (as shown in custom tracks of TTS distributions) and indicates the strength of the TTS.

Supplementary Figure 3 The expression level of CDKN2B-AS1 in 53 tissues.

The picture is obtained from the GTEx Gene track in the UCSC Genome Browser by choosing the GTEx Transcript track in the Expression section to display GTEx genes graphically.

Supplementary Figure 4 The TTS distributions of marmoset lncRNAs in the marmoset CDKN2A/2B locus.

From top to bottom are 14 custom tracks of the TTS distribution of 14 lncRNAs, the track of Ensembl Genes, the custom track of CDKN2B-AS1_Marmoset, and the track of RepeatMasker. Three TTSs at transposable elements of Simple or Low Complexity are marked by three blue vertical lines. The results indicate that, as in humans, many lncRNAs bind to promoters of CDKN2A/2B.

Supplementary Figure 5 The TTS distributions of mouse lncRNAs in the mouse CDKN2A/2B locus.

Some TTSs at promoters and CpG islands (in green) are marked in yellow, and some TTSs at transposable elements (in the RepeatMasker track) and repetitive elements (in the SimpleRepeats track) are marked in blue. Some lncRNAs have TTSs only at transposable and/or repetitive elements.

Supplementary Figure 6 The TTS distributions of H19 and other human lncRNAs in the human IGF2 region.

From top to bottom are custom tracks of the TTS distribution of 16 lncRNAs, UCSC Genes, CpG Islands, ENCODE DNA Methylation (the colored lines indicate DNA methylation signals), and ENCODE Histone Modification (the colored areas indicate histone modification signals). This figure indicates that many lncRNAs may bind to the IGF2 region at the site H19 binds to.

Supplementary Figure 7 Exons of the orthologue of human CDKN2B-AS1 in marmoset.

This webpage shows the coordinates of all exons of the orthologue of human CDKN2B-AS1 in marmoset.

Supplementary Figure 8 The format of the custom gene track file marmoset-CDKN2B-AS1.

The gene name is CDKN2B-AS1_Marmoset as shown in Supplementary Figure 4. All custom gene track files should follow the same format, but can adopt any file name.

Supplementary Figure 9 The results of a M:N case of genome-wide prediction.

The figure shows some of the records in the Excel file that reports TTSs of lncRNA transcripts at the genomic regions of protein-coding transcripts. Here peak_area and TTS_area are defined as in Supplementary Figure 2.

Supplementary Figure 10 Two ENST ID lists.

The two ENST ID lists are the inputs of the M:N case of genome-wide prediction shown in Supplementary Figure 9. These Ensembl ENST IDs are differentially expressed lncRNA transcripts and differentially expressed protein-coding transcripts from an RNA-seq analysis we made that compare the gene expression in 12 human colorectal cancer tissues with the gene expression in 3 normal colorectal tissues (unpublished observations [Sha He, Yujian Wen, Hao Zhu]). These transcripts were assembled using reads of RNA-seq by the StringTie program (Nat. Biotechnol. 33, 290–295; 2015) and differential expression was determined by the EdgeR program (Genome Biol. 17, 75; 2016).

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Supplementary Figures 1–10 and Supplementary Methods

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Lin, J., Wen, Y., He, S. et al. Pipelines for cross-species and genome-wide prediction of long noncoding RNA binding. Nat Protoc 14, 795–818 (2019).

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