Testing the complementarity requirements of plant microRNA-target interactions

Open Access
Author:
Liu, Qikun
Graduate Program:
Plant Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
May 28, 2014
Committee Members:
  • Michael Axtell, Dissertation Advisor
  • Sarah Mary Assmann, Committee Member
  • Ying Gu, Committee Member
  • Surinder Chopra, Committee Member
Keywords:
  • plant
  • microRNA
  • target
  • complementarity
  • afro-infiltration
  • Nicotiana benthamina
  • arabidopsis
  • small RNA annotation
Abstract:
Small RNAs are a group of regulatory RNAs of 20-30 nucleotides (nt) that are involved in diverse cellular processes across multiple eukaryotic kingdoms, including animals, plants and fungi. In complex with Argonaute (AGO) effector proteins, they recognize target RNA transcripts (coding and non-coding) based on sequence complementarity, and function as negative regulators at both transcriptional and post-transcriptional levels. It is widely accepted that plant microRNA-target interactions usually require a high degree of complementarity to trigger target cleavage. However, the sequence requirements for target repression at protein level through slicing independent mechanisms have never been systematically studied. Utilzing a self-designed dual-luciferase reporter system, I systematically examined the complementarity requirements for microRNA function through Nicotiana benhtamiana transient assay. I found that changes of the mRNA accumulation accounts for almost all observed regulatory effects. Comparison among those naturally occurring targets indicated that mismatches located near the miRNA 5’ends were more disruptive to miRNA function than those occurred near miRNA 3’ends. Actually, certain naturally occurring targets with unpaired bases only present at the miRNA 3’ends can even carry stronger regulatory efficiency than perfectly paired sites when position in 3’-untranslated region (UTR), but not open reading frame (ORF). I found that this was largely due to different behavior of perfectly paired sites, when placed in different contexts (ORF vs. 3’-UTR). While up to 3 mismatches could be tolerated at the 3'end without affecting the target efficacy, base pairing patterns that are typical of the known configuration of animal microRNA-target duplex did not trigger any detectable level of target down-regulation. Through collaboration we identified a non-canonical plant miRNA-target pairing pattern, where a 6-nt bulge on the target in between nucleotide 6 and 7 relative to the miRNA was tolerated. I demonstrated that this is very unique in terms of both the miRNA involved and the position of the bulge. De novo annotation and quantification of small RNA producing loci were also carried out in N. benthamiana. A total of 43652 small RNA producing loci, including 177 MIRNA, 4573 hairpin_RNA (hpRNA), and 38902 siRNA loci were identified. 95 MIRNA loci belonging to 37 plant known microRNA families were found. Comparing to the novel MIRNAs, these known MIRNA genes are processed with higher accuracy, and expressed more abundantly. Although small RNA clusters mainly producing 23 to 24 nt siRNA dominated hpRNA and siRNA loci, and 24-nt small RNAs are the most abundant small RNA species, 20 to 21 nt siRNAs are more robustly generated from certain highly active loci. Loci that give rise to longer small RNAs (23-24 nts) are generally less repetitive than those producing short small RNAs (20-22 nts). This is true for all types of annotated loci (MIRNA, hpRNA, and siRNA). MIRNA loci are enriched in genic regions and depleted in repeat regions, whereas siRNA loci, especially 23-24 nt groups, tend to occupy regions enriched for repetitive elements.