The hidden rule in argonaute protein loading : sequence specificity

• Main Author: Goh, Eling
• Other Authors: Katsutomo Okamura
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences::Genetics
• Online Access: http://hdl.handle.net/10356/73227
• Institution: Nanyang Technological University

Argonaute (AGO) proteins play an important role in gene regulation with small RNAs serving as guides to targets. The small RNA duplex structure and 5’ terminal nucleotide are the only known determinants of AGO loading efficacy, and AGOs are believed to load small RNAs independently of their sequences. Challenging this notion, we developed HIgh-throughput Sequencing mediated Specificity Analysis (HISSA), a method that allows massively parallel analysis of AGO loading by using randomized single-stranded oligos in in-vitro loading assays and quantifying AGO-loaded RNA sequences with deep-sequencing. Intriguingly, HISSA divulged sequence specificity as the hidden rule governing fly AGO1 loading when 5’ Guanine-Adenine-Cytosine (GAC) motif was strongly preferred over the classic 5’ Uridine (U). Trends of ‘GAC’ motif enrichment in the 5’ ends of AGO1-loaded messenger RNA (mRNA) fragments are in line with our HISSA results. Our findings together with HISSA may have implications in therapeutic single-stranded RNAi designs.