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GENOME-WIDE ANNOTATION OFMICRORNA PRIMARY TRANSCRIPTSTRUCTURES REVEAL NOVELREGULATORY MECHANISMS
Tsung-Cheng Chang, Mihaela Pertea, Sungyul Lee,
Steven L. Salzberg and Joshua T. Mendell
presented by Cong Ma
2016/09/19
Outline
1. Background
2. Methods
3. Result
4. Conclusion
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BACKGROUND
miRNA
# ∼18-24 nt RNA
# negatively regulatetranslation andstability of mRNA
Julia Winter et. al., Nature Cell Biology 11, 228 - 234 (2009)
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Goal of this paper
# Much less is known about how miRNA expression isregulated.
# Incomplete miRNA gene (pri-miRNA) structure.
# pri-miRNAs are poorly represented in RNA-seq.
# Goal: Reconstruction of pri-miRNA structures, analyzeregulatory mechanism for miRNAs.
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METHODS
Reconstruction Methods
# Goal: increase pri-miRNAproportion in RNA-seq.
# By slowing down or disruptingDROSHA/DGCR8 activity.
# By transfectingtrans-dominant-negativeDROSHA mutant protein(TN-DROSHA).
# Then, use traditionaltranscriptome assemblymethod (stringtie software).
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Validation Methods
Stringtie will predict transcript by analyzing RNA-seqalignment.
# Does this transcript produce miRNA?◦ Compare with miRBase record
# Is this transcript correct in structure (TSS, exon, etc.)?◦ RT-PCR for exon-exon junction.◦ Colocalizaion with H3K4me3 promoter marks.◦ RACE for transcript start and end site.
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RESULT
Effectiveness of DROSHA inhibition
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Classification of miRNA gene structures
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Regulatory mechanisms - Alternative promoters
Alternative transcription start sites were differentially utilizedin a cell-line-specific manner.
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Regulatory mechanisms - Clustered miRNAs
MiRNAs in polycistronic clusters can be uncoupled throughalternative promoters.
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Regulatory mechanisms - Alternative splicing
Using different splicing sites can determine whether themiRNA is generated.
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CONCLUSION
Conclusion
# A multi-step strategy is developed to enable genome-widepri-miRNA reconstruction.
# Pri-miRNA structure revealed previously unrecognizedpotential regulatory mechanisms for miRNAs.
# Pri-miRNA annotation is promising for ongoing efforts todissect mechanisms of miRNA regulation.
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Thank you
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