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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