Poster abstracts

Poster number 52 submitted by Yi-Hui Wang

Identification and characterization of novel proteins in the biogenesis of C. elegans PIWI-interacting RNAs

Yi-Hui Wang (Department of Biological Chemistry and Pharmacology), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology), Benjamin Pastore (Department of Biological Chemistry and Pharmacology; The Center for RNA Biology), Wen Tang (Department of Biological Chemistry and Pharmacology; The Center for RNA Biology)

Abstract:
The piRNA (PIWI-interacting RNAs) pathway plays a crucial role in maintaining genome integrity in Metazoan. Caenorhabditis elegans piRNAs are derived from over 16,000 discrete transcription loci, make it the largest gene family discovered to date. However, it remains unclear how piRNA expression is controlled at the transcriptional level. To address this gap, we employed a proximity-based labeling approach in combination with mass spectrometry to identify the proteins associated with piRNA genes (Hertz et al., 2022). This search captured all known transcription factors for piRNA transcription (Goh et al. 2014; Kasper et al. 2014; Weick et al. 2014). Remarkably, it also identified 19 additional proteins, including two previously uncharacterized protein—ATTF-6 and M01G5.1. Small RNA sequencing analysis showed the depletion of ATTF-6 or M01G5.1 led to the collapse of mature piRNAs and piRNA precursors. ATTF-6 belongs to the AT-hook family that preferentially binds to AT-rich DNA regions. Data from confocal microscopy and ChIP-Seq test the model that ATTF-6 drives the transcription of piRNA genes by binding to AT-rich regions at their promoter. Structural comparison using Alphafold2 revealed that M01G5.1 is structurally homologous to the cap-binding protein (Lee et al., 2016). My APBS electrostatics analysis further showed that M01G5.1 possesses a putative RNA binding channel with a highly positive charge. Integrating genetic and biochemical approaches, I am testing the hypothesis that M01G5.1 acts as a novel cap-binding protein, binding and stabilizing piRNA precursors. My ongoing research aims to elucidate the function of these novel factors and provide insights into piRNA transcription and processing.

References:
1. Goh, W. S. S., Seah, J. W. E., Harrison, E. J., Chen, C., Hammell, C. M., & Hannon, G. J. (2014). Genes & development, 28(7), 797-807.
2. Kasper, D. M., Wang, G., Gardner, K. E., Johnstone, T. G., & Reinke, V. (2014). Developmental cell, 31(2), 145-158.
3. Weick, E. M., Sarkies, P., Silva, N., Chen, R. A., Moss, S. M., Cording, A. C., ... & Miska, E. A. (2014). Genes & development, 28(7), 783-796.
4. Hertz, H. L., Price, I. F., & Tang, W. (2022). Bio-protocol, 12(8), e4386-e4386.
5. Lee, A. S., Kranzusch, P. J., Doudna, J. A., & Cate, J. H. (2016). Nature, 536(7614), 96-99.

Keywords: piRNA biogenesis, AT-hook transcription factor, Cap-small RNA binding protein