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Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery

Li, Na, You, Xintian, Chen, Tao, Mackowiak, Sebastian D., Friedländer, Marc R., Weigt, Martina, Du, Hang, Gogol-Döring, Andreas, Chang, Zisong, Dieterich, Christoph, Hu, Yuhui, Chen, Wei·2013·Nucleic Acids Research
DOI 10.1093/nar/gkt072PMC PMC3616697
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Figure 1. Genome annotation of sequencing reads from 10 mouse tissues. The pie charts depicted the percentage of 10–40 nt short reads (A) and 50–100 nt long reads (B) that were mapped to the indicated genome features based on miRBase v16.0 (34) and Ensembl Genes 59 (www.biomart.org) (37). ‘Other ncRNA’ included rRNA, tRNA, scRNA, snRNA and srpRNA.
Figure 1. Genome annotation of sequencing reads from 10 mouse tissues. The pie charts depicted the percentage of 10–40 nt short reads (A) and 50–100 nt long reads (B) that were mapped to the indicated genome features based on miRBase v16.0 (34) and Ensembl Genes 59 (www.biomart.org) (37). ‘Other ncRNA’ included rRNA, tRNA, scRNA, snRNA and srpRNA.CC BY-NC · PubMed Central
Figure 2. Distribution of the miRNA and pre-miRNA abundance. (A) Histogram of the log2 transformed expression level (sum of RPMs from 10 tissues, RPM: Reads Per Million total miRNA reads) was shown for the known miRNAs. The miRNAs with precursor detected (black) expressed at a higher level than those without (grey). (B) Comparison of the miRNA and pre-miRNA abundance. Each circle represented one pre-miRNA locus. X-axis denoted the log2 transformed sum of miRNA/miRNA* RPM derived from the pre-miRNA locus in 10 tissues, Y-axis denoted the log2 transformed number of pre-miRNA sequencing reads.
Figure 2. Distribution of the miRNA and pre-miRNA abundance. (A) Histogram of the log2 transformed expression level (sum of RPMs from 10 tissues, RPM: Reads Per Million total miRNA reads) was shown for the known miRNAs. The miRNAs with precursor detected (black) expressed at a higher level than those without (grey). (B) Comparison of the miRNA and pre-miRNA abundance. Each circle represented one pre-miRNA locus. X-axis denoted the log2 transformed sum of miRNA/miRNA* RPM derived from the pre-miRNA locus in 10 tissues, Y-axis denoted the log2 transformed number of pre-miRNA sequencing reads.CC BY-NC · PubMed Central
Figure 3. Probabilistic score distribution of pre-miRNA candidates. Histogram showed the probabilistic score distribution of the potential pre-miRNA candidates using a Naïve Bayesian classifier with six features of known miRNA, including pre-miRNA minimal folding free energy, thermodynamic stability, 5′ and 3′ end duplex overhang as well as pre-miRNA and mature miRNA length. Known pre-miRNAs were shown in black, the remaining in grey.
Figure 3. Probabilistic score distribution of pre-miRNA candidates. Histogram showed the probabilistic score distribution of the potential pre-miRNA candidates using a Naïve Bayesian classifier with six features of known miRNA, including pre-miRNA minimal folding free energy, thermodynamic stability, 5′ and 3′ end duplex overhang as well as pre-miRNA and mature miRNA length. Known pre-miRNAs were shown in black, the remaining in grey.CC BY-NC · PubMed Central
Figure 4. Abundance of known and novel mouse miRNAs. Histogram of the log2 transformed expression level (sum of sequencing reads in 10 mouse tissues) of known miRNAs (grey) and novel miRNAs (black) in 10 mouse tissues. Compared with known miRNAs, novel miRNAs expressed at a much lower level.
Figure 4. Abundance of known and novel mouse miRNAs. Histogram of the log2 transformed expression level (sum of sequencing reads in 10 mouse tissues) of known miRNAs (grey) and novel miRNAs (black) in 10 mouse tissues. Compared with known miRNAs, novel miRNAs expressed at a much lower level.CC BY-NC · PubMed Central
Figure 5. Examples of novel pre-miRNAs discovered in this study. (A) A novel mouse pre-miRNA within the intron of the protein-coding gene Camk2a. (B) A novel mouse 5′ tailed mirtron derived from the protein-coding gene Ankrd23. (C) A novel C. elegans pre-miRNA from the 5′UTR of the protein-coding gene F35G12.2.
Figure 5. Examples of novel pre-miRNAs discovered in this study. (A) A novel mouse pre-miRNA within the intron of the protein-coding gene Camk2a. (B) A novel mouse 5′ tailed mirtron derived from the protein-coding gene Ankrd23. (C) A novel C. elegans pre-miRNA from the 5′UTR of the protein-coding gene F35G12.2.CC BY-NC · PubMed Central
Figure 6. Novel miRNAs are dependent on Dicer for expression. RNA interference was used to silence Dicer in a mouse N2a cell line. Log2 fold-changes of small RNA expression after Dicer knockdown were noted for known miRNAs (A), novel miRNAs (B), tRNAs (C) and rRNAs (D).
Figure 6. Novel miRNAs are dependent on Dicer for expression. RNA interference was used to silence Dicer in a mouse N2a cell line. Log2 fold-changes of small RNA expression after Dicer knockdown were noted for known miRNAs (A), novel miRNAs (B), tRNAs (C) and rRNAs (D).CC BY-NC · PubMed Central
Figure 7. Novel miRNAs show similar Ago2-binding profile as known miRNAs. X-axis denoted the abundance (log10 transformed RPM value) of known miRNAs (light blue dots), novel miRNAs (red dots), tRNAs (black solid squares) and rRNAs (dark blue solid triangles) in the input sample. y-axis denoted their abundance in Ago2 IP sample. Novel miRNAs showed similar Ago2-binding profile as known miRNAs, whereas both tRNAs and rRNAs were significantly depleted in Ago2 IP sample.
Figure 7. Novel miRNAs show similar Ago2-binding profile as known miRNAs. X-axis denoted the abundance (log10 transformed RPM value) of known miRNAs (light blue dots), novel miRNAs (red dots), tRNAs (black solid squares) and rRNAs (dark blue solid triangles) in the input sample. y-axis denoted their abundance in Ago2 IP sample. Novel miRNAs showed similar Ago2-binding profile as known miRNAs, whereas both tRNAs and rRNAs were significantly depleted in Ago2 IP sample.CC BY-NC · PubMed Central
Figure 8. Experimental validation of the two novel miRNA candidates. (A) The RNA with size of 10–200 nt was separated using flashPAGE Fractionator (Ambion) and loaded onto the denaturing PAGE gel for northern blot. Northern blot of two novel miRNAs (CandidateMMU32 and CandidateMMU33) revealed bands corresponding to the mature miRNA (marked as *). (B) In vitro rDicer processing. 32P-labelled CandidateMMU32 pre-miRNA (lane 3–5) and CandidateMMU33 pre-miRNA (lane 8–10) were incubated with rDicer for an increasing time. Lane 1 and 6 showed the size marker. Lane 2 and 7 were templates without adding rDicer. Both candidates could be efficiently processed by rDicer. (C) Luciferase assay. The sequences complementary to candidate miRNAs (Target) or scrambled control sequences (Scramble) were inserted into the 3′UTR of the Renilla luciferase gene in psiCHECK-2 plasmid. HEK293 cells were transfected with the plasmid alone or together with candidate pre-miRNA in vitro transcripts. All of the experiments were preformed in triplicates. Compared with scrambled controls, presence of target sequences of the candidate miRNAs in 3′UTR resulted in significant reduction of protein translation.
Figure 8. Experimental validation of the two novel miRNA candidates. (A) The RNA with size of 10–200 nt was separated using flashPAGE Fractionator (Ambion) and loaded onto the denaturing PAGE gel for northern blot. Northern blot of two novel miRNAs (CandidateMMU32 and CandidateMMU33) revealed bands corresponding to the mature miRNA (marked as *). (B) In vitro rDicer processing. 32P-labelled CandidateMMU32 pre-miRNA (lane 3–5) and CandidateMMU33 pre-miRNA (lane 8–10) were incubated with rDicer for an increasing time. Lane 1 and 6 showed the size marker. Lane 2 and 7 were templates without adding rDicer. Both candidates could be efficiently processed by rDicer. (C) Luciferase assay. The sequences complementary to candidate miRNAs (Target) or scrambled control sequences (Scramble) were inserted into the 3′UTR of the Renilla luciferase gene in psiCHECK-2 plasmid. HEK293 cells were transfected with the plasmid alone or together with candidate pre-miRNA in vitro transcripts. All of the experiments were preformed in triplicates. Compared with scrambled controls, presence of target sequences of the candidate miRNAs in 3′UTR resulted in significant reduction of protein translation.CC BY-NC · PubMed Central
Figure 9. Northern Blot validation of six novel miRNAs in C. sp. 11. *Signals denoted the putative mature miRNAs. Note that CandiCsp11_37 and CandiCsp11_49 have corresponding homologues in C. elegans as Cel-mir-58 and Cel-let-7, respectively.
Figure 9. Northern Blot validation of six novel miRNAs in C. sp. 11. *Signals denoted the putative mature miRNAs. Note that CandiCsp11_37 and CandiCsp11_49 have corresponding homologues in C. elegans as Cel-mir-58 and Cel-let-7, respectively.CC BY-NC · PubMed Central

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Metadata

Authors
Li, N., You, X., Chen, T., Mackowiak, S. D., Friedländer, M. R., Weigt, M., Du, H., Gogol-Döring, A., Chang, Z., Dieterich, C., Hu, Y., & Chen, W.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2013-02-08
Volume
41
Issue
6
Pages
3619-3634
ISSN
  • 1362-4962
  • 0305-1048
DOI
10.1093/nar/gkt072
PMC
PMC3616697
Cited by
57
References
1
Language
en
Licence
creativecommons.org/licenses/by-nc/3.0/ (vor, 1554 d)

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