Beyond the seed: the identification of microRNA target sites in Caenorhabditis elegans
- Author(s): Broughton, James Paul
- Advisor(s): Pasquinelli, Amy
- et al.
MicroRNAs (miRNA) are critical regulators of development, cell differentiation, and the stress response. Mature miRNAs are small (~22 nucleotides) RNA molecules that post-transcriptionally regulate their targets by acting as guides for Argonaute (AGO) proteins. Despite the importance of these small RNAs in many pathways, the rules of miRNA target site recognition remain unclear. Currently, nucleotides 2-8 of the miRNA, termed the ‘seed’ sequence, are known to be critical for miRNA targeting. However, it is unclear if families of miRNAs, which share the same seed sequence, functionally target the same target sites. To improve our understanding of miRNA targeting, I generated a unique and reproducible dataset through the ligation of miRNAs to their target sites using individual nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) in the nematode worm Caenorhabditis elegans, which is detailed in Chapter 2.
As reviewed in Chapter 3, these ligated RNAs, called miRNA-target chimeras, provide biochemical evidence of which miRNAs are bound at a specific target site. Chapter 4 details my analysis of these chimeric data. Interestingly, the majority of miRNA target sites had the potential to support 3’ end interactions. In contrast to my prediction that these highly related miRNAs would bind the same sets of targets, family members from multiple miRNA families primarily targeted specific sets of transcripts. To confirm the importance of the 3’ end in vivo, I carried out in vivo rescue experiments that demonstrate that seed pairing is insufficient to mediate targeting in the absence of additional complementarity to nucleotides in the 3’ end of the miRNA. To confirm that 3’ end interactions direct binding specificity, I developed a novel method called ChimeraPCR (ChimP) that allowed for the detection of miRNA-target chimeras without the need for analyzing sequencing datasets.
To understand the role of AGO proteins in regulating lifespan in C. elegans, I generated several genetic tools, which are described in Chapter 5. These tools facilitated studies of how the highly related AGO proteins, ALG-1 and ALG-2, have opposing roles in controlling lifespan.